SCHOOL OF NATURAL SCIENCES

BACTERIOLOGY
CLBACT1

A Self-regulated Learning Module

A Learning Module in

Bacteriology
Antonina Guerrero, RMT, MAED
Bernadette P. Egtapen, RMT, MPH
Author

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 Bacteriology module
COURSE CODE: CLBACT1
COURSE TITLE: BACTERIOLOGY
COURSE DESCRIPTION:
The course encompasses a systematic study of the basic topics in Bacteriology. It is made up of
sufficient lectures and related laboratory experiments that describe bacteria‘s morphological
characteristics, physiology, and occurrences in nature. It also includes the study on its beneficial and
detrimental effects in man and the environment with emphasis on bacterial characteristics useful in the
isolation, identification, susceptibility to different antimicrobial drugs and the clinical application of
Bacteriology that will help the physician in the diagnosis and treatment of diseases.
REQUIREMENTS OF THE COURSE:
1. Regular Attendance to classes: You must attend online classes and live quizzes regularly by
logging in to our scheduled online activities. Online lectures will be done through Google meet
and/or facebook live. Assessments shall be given through Quizziz, Pear Deck, Canvas and/or
Google forms. For offline students, your attendance will be monitored through your responses
to text information and through timely correspondence.
2. Submission of required activities: All required activities (assignments, research work,
laboratory illustrations) should be submitted on or before the given deadline. Deadlines will be
posted by the teacher in the google classroom and messenger group chat. It will also be texted
to offline students. For online students, requirements must be submitted to the teacher‘s email
address which will be provided during the class orientation. For offline students, requirements
must be submitted via mail or express courier (e.g. LBC, JRS) addressed to: Instructor’s name,
School of Natural Sciences, University of Baguio, Baguio City.
 Quizzes using google forms and other media will be announced during scheduled
online lectures.
3. Seventy percent (70%) passing score in all required activities: Quizzes, exams, assignments,
research work, laboratory illustrations.
Computation of grades:
 The Course Grade is obtained by combining the lecture and laboratory grades (50%:50%)
for the subject.
 Laboratory grade shall be computed as 30% enhancement activities (illustrations; research
work; case study; experiments – when possible) plus 70% class standing (quizzes and
exams).
 The cumulative system of computing grades shall be followed. Grades computed for
midterms and finals are considered tentative. The final midterm grade is calculated by getting
1/3 of the first grading grade plus 2/3 of the tentative midterm grade and the final grade is
computed by getting 1/3 of the midterm grade plus 2/3 of the tentative final grade.

4. Study/Learning Guidelines:
a. Manage your time properly. As students of higher education (College), you are expected to
be more responsible in paying attention to course schedules, requirements, and deadlines.
Schedule how you will accomplish all the requirements in all your enrolled courses (reading

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 the modules, reading on research/ enhancement questions, doing assignments and
laboratory illustrations) and focus your attention when doing your tasks.
b. Observe proper conduct. Despite this online mode of learning, you must still maintain
appropriate behavior at all times. All standards of student conduct outlined in the University of
Baguio Student Handbook remain in full effect during this time of distance learning. Be
honest in answering your quizzes and exams. Work independently when accomplishing tasks
and assignments.
c. Stay motivated. Your future depends on what you do today. Maintain a positive attitude
towards learning and enjoy a fun-learning environment despite the current circumstances.
d. Maintain a performance of high standard. Give your best in accomplishing all the assigned
tasks. Do not be complacent with just a 70% passing cut-off score. Remember that this is a
board subject, and the best preparation for the board/licensure examination should be during
these formative years. The board review is but supplementary to the knowledge you have
already learned during your Med Tech education.
e. Communicate properly. Promptly respond to notifications by regularly visiting our google
classroom and messenger group chat. If you have confusions or queries in any part of this
module, I am here to guide you through. Send your academic concerns using the same
online platforms. For offline students, text messages and mobile calls are welcome during
scheduled hours of the day and week. Be guided by this schedule when communicating:
 Respect private hours. I do not always open my laptop/email/messenger 24/7. Send
your queries and/or concerns during regular office hours. For concerns that need
immediate attention, send through mobile text.
 Be patient. Messages received between 8 AM to 8 PM will be responded to within the
same day. Messages received after 8 PM will be answered starting 8 AM the next day.
 Before calling my mobile number, text first for permission for I might be giving an online
lecture or in a meeting or on private moment at that very instance.
 Saturdays and Sundays are for my family and home chores. I shall respond to
queries/messages received during these days within the first office hour of Monday.
f. Show mutual support. Support one another. Let us all be responsible and supportive in
making this new learning process more effective.
g. Live lecture/Video conferencing guidelines:
g.1 Be punctual. Live lectures/Video conferences will be scheduled during the official class
period/time of this course. Log in to the platform at least 5-10 minutes before the class
period. Prepare your learning materials such as this module, pens, papers, etc.
Attendance will be checked during the lecture/video conference.
g.2 Maintain professionalism.
- Wear appropriate clothing and set your gadget in an appropriate area. You may be
asked to turn on your video/camera at any time during the lecture.
- Log in using your UB gmail account. Unidentified names like nicknames, phone
models, etc. will not be allowed in the video conference.

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 - Mute your microphone as soon as you log in to the platform to avoid any excess
background noise. Unmute your microphone when instructed to do so.
- Be courteous. Do not interrupt your teacher or a classmate who is speaking. You may
type your question in the Chat area, or use the ―raise hand‖ feature if available, and
wait until you are allowed to speak.
- Respect privacy. Do not take a screenshot, picture, snapshott, etc. of your teacher or
fellow students, nor make any unnecessary audio or video recordings.
g.3 Remain focused and engaged. Do not be distracted by your gadget. Keep your
videoconference platform open and do not navigate other tabs or webpages unless
directed by your teacher.
LEARNING COMPETENCIES:
COGNITIVE
1. develop technical and judgmental skills in the proper use and handling of the microscope and
different equipment and apparatus in Bacteriology
2. relate the significance of microorganism in the disease process and everyday living
3. recognize and explain terms and origin of microbiology. 4. discuss updates related to
Bacteriology
4. identify and classify bacteria according to its morphology, structure, staining characteristics, and
growth requirements.
5. summarize new and or automated methods used in bacterial identification.
6. identify the role of microorganisms in immunity.
7. discuss serologic and molecular tests
8. develop judgmental, technical, and clinical skills in identifying the members of Pyogenic cocci.
9. discuss the pathogenesis and prevention and control of disease transmission.
10. Differentiate normal flora from transient flora.
11. Compare and contrast health from disease.
12. Relate the etiologic agent, mode of transmission and the host to disease formation
13. Compare and contrast the types of body defenses against infections
14. Identify and illustrate Genus Staphylococcus, Streptococcus and Neisseria
15. Systematize a step-by-step procedure in the identification of pyogenic cocci
16. Describe the diseases caused by pyogenic cocci including its treatment and prevention
17. Explain the pathogenesis of pathogenic Mycobacteria species
18. Explain why Acid-Fast Staining is Required for Mycobacteria
19. Differentiate sporeforming from non-sporeforming Gram Positive bacilli.
20. Differentiate aerobic from anaerobic Gram-Positive bacilli.
21. Describe the diseases caused by Gram + bacilli including its treatment and prevention
22. Explain the different ways of providing the necessary environment for anaerobic culture
23. Differentiate enteric from non-enteric gram-negative bacilli
24. Identify the different gram-negative bacilli according to its biochemical reactions
25. Describe the diseases caused by Gram negative bacilli including its treatment and prevention
26. Recommend ways in preventing bacterial contamination in water and food.
27. Describe the different types of spirochetes
28. Describe the diseases caused by spirochetes including its treatment and prevention
29. Describe the morphology of Rickettsia and Chlamydia species
30. Describe the diseases caused by Rickettsia and Chlamydia species including its treatment and
prevention

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 31. Describe the morphology of Mycoplasma and Ureaplasma species
32. Describe the diseases caused by Mycoplasma and Ureaplasma species including its treatment
and prevention

AFFECTIVE
1. Appreciate the body‘s defense mechanism
2. Have a sense of responsibility for the control and prevention of Tuberculosis in the Philippines
3. Educate the public on the possible diseases caused by the presence of bacterial contaminants

PSYCHOMOTOR
1. develop judgmental, mathematical, clinical, and technical skills in specimen management,
preparation, selection, and use of the different inoculation and sterilization techniques, and
culture media.
2. perform the different identification techniques related to Bacteriology.
3. select, and perform specimen collection and antimicrobial susceptibility test.
4. follow standard precautions, asepsis, and quality assurance program.
5. correctly perform tests for the differentiation and identification of Pyogenic cocci according to
standards.
6. develop analytical and research skills in relation to bacteriology

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 TABLE OF CONTENTS
I. Introduction to
Microbiology
Lesson1. Microscopy Page 9
Lesson 2. Branches Page 21
Lesson 3. Importance Page 21
Lesson 4. History Page 21
Lesson 5. Germ Theory of Disease Page 24
Lesson 6. Taxonomy Page 26

II. Introduction to Bacteriology

Lesson 1. Definition Page 34
Lesson 2. Prokaryote vs. Eukaryote Page 34
Lesson 3. Morphology (size, shape, and arrangement) Page 35
Lesson 4. Cell Structure Page 35
Lesson 5. Microscopic Study of Bacteria Page 54
Lesson 6. Growth and Reproduction Page 57
Lesson 7. Sterilization and Disinfection Page 63
Lesson 8. Cultural study of Bacteria Page 79
Lesson 9. Specimen Management Page 86
Lesson 10. Quality Assurance in the Microbiology Lab Page 100
Lesson 11. Antimicrobial agents Page 103
Lesson 12. Antimicrobial Susceptibility Testing (Automated,Diffusion, Dilution) Page 104
Lesson 13. Principles of disease and Epidemiology Page 113
III. Immunology
Lesson 1. Innate Immunity Page 129
Lesson 2. Specific Host Defense Page 129
Lesson 3. Types of Immunity Page 134
Lesson 4. Practical Application (Antisera& Vaccines Page 135
IV. Medical and Diagnostic
Bacteriology
Lesson 1.Micrococcaceae Page 142
Lesson 2.Streptococcaceae Page 145
Lesson 3. Neisseriaceae Page 156

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Lesson 4. Mycobacteriaceae Page 161
Lesson 5. Aerobic Non-sporeformers Page 168
Lesson 6. Aerobic Sporeformers Page 176
Lesson 7 Anaerobic spore formers Page 179
Lesson 8 Enterobacteriaceae & Non-enteric Bacilli) Page 184
Lesson 9 Spirochetes (Treponema, Leptospira, Borrelia,Spirrilum) Page 205
Lesson 10 Obligate Intracellular Bacteria (Ricketssia, Chlamydia) Page 216
Lesson 11 Atypical Bacteria (Mycoplasma, Ureaplasma) Page 224

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TIME/SCHEDULE: Week 1
TIME ALLOTMENT: 9 HOURS
Topics: lecture
Unit 1: introduction to bacteriology
1. Microscopy
2. Branches
3. Importance
4. History
5. Germ theory of disease
6. Taxonomy

Laboratory
Activity No. 1: Microscopy and Instrumentation
Activity no. 2: The Bacterial Cell

LEARNING OBJECTIVES
At the end of the learning session, the student should be able to
1. manipulate the microscope correctly
2. accurately name the scientists who contributed to the science of microbiology
3. completely understand the germ theory of disease
4. write the scientific name of the bacteria correctly
Lesson 1: MICROSCOPY
TIME ALLOTMENT: 1 hour lecture
The history of microscopy has been closely linked to the beginning of microbiology since 1665,
when Hooke published his treatise Micrographica, which included illustrations of mold forms and the
anatomy of the flea (1). Today, light microscopy is used not only in microbiology, pathology, and cell
biology but also in metallurgy, materials science, computer chip design, and microsurgical applications.
This chapter will attempt to describe the basic concepts of light microscopy as they are practiced in the
microbiology laboratory. (Wiedbrauk, 2015)
Technical Background and Definition of terms
Activity 1: Define the following terms
1. Aberration 7. Köhler Illumination
2. Contrast 8. Mechanical tube length
3. Depth of field 9. Numerical aperture
4. Depth of focus 10. Refractive index
5. Immersion fluid 11. Resolution
6. Working distance 12. Parfocal

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SIMPLE MICROSCOPE
- A simple microscope contains a single bi-convex magnifying lens which is thicker in the center
than at the periphery.
- It produces a magnified image that is in the same orientation as the original object.
- Disadvantages:
o Have limited resolution and magnifying power because of their low NA
o Most commercial magnifiers are able to produce a ×2 to 30 magnification, and the better
lenses will have a resolution of about 10 μm.
- Uses: dissection, examination of bacterial colonies, and interpretation of agglutination reactions.
- Examples: jeweler‘s loupes, photographic slide viewers, and simple magnifying or reading
glasses
COMPOUND MICROSCOPE
The first compound microscopes were constructed around 1590 by Dutch spectacle makers
Zaccharias Janssen and Hans Janssen. The Janssen microscope consisted of an object lens
(objective) that was placed close to the specimen and the eye, or an ocular lens that was placed close
to the eye. The lenses were separated by a body tube.
- The objective lens projects a magnified image into the body tube and the eyepiece magnifies
the projected image
- It produces a two-stage magnification
The Light Microscope

- The light microscope is an instrument for visualizing fine detail of an object. It does this by
creating a magnified image through the use of a series of glass lenses, which first focus a beam
of light onto or through an object, and convex objective lenses to enlarge the image formed.
(sciencedirect.com)
- The light microscope is named as such because it uses visible light to detect small objects
Types of light microscope
1. Bright field
2. Dark field
3. Phase contrast
4. Fluorescence
BRIGHT FIELD MICROSCOPE

- An ordinary type of microscope

- It forms a dark image against a brighter background
Parts of the bright-field microscope
1. Ocular lens (eyepiece) - This part remagnifies the image formed by the objective lens
- The lens should be small to achieve magnification with good resolution
2. Body tube - It transmits the image from the objective lens to the ocular
3. Arm - It is used for holding the microscope
4. Nosepiece - It holds the objectives
5. Objective lenses - These are the primary lenses that magnify the specimen

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 - It is the most important part of the microscope
6. Mechanical stage - It holds the slide in place
7. Condenser - Focuses light through the specimen
8. Diaphragm - Controls the amount of light entering the condenser
9. Coarse adjustment knobs - Focuses the image under low power and usually move the stage
10. Fine adjustment knob - It sharpens the image under all types of objectives
11. Illuminator - Light source
12. Base – supports the microscope
Resolution

- The ability of a lens to separate or distinguish between small objects that are close together
Ernst Abbe

- Formulated the Abbe sine equation which shows the maximum resolution for a microscope

d = 0.5 λ * As d becomes smaller, the resolution increases

n sin θ

Where:
λ = wavelength of light
n sin θ = numerical aperture
d = min. distance bet. 2 objects
wavelength

- Important factor
- Must be shorter than the distance between 2 objects
- The shorter the wavelength, the higher the resolution
- Use blue end of visible spectrum (450 – 500 nm)
Numerical Aperture (n sin θ)

- θ – ½ the angle of the cone of light entering an objective

- n – refractive index
o The n of air is 1.00

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 o No lens working in air can have NA of greater than 1
o To increase the refractive index, use immersion oil (a liquid with the same refractive
index as glass)
o Air is replaced in immersion oil

(https://microscope-microscope.org/microscope-info/numerical-aperture/)

• The maximum theoretical resolving power of a

microscope with an oil immersion objective (NA 1.25)
and blue green light is approximately 0.2 um
d = (0.5)(530nm) = 212 nm or 0.2 um
1.25
* A bright field microscope can distinguish between 2
dots around 0.2 um apart
(http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html)

Working Distance

- Distance between 2 front surface of the lens and the surface of the cover glass (if one is used)
or the specimen when it is in sharp focus
- Objectives with large numerical apertures and great resolving powers have short working

distances
(https://dovermotion.com/applications-capabilities/automated-imaging/microscope-calculations/)

DARK-FIELD MICROSCOPE

- It uses darkfield condenser that blocks light that would enter the objective directly
- It directs the light to hot the specimen at an oblique angle

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 o All the other light that passes through the specimen will miss the objective, thus making
the background a dark field.
o Organisms will appear extremely bright against a dark field
- It is used to view spirochetes (Treponema pallidum)
- It is used to examine unstained microorganisms suspended in liquid, living organisms that are
invisible in ordinary light.

(https://microbeonline.com/dark-field-microscopy-principles-use-advantages-and-limitations/)

Phase contrast microscope

- Converts slight differences in refractive index and cell density into easily detected variations in
light intensity
- The phase differences are seen through the
microscope as different degrees of
brightness
- Staining is not part of phase-contrast
microscopy
- It permits detailed examination of internal
structures in living organisms
- It is used to identify medically important fungi
grown in culture
- Detection of bacterial components
(endospore and inclusion bodies containing β
– hydroxy butyrate, polymetaphosphate,
sulfur or other substances)
- Condenser: annular stop – opaque disk with
a thin transparent ring which produces a
hollow cone of light
- The background is bright while the unstained
object appears dark as well as well-defined
(Dark Phase microscopy)
(https://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phase.html)

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Fluorescence Microscope

- When some molecules absorb radiant energy, they become excited and release much of the
trapped energy as light
- Exposes the specimen to UV light, violet or blue light and forms an image of the object with the
resulting fluorescence light
- Fluorochromes – stains used in this technique
o Fluoresce brightly upon exposure to light of a specific wavelength
o The microscope forms an image of the fluorochrome-labeled microorganisms

(https://microbenotes.com/fluorescence-microscope-principle-instrumentation-applications-advantages-limitations/)

Electron Microscope

- is one of the most advanced and important types of microscopes

- highest magnifying capacity.
- electrons are used to illuminate the tiniest particles.
- more powerful tool than light microscopes
How the EM works
- Energy source - a beam of electrons.
- Electrons
o have exceptionally short wavelength
o strikes most objects in its path
o travel in a vacuum to avoid contact with deflecting air molecules
o magnets focus the beam on the object to be viewed.
o An image is created on a monitor and viewed by the technologist
o increases the resolution of the microscope significantly
Transmission Electron Microscope (TEM)
- The more traditional form of electron microscope.

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 - ultrathin slices of microorganisms or viruses are placed on a wire grid
- Stain: gold or palladium
- The densely coated parts of the specimen deflect the electron beam, and both dark and light
areas show up on the image
The Scanning Electron Microscope (SEM)

- The more contemporary form electron microscope.

- this microscope gives lower magnification than the TEM
- permits three-dimensional views of microorganisms and other objects.
- Whole objects are used
- Stain: gold or palladium
The Scanning tunneling Microscope (STM)

- a type of electron microscope

- shows three-dimensional images of a sample
- the structure of a surface is studied using a stylus that scans the surface at a fixed distance
from it.
- electrons tunnel between the surface and the stylus, producing an electrical signal.
- stylus tip is formed by one single atom
- it scans across the surface at a distance of only an atom's diameter.
- The vertical movement of the stylus is recorded
ASSESSMENT: TBA

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LABORATORY:
TIME ALLOTMENT: 2 HOURS

Name: ______________________________________________
Group No. ___________________________________________

Exercise No. 1

MICROSCOPY AND INSTRUMENTATION

Introduction
The microscope is an indispensable tool in microbiology. It magnifies the minute details of the
object under study. The microscope provides the technologist with daily opportunity to familiarize with
the microscopic structure, morphology and staining characteristics of microorganisms.
Objectives

1. To introduce students of one of the chief scientific tools in microbiology

2. To impress students with the importance of proper care and maintenance of such a precision
instrument
Materials:

A. Equipment
a. Microscope
b. Incubator
c. Autoclave
d. Hot air oven
e. Water bath
f. Centrifuge
B. Glassware
a. Test tubes
1. Plain
2. Screw-capped tubes
3. Centrifuge tubes
b. Fermentation tubes
1. Smith fermentation tubes
2. Durham fermentation tubes
c. Petri dishes
1. Plain
2. With division
d. Flasks
1. Erlenmeyer flasks
2. Volumetric flasks
e. Serological pipettes
f. Beaker
g. Funnel
h. Watch glass
i. Glass slide and cover slip
j. Graduated cylinder

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 C. Apparatus
a. Alcohol lamp/Bunsen burner
b. Inoculating loop
c. Inoculating needle
d. Staining rack
e. Forceps
f. Tripod
g. Wire gauze
Procedure A: Microscope
Use and Care for the microscope:
Watch:
A. https://www.youtube.com/watch?v=vlwtTLKWYSY
B. https://www.youtube.com/watch?v=SUo2fHZaZCU
C. https://www.youtube.com/watch?v=eZX9U15F5Q8
Parts of the Microscope
1. Place the microscope in proper working position on a table at a convenient height. As you look
into the microscope, be sure you are comfortably seated and that you do not have to stretch or
that you are not cramped,
2. Keep both eyes open when looking through a monocular microscope. With a little practice, you
can do this easily.
3. Locate all parts of the microscope
4. Place a prepared microslide on the stage of the microscope. Be sure that the slide lies flat on
the stage
5. Elevate the top of the condenser so that it is flush with the surface of the stage
6. Focus on the specimen with the lowest powered objective (scanner)
Illumination

Note: for maximum performance from the microscope, the lighting must be efficient.
1. Many modern microscopes have built-in base illuminators. Adjust the illumination so that it is
even throughout the microscopic field of view.
2. When using low powered objectives such as LPO and scanner, adjust the illumination to a
lesser degree, on the other hand, for HPO and OIO, use a higher degree of illumination.
Contrast: The iris diaphragm
1. Observe changes when the iris diaphragm is opened or closed
2. Note changes in contrast of the specimen image in the microscope as well as subtle changes in
resolution
Focus: The coarse and fine adjustments
1. Observe the changes in focus when the coarse and fine adjustments are manipulated
2. Note the difference between the results obtained with the coarse and fine adjustments

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Resolution: Function of the three objectives
1. To focus with the low power objective, position the objective with the coarse adjustment until it‘s
nearly (but not quite) touches the cover glass or the upper surface of the mounted specimen.
2. Bring the specimen into view by moving the objective away from the specimen. To some
microscopes, this means moving the objective up from the stage, in other models, the stage is
lowered away from the objective.
Note: the distance from the specimen to the objective lens at which the specimen is in focus is
the focal length. For the low power objective lens, this is 16 mm.
3. Complete focusing, that is, clear the specimen image with the fine adjustment.
4. To use the high power (high dry) objective, first bring the specimen into view and center it with
the low power objective. Then move the high-power objective into position.
5. Complete focusing with the fine adjustment. Center specimen image if necessary.
Note: the constant movement of the fine adjustment is essential in producing a three-
dimensional effect on the specimen image in microscopy. The fine adjustment is rotated slightly
clockwise and the slightly counterclockwise
6. To use the oil immersion objective, first place a drop of oil immersion oil on the cover glass of
the microslide or on the upper surface of the specimen preparation.
7. Lower the oil immersion objective with the coarse adjustment into the drop of oil until the
objective engages the drop and spreads it somewhat. The objective does not quite touch the
surface of the microslide.
8. Complete focus with the fine adjustment.
Note: if the immersion objective comes out of the oil in the process of focusing, the focal length
of the lens has been exceeded.
Care of the Microscope
1. Clean the microscope when the experiment is ended. Be sure to remove oil from the oil
immersion objective. Use only lens paper on the optical glass parts (objectives and oculars) of
the microscope.
2. Leave the microscope with the lowest power objective in the working position. (if the optical
system is accidentally jammed down, the least expensive objective will be damaged,)
3. Place a plastic cover over the microscope or place it in tis case. Return it to the locker.
4. Carry the microscope with the arm of the scope in one hand the palm of the opposite hand
supporting the base.
5. Remove immersion oil from prepared slides.
Precautions in handling the microscope

1. Keep the microscope clean and handle all parts with care
2. Do not allow chemical to contact the microscope since they may damage the finish
3. Clean the mechanical parts by applying recommended oils and the oils may be wiped out with
lens paper.
4. To remove immersion oil from the optical glass parts, wipe with lens paper moistened with xylol.
Discard the lens paper and wipe rapidly to prevent damage to the optical glass settings.

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Procedure B: illustrations and uses
Equipment:

1. Thoroughly scan the internet for a good illustration/photo of the abovementioned equipment.
Choose photos with clear labels. You may also draw if you choose so.
2. Below each clear photo of the equipment, state its use and the principle behind it.
3. A maximum of 2 photos of the equipment per page is recommended except for the microscope
which should be illustrated on a separate page.
Glassware and apparatus.
1. Carefully scan the internet for a clear illustration of the abovementioned glassware and
apparatus.
2. Below each photo, state its use in the bacteriology laboratory.
3. A maximum of 4 glassware and apparatus per page is recommended.

Questions for Research:

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. Describe the quality control measures for the following

a. Microscope
b. Incubator
c. Hot air oven
d. Autoclave
e. Water bath
f. Inoculating loop
2. Differentiate the types of biological safety cabinets
3. Explain the use of the low-power, high dry and oil immersion objectives. What is meant by
―immersion objective‖?
4. Compute for the total magnification of the following objectives of a microscope given the
following:

Objectives Ocular
Designation Magnification Focal Numerical Final magnification with
length aperture 8x 10x 12.5x 15x 20x
(NA)
Scanning 4x 46 mm 0.1

Low power 10x 16 mm 0.25

High dry 43x 4 mm 0.55

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 Oil 97x 1.8 mm 1.30
immersion

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Lesson 2: Microbiology and its branches
TIME ALLOTMENT: 0.5 hour
Microbiology
- Study of organisms and agents too small to be seen by the unaided eye
- Study of microorganisms
Branches of Microbiology

- Bacteriology – study of bacteria

- Mycology – study of fungi
- Virology – study of viruses
- Phycology – study of algae
- Protozoology – study of protozoans
Activity 1: Describe the following scopes of microbiology.
1. Medical microbiology
2. Public health microbiology
3. Immunology
4. Agricultural microbiology
5. Microbial ecology
6. Food and dairy microbiology
7. Industrial microbiology
8. Microbial physiology and biochemistry
9. Microbial genetics and molecular biology
Lesson 3: Importance of Microbiology

In your own words, state the importance of microbiology.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
________________________________________________________________

Lesson 4: History and the Germ theory of disease

TIME ALLOTMENT: 1.5 hours
Microbiology has been defined as the study of organisms that are too small to be seen by the
naked eye. In the beginnings of this study, great minds have contributed to the discovery and evolution
of microbiology, and its relationship to medicine and other areas of biology.
The Discovery of Microorganisms
Roman Philosopher Lucretius (98 – 55 BC) and

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Girolamo Fracastoro (1478 – 1553)
- Suggested that diseases were caused by ―invisible living creatures‖
Francesco Stelluti (1625 and 1630)
- He made the earliest microscopic observations on bees and weevils using a microscope
probably supplied by Galileo
Anton van Leeuwenhoek (1632 – 1723)

- The ―first true microbiologist‖

- He was the first person to observe and describe microorganisms accurately
- He described the microorganisms from the pond water as ―animalcules‖
- He used his self-made single lens microscope with 50 – 300x magnification
Spontaneous Generation
People (including scientists) believed that simple living organisms could come into being by
spontaneous generation. This was the idea that non-living objects can give rise to living organisms. It
was common "knowledge" that simple organisms like worms, beetles, frogs, and salamanders could
come from dust, mud, etc., and food left out, quickly "swarmed" with life.
(https://www2.nau.edu/gaud/bio301/content/spngen.html)

Aristotle (384 – 322 BC)

- Simpler invertebrates could arise from spontaneous generation

Francesco Redi (1626 – 1697)
- In 1668, even before Leeuwenhoek‘s discovery of the microscope, he demonstrated that
maggots do not arise spontaneously from decaying meat
- His results were a serious blow to the long-held belief that large forms of life could arise from
non-life
John Needham (1748)
- He observed that boiled mutton broth (tightly sealed) eventually became cloudy with
microorganisms
- He proposed that organic matter possessed a ―vital force‖ that could give rise to life.
Lazzaro Spallanzani (1729 – 1799)
- He improved the previous experiments of Needham by using sealed boiled water and seeds
- He observed that no growth took place as long as the flasks remained sealed.
- He proposed that air carried microorganisms to the culture medium and that external air might
be essential for the growth of animals present already in the medium
Biogenesis
Biogenesis is any process by which lifeforms produce other lifeforms. For example, a spider
lays eggs that become other spiders. This premise historically contrasted with the ancient belief in
spontaneous generation, which held that certain inorganic substances, left alone, give rise to life (such
as bacteria, mice and maggots) in a matter of days. The premise of biogenesis had been suspected

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long before being definitively demonstrated. A demonstrative experiment, which showed biogenesis
right down to the bacterial level, was devised by Louis Pasteur in 1859. (https://sciencing.com/theory-biogenesis-
5419233.html)

Rudolf Virchow (1858)

- He challenged spontaneous generation with the concept of biogenesis
Theodore Schwann (1810 – 1882)
- He observed that no growth occurred in a flask containing nutrient solution after allowing air to
pass through a red-hot tube
Georg Friedrich Schroder and Theodore van Dusch

- They observed that no growth occurred after allowing air to pass through sterile cotton wool
placed in a flask of the heat-sterilized medium
Louis Pasteur (1822 – 1895)
- He resolved the issue of spontaneous generation
- He stated that microorganisms are indeed present in the air and can contaminate seemingly
sterile solutions, however the air itself does not create microbes
- He showed that microorganisms can be present in non-living matter
- He stated that microbial life can be present in non-living matter
- He stated that microbial life can be destroyed by heat (basis of the aseptic technique –
techniques to prevent contamination by unwanted microorganisms)
- He provided evidence that microorganisms cannot originate from mystical forces present in non-
living materials
 However, according to Ferdinand Cohn, no matter how long some flasks were
boiled, they always produce certain growth – heat resistant bacterial spores.
John Tyndall (1820 – 1893)
- He showed that dust carry germs which contaminates sterile broth
- ―tyndallization‖ – form of sterilization for three consecutive days
Fermentation and Pasteurization
- Theodore Schwann stated that yeast cells were responsible for the conversion of sugars to
alcohol, however he said that fermentation was not due to microorganisms but to a chemical
instability that converted sugars to alcohols
- Pasteur described that certain microorganisms knows as ―yeast‖ converts sugar to alcohol in the
absence of air (fermentation)
- Souring and spoilage are caused by different microorganisms called bacteria
- In the presence of air, bacteria change the alcohol in the beverage into vinegar (acetic acid)
- Heating the bear and wine just enough to kill most of the bacteria (pasteurization).

Pasteur’s contribution to science

o He disproved the theory of spontaneous generation
o He developed vaccines against anthrax (1861) and rabies (1885)
o He improved the wine industry (theory of fermentation)

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 Charles Chamberland
o Created a porcelain bacterial filter (1884) and developed anthrax vaccine together with
Pasteur
The Germ Theory of Disease
At the same time Pasteur began his fermentation studies, he adopted a related view on the
cause of diseases. He and a minority of other scientists believed that diseases arose from the activities
of microorganisms—germ theory. Opponents believed that diseases, particularly major killer diseases,
arose in the first instance from a weakness or imbalance in the internal state and quality of the afflicted
individual. In an early foray into the causes of particular diseases, in the 1860s, Pasteur was able to
determine the cause of the devastating blight that had befallen the silkworms that were the basis for
France‘s then-important silk industry. Surprisingly, he found that the guilty parties were two
microorganisms rather than one. (https://www.sciencehistory.org/historical-profile/louis-pasteur)
Joseph Lister (1827 – 1912)
- He developed the antiseptic system of surgery
- He demonstrated the use of phenol for treating surgical wound and also sprayed phenol over
the surgical area
Robert Koch (1843 – 1910)
- He established the first proof that bacteria indeed cause diseases
- He discovered Bacillus Anthracis – causes Anthrax (1876 – 1877)
- He discovered Mycobacterium tuberculosis (1882)
- He was the first to culture bacteria on boiled potatoes, gelatin and used meat extracts and
protein digests for cultivation
- He developed culture media for observing growth of bacteria isolated from the human body.

Koch’s Postulate
o The microorganism must be present in every case of the disease but absent from
healthy organisms
o The suspected microorganism must be isolated and grown in a pure culture
o The same disease must result when the isolated microorganism is inoculated into a
healthy host
o The same organism must be isolated again from the diseased host
 Fannie Eilshemius Hesse – suggested the use of agar as a solidifying agent
 Richard Petri – developed the petri dish (plate)
 Martinus Beijerinck and Sergie Winogradsky – developed the enrichment-culture
technique and the use of selective media
Immunological Studies – Vaccination
- Edward Jenner (1749 – 1823) – found a way to protect people from small pox
- Pasteur used the term ―vaccine‖ (Latin ―vacca‖ -cow)
- Emil von Behring – prepared antitoxins for diphtheria and tetanus

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Modern Therapy” “Magic Bullet”
Chemotherapy

- The treatment of disease using chemical substances

- It also refers to chemical treatment of non-infectious diseases, such as cancer
o Synthetic drugs – prepared from chemical in the laboratory
o Antibiotics – produced naturally by bacteria and fungi to act against microorganisms
- Paul Ehrlich – discovered salvarsan (arsphenamine) for treatment of syphilis
- Alexander Fleming – discovered penicillin (Penicillium notatum)
- Howard Florey and Ernst Chain - made the purification process for penicillin

Activity: Fill up the following table with the correct data

Important events in the development of Microbiology

YEAR SCIENTIST CONTRIBUTION/EVENT

1676 Discovered animalcules
1786 First classification of bacteria
1798 Edward Jenner
1838 - 1839 Schleiden and Schwann
1858 Theory of Biogenesis
1867 Antiseptic surgery
1881 Developed Anthrax vaccine
Robert Koch Cultured bacteria on Gelatin
1882 Robert Koch Discovered tubercle bacilli
1884 Robert Koch First publication of Koch‘s postulates
Discovered phagocytosis
NA Autoclave was developed
Hans Christian Gram
1885 Louis Pasteur Developed the rabies vaccine
1886 Richard Petri Developed the Petri dish
1890 Von Behring Prepared antitoxins for Diphtheria and Tetanus
1895 Discovered complement
1896 Ronald Ross Malaria parasite is carried by mosquitoes
1900 Walter Reed
1901 Discovered blood group system
1906 T. pallidum causes syphilis
1907 NA Role of protozoa in the disease process
1910 Paul Ehrlich Developed therapeutic agent of Syphilis
1923 NA First edition of Bergey‘s Manual
1933 Developed the first Transmission electron microscope
1937 Edouard Chatton Divided organisms into prokaryotes and eukaryotes
1944 Selman Waksman
1945 Fleming and Chain Discovered penicillin and its therapeutic use

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 1951 Developed yellow fever vaccine
1953 NA Development of the phase contrast microscope
1954 Developed the radio immunoassay technique
1962 Discovered the structure of the DNA
1975 NA Lime disease was discovered
1976 NA Archaeobacteria as a distinct microbial group
1980 NA Development of Scanning Tunneling microscope
1983 - 1984 Gallo and Montagnier
PCR
1986 NA 1st hepatitis vaccine from genetic engineering
1995 NA Chicken pox vaccine was approved for US use

Lesson 5: Taxonomy
Time allotment: 1 hour lecture
Bacterial taxonomy is concerned with the naming of bacterial organisms and with organizing
these names according to various criteria. Overall, classification involves the recognition of similarities
and relationships as a basis for the arrangement of the bacteria into taxonomic groups or taxa. The basic
taxon is the species. Identification also involves the recognition of a bacterium as a member of one of the
established taxa, appropriately named, by the comparison of a number of characters with those in the
description. (https://www.accessscience.com/content/bacterial-taxonomy/069700)
Taxonomy
- Greek
o Taxis – arrangement or order
o Nomos – law
o Nemein – to distribute or govern
- Taxonomy is the science of biological classification

3 parts of taxonomy
- Classification
o Arrangement of organisms into groups or taxa (sing. taxum)
o Based on mutual similarity or relatedness
- Nomenclature
o Branch of taxonomy concerned with assignment of names to taxonomic groups in
agreement with published rules
- Identification
o Process of determining that a particular isolate belong to a re-organized taxon
Systematics
- The scientific study of organisms with the ultimate object of characterizing them in a orderly
manner
- Encompasses
o Morphology
o Ecology
o Epidemiology
o Biochemistry

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 o Molecular biology
o Physiology
Bergey’s Manual of Systematic Bacteriology
- This is the most widely followed classification and nomenclature system in the US and was
first published in 1974.
- The American Society for Microbiology (originally the Society of American Bacteriologists)
has for decades published a compilation of known bacteria, first as Bergey's Manual of
Determinative Bacteriology and more recently as Bergey's Manual of Systematic
Bacteriology.
- Multiple editions of Bergey's Manual of Determinative Bacteriology, published between 1923
and 1994, organized bacteria in groups by phenotypic characteristics, with no attempt to sort
out higher phylogenetic relationships.
- They were very useful for identifying unknown bacterial cultures
- The first edition of Bergey's Manual of Systematic Bacteriology, which came out in four
volumes from 1984 through 1989, attempted to organize bacterial species according to
known phylogenetic relationships, (https://www.ruf.rice.edu/~bioslabs/BIOC318/bergeys.asp)

Bacterial Classification
Kingdoms

2-kingdom classification by Aristotle (384 – 322 BC)

- Plants
- Animals
3-kingdom classification by Ernst Haeckel (1735)
- Plants
- Animals
- Protista – includes singles celled organisms, bacteria, protozoa, fungi and algae
4-kingdom classification by Herbert Copeland (1938)
- Plants – includes fungi
- Animals
- Protista – amoebas, diatoms, some unicellular and multicellular eukaryotes
- Monera/Prokaryotes – bacteria
5-kingdom classification by Robert Whittaker (1957)
- Plants
- Animals
- Protista – algae and protozoa
- Monera/Prokaryotae – bacteria and archaeans
- Fungi

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Taxonomic Ranks (descending)
- Kingdom
- Division
- Phyla
- Classes
- Orders
- Families
- Genera
- Species
Linnaean Scheme of Classification

- Bacteria may be classified as to:

o Kingdom
o Class (-aceae)
o Order (-ales)
o Family (-aceae)
o Tribe (-ieae)
Binomial System – by Carl Von Linne/ Carolus Linnaeus
- Latinized, italicized name
- Generic name
o First part
o Capitalized
o May be changed
- Specific epithet
o Second part
o Stable
o The oldest epithet for a particular organism takes precedence and must be used
- Names may be shortened by abbreviating the genus name with a single capital letter
o e.g. E. coli
Terminologies
- Species
o The basic taxonomic group in microbial taxonomy
- Bacterial Species
o Collection of strains that share many stable properties and differ significantly from other
groups of strains
- Strain
o population of organisms that descends from a single organism or pure culture isolates
- Type strain
o One strain of species that is more fully characterized than other strains
- Biovars
o Variant bacterial strains
o Characterized by biochemical physiological differences
- Morphovars
o Differ morphologically

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 - Serovars
o Distinctive antigenic property
- Genus
o Well-defined group of one or more species that is clearly separate from other genera
Characteristics used in Taxonomy
- Morphological characteristics
- Physiological and metabolic characteristics
- Ecological characteristics
- Genetic analysis
- Molecular characteristics
o Comparison of proteins
o Nucleic acid base composition
o Nucleic acid hybridization
o Nucleic acid sequencing
Morphological Characteristics
Reasons
a. Easy to study and analyze
b. Structural features depend on the expression of many genes
c. Usually genetically stable
d. Normally do not vary greatly with environmental changes
e. Morphological similarity is a good indication of phylogenetic relatedness
Morphological Features

- Cell shape
- Cell size
- Colonial morphology
- Ultrastructural characteristics
- Staining behavior
- Cilia and flagella
- Mechanism of motility
- Endospore shape and location
- Cellular inclusions
- color
Physiological and metabolic characteristics
- Directly related to the nature and activity of microbial enzymes and transport proteins
- Provides indirect comparison of microbial genomes
- Some important physiological and metabolic characteristics used for classification and
identification
o Carbon and Nitrogen content
o Cell wall constituents
o Energy sources
o Fermentation products
o General nutritional status

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 o Growth temperature optimum and range
o Luminescence
o Mechanisms of energy conversion
o Motility
o Osmotic tolerance
o Oxygen relationships
o pH optimum and growth range
o Photosynthetic pigments
o Salt requirements and tolerance
o Secondary metabolites formed
o Sensitivity to metabolic inhibitors and antibiotics
o Storage inclusions
Ecological characteristics
- Life cycle patterns
- Nature of symbiotic relationships
- Ability to cause disease in a particular host
- Habitat preferences
o Requirement for temperature, pH, oxygen, and osmotic concentration
Genetic Analysis
- Chromosomal gene exchange thru
o Transformation
 Can occur between different bacterial species but only rarely between genera
o Conjugation
o Plasmids
Molecular characteristics
1. Comparison of Proteins
- Amino acid sequences are direct reflections of mRNA sequences
- closely related to the structure of the genus coding for their synthesis
2 methods
- Direct approach
o determine amino acid sequence of proteins with the same function
o slow and expensive
- 2. Indirect approach
o Electrophoretic mobility
 Relationships at species and subspecies level
o Antibodies
 Can discriminate between very similar proteins
o Immunologic
 Compare proteins from different microorganisms
o Enzymes
 Physical, kinetic and regulatory properties

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  Enzyme behavior reflects amino acid sequence
2. Nucleic Acid base composition
- G + C content
o reflects the base sequence
o Determined from melting temperature (Tm) of DNA
o DNA with a greater G + C content will have a higher melting point
o use spectrophotometer – absorbance of 260 nm UV light by DNA‘s increase during
DNA separation
- Importance
o Can confirm a taxonomic scheme developed using other data
o Useful in characterizing bacterial genera since the variation within a genus is usually
less than 10%
3. Nucleic acid hybridization
- compare similarity between genomes
- If a mixture of single stranded DNA formed by heating dsDNA is cooled and held at a
temperature about 25C below the Tm, strands with complementary base sequences will
reassociate to form stable dsDNA, while non-complementary strands will remain single
- incubation of the mixture at 30 – 50C below the Tm will allow hybrids of more diverse ssDNA‘s
to form
4. Nucleic acid sequencing

- genome structure can only be directly compared by sequencing DNA and RNA
- rRNA‘s are most ideal for studies of microbial evolution and relatedness since they are essential
to a critical organelle found in all microorganisms
Numerical Taxonomy
- Quantitative approach
- Grouping by numerical methods of taxonomic units into taxa on the basis of their character
states
- Based on general similarity as judged by comparison of many characteristics, each given equal
weight

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LABORATORY
TIME ALLOTMENT: 2 hours

Name: _______________________________________
Group No. ____________________________________

Exercise No. 2

THE BACTERIAL CELL

Introduction
The shape of a cell is referred to as its morphology. Several distinct shapes of bacteria can be
recognized and have been given different names. A bacterium that is spherical or ovoid in morphology
is called a coccus (plural, cocci). A bacterium with a cylindrical shape is called a rod. Some rods are
curved, frequently forming spiral-shaped patterns and are then called spirilla.
In many prokaryotes, the cells remain together in groups or clusters after division, and the
arrangements in these groups are often characteristic of different organisms. For instance, cocci and
rods may occur in long chains. Some cocci may form thin sheets of cells, whereas others occur in
three-dimensional cubes or irregular cube-like clusters. Several groups of bacteria are immediately
recognizable by their unusual shapes. Examples include spirochetes, which are tightly-coiled bacteria,
appendaged bacteria, which possess extensions of their cells as long tubes or stalks, and filamentous
bacteria which form long, thin cells or chains of cells.
Bacteria can be divided into two groups, called gram-positive and gram-negative. The original
distinction between gram-positive and Gram-negative was based on a special staining procedure, the
Gram stain, but differences in cell wall structure are at the base of these differences in the Gram-
staining reaction. Gram-positive and gram-negative cells differ markedly in the appearance of their cell
walls. The gram-negative cell wall is a multilayered structure and quite complex, whereas the gram-
positive cell wall is often much thicker. (Brock, 2017)
Objectives
At the end of the laboratory session, the student should be able to
1. know the different characteristic morphology and arrangement of common bacterial pathogen
accurately
2. differentiate correctly the characteristics of a Gram-positive cell wall and Gram-negative cell wall
as well as an acid-fast bacillus cell wall
3. to illustrate clearly the different types of flagellation and spore locations
Materials:

Microscope Reference materials

Prepared slides colored pencils
Oil immersion oil pencils
Procedure:
1. Carefully scan reference materials/books and/or the internet and look for clear and labeled
photos of the following:
a. Eukaryotic and Prokaryotic cell
b. Gram-positive and Gram-negative cell wall

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 c. Typical bacterial cell
d. Different bacterial morphology and arrangement
e. Different types of flagellation and parts of bacterial flagella
f. Different spore location
g. Stained smears of
- Staphylocccus aureus
- Streptococcus pyogenes
- Streptococcus pneumoniae
- Neisseria gonorrheae
- Bacillus anthrachis
- Corynebacterium diphtheriae
- Escherichia coli
- Vibrio cholerae
- Borrelia recurrentis
- Treponema pallidum
2. Download these photos and study them carefully.
3. Attach photos ―a to f ―on a separate page/s. Make sure that they are not crowded so that labels
are still readable. You may also draw if you choose.
4. For letter ―g‖, caption each photo with the name of the organism, shape and arrangement
- follow the format below

Photo of
organism
Scientific name: ________________________________
Shape: _______________________________________
Arrangement: __________________________________

Questions for Research:

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. Tabulate the difference between a gram-positive and gram-negative cell wall

2. Why are compounds like sterols and hopanoids good at stabilizing the cytoplasmic membrane
3. What is the function of porins and where are they located in a gram-negative cell wall?
4. Why does alcohol readily decolorize gram-negative bacteria?
5. What is a nucleoid?
6. What laboratory tests can motility be detected?

Page 33 of 227
TIME/SCHEDULE: Week 2
TIME ALLOTMENT: 9 HOURS
Lecture: 5 hours
Laboratory: 4 hours

TOPICS: Lecture

Unit II. Introduction to Bacteriology

Definition of terms
Prokaryote vs. Eukaryote
Morphology (size, shape, and arrangement)
Cell Structure
4.1 Cell wall
4.2 Cell membrane
4.3 Cytoplasm
4.4 Specialized structures
Microscopic Study of Bacteria
Growth and Reproduction

Laboratory
Activity no. 3: Factors Affecting Bacterial Growth
Activity no. 4: Microscopic Study of Bacteria
Learning Objectives

At the end of the learning session, the student should be able to

1. Differentiate accurately a prokaryote from eukaryotes
2. Correctly use terms related to bacterial morphology
3. Prepare bacterial smears confidently
4. Apply bacterial stains onto prepared slides correctly
5. Understand completely the different factors that can affect bacterial growth

Lesson 1: Bacteriology
Time Allotment: 2.5 hours
Activity 1: differentiate microorganisms from germs
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
___________________________________________________________________
Prokaryotes vs. eukaryotes

Prokaryotes Eukaryotes
General description PRO: primitive nucleus EU: true nucleus
Size of cell 0.20 – 2 um 10 – 100 um
(diameter in um) Ave: < 5 um Ave: > 10 um

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Nucleus Non-membrane bound single True Nucleus within a membrane
stranded DNA
Divide by binary fission Divide by mitosis
Single, circular and naked More than one chromosome
chromosome
DNA not complexed with histones DNA complexed with histones
Cytoplasm No cytoskeleton or cytoplasmic Cytoskeleton; cytoplasmic
streaming streaming
ER Absent Present
Golgi apparatus Absent Present
Mitochondria Absent Present
Lysosomes Absent Present
Ribosomes 70 s in the cytoplasm 80 s in ER and cytoplasm
70S in organelles
Plasma membrane Sterols usually absent except in Contains sterols;
mycoplasma; CHO serve as receptors
No CHO
Cell wall Chemically complex, Chemically simple; if present
mucopolypeptides containing consists of simple
muramic acid and diaminopimelic polysaccharides or inorganic
acid (DAP) or lysine substances (cellulose and chitin)
Capsule Frequently present Absent

THE BACTERIAL CELL

Morphologic Classification
Size: the bacterial cell is measured in micrometer that is equal to 1/25,000 of an inch or 0.001 nm

• Haemophilus: smallest pathogenic bacillus with cell wall (0.2 x 0.5 um)

• Mycoplasma: smallest pathogenic bacteria without cell wall (0.20 um)

• Bacillus Anthracis: largest pathogenic bacillus (1 x 3-10 um)

Shape and Arrangement

Morpholo Arrangement
gy
Cocci
(spherical)

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Bacilli
(rods)

Spiral
shape

Chemical components
- Water: 70%
- Dry weight: 30% composed of
o DNA – 5% MW 2,000,000,000
o RNA – 12%
o Protein – 70% found in
 Ribosomes (10,000) – RNA
 Protein particles – MW 3,000,000
 Enzymes
 Surface structures
o Polysaccharides – 5%
o Lipids – 6%
o Phospholipids – 4%
Parts of the Bacterial Cell Wall
A. Parts of the Outside cell wall

Glycocalyx: classically associated with the cell wall

 Types
 Capsule: tightly attached
 Slime layer: loosely attached
 Function
Virulence factors protect bacteria from
phagocytosis
Attachment to host surfaces

Capsule Slime layer

Attachment to the cell wall Firmly attached Loosely attached
Component Polysaccharide Exopolysaccharide,
glycoproteins and glycolipids

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Size Thick Thin
Organization Organized and difficult to be Unorganized and easily washed
washed off off
Functions - Virulence and antigenicity
- Bacterial protection (antibiotic barrier
- Vaccine preparation
- Bacterial identification

Encapsulated cells form smooth or mucoid colonies

Encapsulated cells form rough colonies

Lab ID
Colonial appearance: Slimy mucoid
Quellung test: Utilizes anti-capsular antiboides that bond and induce capsular swelling, which can be
visualized by light microscopy
Staining Techniques: Wadsworth, Welch, Anthony, Tyler, Hiss, Muir‘s, MacNeal, Gin, Lawson

Work, work and take hot meal and milk that gives life

Examples: Streptococcus pneumoniae, Klebsiella pneumoniae,

Haemophilus influenzae, Pseudomonas aeruginosa, Neisseria

Some killers have pretty nice capsules

meningitides, Cryptococcus neoformans

Flagella
- long filamentous appendage originating in a spherical body
or basal granules
Three morphological regions
a. helical filament: long outermost region
b. hooked or curved area: where the filament is attached

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 c. basal body: terminal portion and anchors the flagellum to the cell wall and plasma membrane

- Function:
o Locomotion
o Antigenicity (flagellar or H antigen) useful in the serological identification of serotypes of
Salmonella organisms
Lab ID:
Stainning technique: Fisher-Conn, Gray, Loeffler, Leifson, Caesares-Gill, Van Ermenger

A fisherman has a Gray loafer and a lovely comfortable Vans

Evaluation of Motility
- Wet mount
- Hanging drop
o Darting: V. cholera
o Tumbling: Listeria at 20 – 25C
o Stately: Clostridia
o Corkscrew: T. pallidum
o Lashing: Borrelia
o Gliding: Mycoplasma
o Swarming: Proteus mirabilis, P. vulgaris
- Semi-solid medium using motility medium (e.g. SIM)
o Young culture with at least 18 h growth
o Best at 25C
o 35 – 37C may be inhibitory
Activity 2: describe the following types of Flagellation
1. Atrichous: ____________________________________________________________________
2. Monotrichous: ________________________________________________________________
3. Amphitrichous: ________________________________________________________________
4. Lophotrichous: ________________________________________________________________

Peritrichous and monotrichous are most common in pathogens

Spirochetes move with periplasmic flagella

5. Peritrichous: __________________________________________________________________
Fimbriae and Pili
- Hair-like appendages that are shorter, straighter and thinner than flagella

Fimbriae Pili
Located at the poles of the bacterial cell or can Longer than fimbriae and there is only one or two
be evenly distributed over the entire surface of per cell

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the cell
Genetically determined by a fertility factor called
F factor which is carried on an episome (sex of F
For bacterial adherence pilus)

Functions:
- Sites of adsorption fo RNA and DNA viruses
- Act as a means of genetic transfer between similar or related Gram-negative enteric species
- Provide the channel through which DNa from the donor (male) cells is transferred to the
recipient (female cell)

B. The Cell Wall

- this is the major structural
component of bacteria. It is a
strong and rigid structure
that protects and supports
the weaker and
biochemically more active
parts of the cell
- functions
o maintain the cell‘s
characteristic shape
o countering the effects
of osmotic pressure
o provides attachment
sites for
bacteriophages
o provides rigid platform for surface appendages

C. Plasma (Cytoplasmic membrane or inner membrane)

- Thin structure lying inside the cell wall and enclosing the cytoplasm of the cell
- Consists primarily of phospholipids which are the most abundant in the membrane and proteins
o Functions:
 Encloses intercellular contents and structures within the cytoplasm
 Controls transport of nutrients from the external environment of the cell to the
cytoplasm of the cell
 Provides enzymes necessary for capsules, cell wall and cell membrane synthesis
 Serves as the site for enzymes involved in electron transport and metabolism

D. Nuclear Area or nucleoid

- Bacterial chromosomes – carries all the information required for the cell structures and
functions
- Plasmids – extrachromosomal gene that replicates independently from chromosomal DNA and
can be transferred from one bacterium to the other. May carry genes for such activities as
o Antibiotic resistance
o Tolerance to toxic metals

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 o Production of toxins
o Synthesis of enzymes
- Ribosomes – sites for protein synthesis

E. Inclusions: reserve deposits

- Metachromatic granules – spherical granules that are not membrane bound. Contains
relatively large amound of trichloroacetic acid-insoluble polymetaphosphate or polymerized
phosphoric acid or polymerized polymetaphosphate or volutin
 Function: for energy storage
 Example: Corynebacterium diphtheriae: Babes-Ernst granules
Mycobacterium: Much granules
Lab ID:
Staining Technique: have high affinity to basic dyes
 Loeffler‘s Alkaline methylene blue, Ljubinsky, Albert‘s, Neisser‘s, Gohor

Live love and not be greedy

- Polysaccharide granules – consists of glycogen and starch

o glycogen granules – reddish brown with iodine
o starch granules – blue with iodine
- lipid inclusions – appear in various species of Mycobacterium, Bacillus, Spirillum
- Sulfur granules – sulfur bacteria (Genus Thiobacillus) deposit sulfur granules in the cell, where
they serve as an energy reserve
- Carboxysomes – required by bacteria for carbon dioxide fixation during photosynthesis

F. Endospores
- A refractile oval body formed within the bacterial cell found intracellularly and extracellularly in
the usual stained smear
- Function: protect the organism from adverse environmental conditions
- Composition: Bacterial DNA with sparse cytoplasm, cell membrane and peptidoglycan, thick-
keratin-lie coat and dipicolinic that is found in the core
- Found in
o Genus Bacillus: Aerobic sporeforming rods
o Genus Clostridium: Anaerobic sporeforming rods
o Sporosarcinae: gram positive coccus
o Coxiella Burnetti
Lab ID:
Staining techniques: heat and acetic acid, Schaefer-Fulton, Dorner‘s, Wirtz, and
Conklin
Examples: Bacillus (aerobic, catalase +), Have a safe fine day walk carefully
Clostridium (anaerobic; catalase -)

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 - Spore location
o Terminal
o Central
o subterminal

Process of Sporulation
- Axial Filament formation
- Forespore Septum formation
o infolding of the cell membrane to produce a double membrane
- Engulfment of Forespore
o results to the synthesis of 2 special layers forming the cell envelope
- Cortex Synthesis
- Coat Deposition
- Maturation
- Lysis of Mother Cell

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Presence or Absence of Cell Appendages
Motile Bacteria Non-Motile Bacteria
1. Alcaligenes 1. Bacillus anthracis
2. Bacillus cereus 2. Bordetella
3. Bacillus subtilis 3. Brucella
4. Campylobacter 4. Clostridium perfringens
5. Clostridium botulinum 5. Corynebacterium diphtheriae
6. Clostridium tetani 6. Erysipelothrix
7. Escherichia coli 7. Hemophilus
8. Listeria 8. Klebsiella pneumoniae
9. Proteus 9. Mycobacterium tuberculosis
10. Providencia 10. Neisseria
11. Pseudomonas 11. Pasteurella
12. Salmonella 12. Shigella
13. Vibrio 13. Staphylococcoci
14. Streptococci

With Capsule With Spore

1. Bacillus anthracis 1. Bacillus
2. Klebsiella pneumoniae 2. Clostridium
3. Pasteurella
4. Streptococcus pneumoniae

With Inclusion bodies or Granules

1. Corynebacterium - Babes-Ernst Bodies
2. Mycobacteria - Much granules
3. Nocardia and Actinomyces - Sulfur granules
4. Pasteurella and Bordetella - Bipolar bodies

With pili
1. Neisseria
2. Pseudomonas

BACTERIAL GENETICS
Activity 1: Define the following terms

1. Genetics
2. Gene
3. Genome
4. Chromosome
5. Nucleoside
6. Nucleotide
7. Nucleic acid
8. DNA
9. RNA

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Replication of Genetic Information
• involves a complicated process mediated by the enzyme DNA polymerase
• Unwinding
• for enzymes and cofactors to act on the DNA molecule
• Unzipping
• DNA polymerase opens the replication fork
• Synthesis of the new DNA
• each parent strand serves as a template from which a complementary strand is
produced
• Termination of Replication
• occurs when 2 replication forks meet

Bacterial Diversity

Ways in which bacteria acquire new genetic information

1. Mutation
• A mutation is a change in the DNA base sequence that alters the structure and function of the
protein in the cell
• Change is transmissible to offspring
• Mutagen – substance that increases the rate of mutation
• Mutant – organism containing the mutation

3 categories of Mutation
• A. Beneficial mutation
• Mutation that is of benefit to the organism
• B. Harmful mutation
• Mutation that leads to the production of a non-functional gene
• Lethal mutation – leads to the death of the organism
• C. Silent Mutation
• mutation that has no effect on the cell
• Mutation that causes no change in function

Factors that Cause Mutation

a. Viruses
b. Chemicals (industrial chemicals, pesticides, food additives, hair dyes and cosmetics)
c. Ultraviolet light (overexposure to sum)
d. X-rays

Mutagen
- the agents that cause mutation which include chemical agents or various types of radiation
- Ames test – developed by Bruce Ames, is the standard test for mutagenicity

2. Recombination
- The process in which a new recombinant chromosome, one with a genotype different from
either parent, is formed by combining genetic material from two organisms
- Types of Recombination
o General recombination
 The most common form
 Involves a reciprocal exchange between a pair of homologous DNA sequences
o Specific recombination

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  The genetic material is not homologous with the chromosome it joins
 Enzymes responsible for this event are specific for the particular virus and its
host
o Replicative recombination
 Accompanies the replication of genetic material and does not depend on
sequence homology
 Used by genetic elements that move about the chromosome

3. Lysogenic Conversion
- Happens when a bacterial cell has acquired new phenotypic characteristics as a result of
lysogeny
- Term applied to the new properties that a bacterium acquires as a result of the expression of the
integrated lysogenic process
- Involves bacteriophages and the acquisition of new viral genes
- Bacteriophage – a virus that infects bacteria
- Types of Bacteriophage
o Virulent phage
 Phages that lyse their host
 Always cause the lytic cycle to occur ending with the destruction (lysis) of the
bacterial cell
o Temperate or lysogenic phages
 Integrate their genomes into the host genome
 Lysogeny – when the bacteriophage DNA is integrated into the bacterial
chromosome
 The bacteriophage DNA replicates along with the chromosome
 Lysogenic cycle – happens when the viral DNA becomes integrated in the host
cell chromosome and no progeny virus particles are produced at that

4. Bacterial Conjugation
- Transfer of genetic information by direct cell to cell contact
- Involves a specialized type of pilus called sex pilus
- The sex pilus is used to attach to another sex pilus of another bacterial cell
- The genetic material is then transferred thru the hollow sex pilus

5. Transformation
- The uptake of a cell by a naked DNA molecule or fragment from the medium and the
incorporation of this molecule into the recipient chromosome in a heritable form
- When bacteria lyze, they release considerable amounts of DNA into the surrounding
environment.
- If a fragment contacts a competent cell (one able to take up DNA and be transformed), it can
be bound to a cell and taken inside

6. Transduction
- The transfer of bacterial genes by viruses
- Bacterial genes are incorporated into a phage capsid because of errors made during the virus
life cycle
- May be the most common mechanism for gene exchange and recombination in bacteria
- Types of Transduction
o Generalized transduction
 Occurs during the lytic cycle of virulent or temperate phages and can transfer any
part of the bacterial genome

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  Occurs during the assembly stage of viral replication
 When viral chromosomes are packaged into protein capsids, random fragments
of the partially degraded bacterial chromosome also may be packaged by
mistake
 The virus then carries the bacterial DNA when it infects another bacteria
o Specialized transduction
 AKA restricted transduction
 The transducing particle carries only specific portions of the bacterial genome
 Made possible by an error in the lysogenic life cycle
 When a prophage is induced to leave the host chromosome, excision is
sometimes carried out improperly – the resulting phage genome containns
portions of the bacterial chromosome
Plasmids
- Small, circular DNA molecules that can exist independently of host chromosomes
- Replicon – a DNA molecule or sequence that has a replication origin and is capable of being
replicated

Classification of Plasmids
- Episome
o A plasmid that can exist either with or without being integrated into the host‘s
chromosome
- Conjugative plasmids
o Have genes for pili and can transfer copies of themselves to other bacteria during
conjugation
- Fertility factor F factor
o Plays a major role in conjugation
o Bears genes responsible for cell attachment and plasmid transfer between specific
bacterial strains during conjugation
- Resistance factors or R factor
o Have genes for enzymes capable of destroying or modifying antibiotics
o Genes coding for resistance to antibiotics such as ampicillin, chloramphenicol and
kanamycin have been found in plasmids
o They are also conjugative plasmids, thus they can spread throughout a population,
although not as rapidly as the F factor
o These plasmids are readily transferred between species that further promotes the
spread of resistance
- Col plasmids
o Plasmids with genes that may give them a competitive advantage in the microbial world
o Code bacteriocins (colicins – produced by E. coli) that destroy other bacteria
- Virulence plasmids
o Makes host more pathogenic because the bacterium is better able to resist host defense
or to produce toxins
- Metabolic plasmids
o Carry genes that degrade substances such as aromatic compounds (toluene), pesticides
(2,4-dichlorophenocyacetic acid), and sugars (lactose)

ASSIGNMENT:
1. Draw a diagrammatic illustration of
a. Lysogenic conversion/cycle
b. Bacterial conjugation
ASSESSMENT: TBA

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Lesson 2: Bacterial Growth
Time Allotment: 2.5 hours

BACTERIAL PHYSIOLOGY
Bacterial Growth
- Defined as an orderly increase of all chemical constituents of the cell. It entails the replication of
all cellular structures, organelles and protoplasmic components from the nutrients present in the
surrounding environment
o Increase in cell size – when nuclear division is not accompanied by cell division
(coenocytic)
o Increase in cell number – when microorganism reproduce processes like budding or
binary fission (one parent cell gives rise to two progeny cells)

- Stages of Bacterial growth

o Lag phase
 Little or not multiplication but enzymes are very active
o Logarithmic (log) phase
 Organisms grow at maximum rate, exponential rate
o Stationary phase
 Plateau
 Growth ceases because nutrients are exhausted or toxic metabolic products
have accumulated
o Decline and death
 Direct microscopic count may remain but viable count slowly decreases
Notes:
 Highest metabolic activity but without cell division: Lag phase
 Fastest growth: Log phase
 Highest sensitivity to β-lactam: Log phase
 Best to perform biochemical test: Log phase

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  Spore formation: Stationary phase
Reproduction
- Bacteria produce asexually by transverse binary fission
(https://socratic.org/biology/the-eukaryotic-cell/binary-fission)

Atmospheric requirement:
- Adequate supply of oxygen enhances
metabolism and growth
- Oxygen acts as hydrogen acceptor in the
final steps of energy production
catalyzed by flavoproteins and
cytochromes.
o Bacteria need 2 enzymes to utilize
oxygen since the use of oxygen
generates 2 toxic molecules: hydrogen
peroxide (H2O2) and the radical
superoxide (O2)
 a. Superoxide dismutase – to
catalyze the reaction:

 2 O2 + 2 H+ H2O2 + O2
 b. Catalase – to catalyze the reaction:
 2 H2O2 2 H2O + O2
o Possible targets for damage by H2O2 and O2 include:
 Specific outer membrane proteins
 Redox active components of the cytoplasmic membrane

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  Enzymes in periplasmic space

- Classification of bacteria based on Oxygen requirement

Group Description Examples

Aerobe Require oxygen for metabolism Nocardia, Bacillus, Pseudomonas,
- Obligate Bordetella, Legionella, Brucella
aerobes
Anaerobe Cannot survive in oxygen Clostridium, Actinomyces,
(85% N2, 10% H2, 5% CO2) Bacteroides
- Obligate
Anaerobes - An organism that strictly does
not require the presence of
- Facultative oxygen and die in the
anaerobe presence of oxygen
- Organisms that do not require
oxygen but grows better in the
presence of oxygen
(ex. E. coli)
- They are the most clinically
significant organisms

* obligate aerobes and facultative

anaerobes contain protective
enzymes against the toxic effect of
oxygen – superoxide dismutase
(SOD) and catalase
Aerotolerant Can grow with or without oxygen, but
only use anaerobic metabolism
Microaerophiles Require low oxygen tension (5% O2, Campylobacter
10% CO2, 85% N)
Capnophiles Microaerophiles that are dependent Neisseria
on carbon dioxide

Capnophiles requires 5 – 10% CO2

Only 0.03% CO2 is needed by aerobic

bacteria

Thermal Requirements
- Bacteria are divided into the following categories according to the temperature at which they
grow best.

Psychodurics Extreme cold Optimum temp: below

Psychrophilic/psychrophiles
loving OC
―cold-loving‖ Grow below 10C (blood Listeria monocytogenes
bank contaminants) Yersenia enterocolitica
Optimum temp: 4 – 8C
Range: 0C to 20 C

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Mesophilic/mesophiles
Thermophilic/ thermophiles
Thermodurics
Most pathogens Optimum temp: 30 –
37C
Range: 20 C to 40C
―heat-loving‖ Grow at 50 – 55C (basis Bacillus
at test for effective stearohtermophilus
autoclaving) Thermus aquaticus
Optimum Temp: 60C
Range: 45 C to 90C
Extreme heat .90C
loving

Thermal Death Time: the lowest temperature required to kill organisms in a constant temperature
Thermal Death Point: the lowest temperature required to kill an organism in a constant time

 Extremophiles: these are prokaryotes that are able to live at unusual conditions
like absence of oxygen, increased temperature and below the Earth‘ surface
(Bacillus infernus)
pH
- Different bacterial species can grow over a broad range of pH levels
Acidophiles
- Live at pH levels as low as 0 and ranging up to 5.0
o Ex. Sulfolubus, Picrophilus, Acontium
Neutrophiles
- Can exist from pH of 5 to 8.5
- pH 6.5 – 7.5 – optimum pH of most pathogenic bacteria
- Most of the human pathogens belong to this group
o Ex. E. coli
Alkalinophiles/Alkaliphiles
- Live at pH levels from about 7.0 to about 11.5
o Ex. Bacillus alcalophilus, Natronabacterium
*diagnostic lab media for bacterial isolation are usually adjusted to a final pH between 7.0 and 7.5

Moisture

- A major constituent of culture media

- Importance:
o For various metabolic pathways
o Loss of water would mean an increase in solute concentration in the media

Osmotic Requirements

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Halophiles
- Require high salt concentration (30%)
Facultative halophiles
- May survive at 2% salt concentration
Non-halophiles
- Grows at 1.5% or less of salt concentration

Nutritional Requirements
- Necessary for the biologic synthesis of new organisms
As to carbon source
1. Photoautotrophs
- Uses photosynthetic energy to reduce carbon dioxide at the expense of water
- It does not require organic matters for growth
o Ex. Cyanobacteria
2. Photoheterotrophs
- also uses photosynthetic energy to reduce carbon dioxide at the expense of water
- requires organic compounds for growth
o Example: Purple and Green bacteria
3. Chemoautotrophs or lithotrophs (lithoautotrophs)
- autotrophs that use an inorganic substrate such as hydrogen of thiosulfate as a reductant and
carbon dioxide as carbon source
- it does not require organic matters for growth
o Example:Archaea and a few bacterial genera
4. Chemoheterotrophs or heterotrophs
- it requires organic carbon for growth
- glucose is usually the source which supports the fermentative and respiratory growth
o Example: Most bacteria and a few Archaea
*saprophytes - require dead organic substances
*parasites – require organic substances from living tissues

Pressure requirement
- Barophilic – organisms that grow in the presence of high pressure
- 600 to 1100 atm – pressure
- Ex. Photobacterium, Shewanella, Colwella
Growth Factors
- Amino acids, purines and pyrimidines and vitamins

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ASSESSMENT: TBA
LABORATORY
TIME ALLOTMENT: 2 HOURS
Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Activity No. 3
FACTORS AFFECTING BACTERIAL GROWTH
Introduction:
The diversity of bacteria plays an important role in its identification in the laboratory. Different
bacteria have different requirements for growth. In this activity, four of the most important requirements
shall be identified and shall be used to classify different bacteria.
Objectives
At the end of the of the laboratory session, the student should be able to
1. Provide the necessary growth requirements of the bacteria being cultured correctly
2. Classify accurately the bacteria on hand based on its growth requirements
Temperature
Temperature is one of the most important factors influencing the activity of bacterial growth.
Unlike warm-blooded animals, bacteria lack mechanisms that conserve or dissipate heat generated by
metabolism, and consequently their enzyme systems are directly affected by ambient temperatures.
Enzymes have minimal, optimal and maximal temperature requirements. At optimum temperature, the
enzymatic reactions progress at a maximum speed. Below the minimum and above the maximum
temperatures the enzymes become inactive. At some point above the maximum temperature,
destruction of specific enzymes will occur.
Materials
Nutrient broth cultures of E. coli
5 uninoculated nutrient broth
Inoculating loop
Alcohol lamp

Procedure:
Note: Use your PPE‘s properly as we are dealing with live microorganisms with a potential to cause
disease.

1. Label the broth tubes as follows: control, 6C, 22C, 37C, and 45C
2. Inoculate the tubes using a wire loop with E. coli following strict aseptic techniques
3. Do not inoculate the control tube.

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 4. Incubate the tubes at the proper designated temperatures for 18 – 24 hours.

Results and Observations

After incubation, shake the broth cultures and compare them, noting the differences in turbidity.
Tubes which appear to have no growth shall be compared with a tube of sterile nutrient broth.

Record the turbidity by visual observation using the following gradation:

Observation Interpretation
No growth – clear No growth or (-)
Least growth – solution is slightly turbid when +
shaken
More growth – solution is turbid ++
Most growth – solution is turbid to opaque +++

*you may also look for any differences in pigment production if the colonies

Your result:
Note: your answers will be based on theory regarding the growth characteristics of E. coli as affected
by temperature.

Temperature Growth Pigment production (if any)

6C
25C
37C
45C

Conclusion:
__________________________________________________________________________________
____________________________________________________________________________

Osmotic pressure
Growth of bacteria can be affected by the amount of water entering or leaving the cell. When the
medium surrounding an organism is hypotonic (low solute content), a resultant higher osmotic pressure
occurs in the cell. In the reverse situation, when bacteria are placed in a hypertonic solution (high solute
content) its growth may be considerably inhibited. The degree of inhibition will depend on the type of
solute and the nature of the organism. In media of growth-inhibiting osmotic pressure, the cytoplasm
becomes dehydrated and shrinks away from the cell wall. Such plasmolyzed cells are often simply
inhibited in the absence of sufficient cellular water and return to normal when placed in an isotonic
solution.
In this activity, the effect of the different concentrations of NaCl in the culture media on the
degree of growth of the bacteria will be tested.
Materials:

1 nutrient agar plate with 0.5 % NaCl

1 nutrient agar plate with 5% NaCl
1 nutrient agar plate with 10% NaCl

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 1 nutrient agar plate with 15% NaCl

Cultures:
Broth culture of Staphylococcus aureus
Broth culture of Escherichia coli

Procedure:
1. With an acetate pen, mark the bottom of the petri dish with a line dividing it into two. Label the
upper half with E. coli and the other half with S. aureus.
2. Streak each organism in a straight line on the agar using a wire loop using strict aseptic
technique.
3. Incubate the plates for 24 hours at 37C.
4. Record your results below

Observations:
Note: note the amount of growth after incubation and grade them as follows
(-) – no growth
(+) – scanty growth
(++) – moderate growth
(+++) – abundant growth

*answers are all theoretical based on the salt requirements of the test organisms
NaCl Concentration S. aureus E. coli
0.5% NaCl
5% NaCl
10% NaCl
15% NaCl

Hydrogen Ion concentration

The hydrogen ion concentration of an organism‘s environment exerts the greatest influence on
its growth. The hydrogen ion concentration (pH) limits the activity of enzymes with which an organism is
able to synthesize protoplasm. As in the case of temperature, there exists for each organism an
optimum concentration of hydrogen ions in which it grows best. The pH values above and below which
an organism fails to grow are respectively referred to as the minimum and maximum hydrogen ion
concentrations.

Oxygen Concentration
The oxygen requirements of bacteria range from the strict (obligate) aerobes that cannot exist
without oxygen to the strict (obligate) anaerobes that die in its presence. In between these extremes
are the facultatives, and microaerophilics. The facultatives are bacteria that have enzyme systems
enabling them to utilize free oxygen or some alternative oxygen source such as nitrates. If oxygen is
present, they tend to utilize it in preference to the alternative. Microaerophiles are organisms that
require free oxygen but only in limited amounts.
In this exercise, fluid thioglycolate medium will be used to determine the oxygen requirements of
your organisms. Fluid thioglycolate medium utilizes glucose, cystine and sodium thioglycolate to lower
its oxidation-reduction potential. The dye resazurin is included to indicate the presence of oxygen. In
the presence of oxygen, the dye become pink. Since the oxygen tension is always higher near the
surface of the medium, the medium will be pink at the top and colorless in the middle and bottom. The
medium also contains some agar that helps to localize the organisms and favor anaerobiosis in the
bottom of the tube.

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Materials:

3 tubes of fluid thioglycolate (FT) medium

Broth cultures of:
Escherichia coli
Staphylococcus aureus
Bacillus spp

Procedure

1. Label the FT tubes with the corresponding organism to be inoculated.

2. Inoculate the tubes strictly following aseptic techniques
3. Incubate the tubes at 37C for 24 hours
4. After the incubation period, carefully remove the test tubes from the incubator, taking care not to
unduly agitate the tubes
5. Determine the oxygen requirement of the organisms based on their growth patterns in the FT
medium

Conclusion: classify the bacteria tested based on their oxygen requirements.

S. aureus: _________________________________________________________________________
E. coli: ____________________________________________________________________________
Bacillus spp: _______________________________________________________________________

Questions for research:

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted
1. Describe the following methods of bacterial acquisition of nutrients
a. Active transport
b. EMP
c. TCA
d. EDP
2. Differentiate aerobic respiration from anaerobic respiration

Lesson 3: MICROSCOPIC STUDY OF BACTERIA

Direct Bacterial Examination in Living State

1. Saline Wet mount – to determine biologic activity of microorganisms, including motility or
reactions to certain chemicals or serologic reactivity in specific antisera.
2. Hanging Drop Procedure - Serves the same purpose as the saline mount, except that there is
less distortion from the weight of the coverslip and a deeper field focus into the drop can be
achieved.

Activity 1: Describe how the hanging drop procedure is done. Include photos

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Activity 2: Types of Motility
- What are the different types of motility that can be observed using the hanging drop procedure
and give specific examples of bacteria that shows that type of movement?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

3. Darkfield Preparation – used to visualize certain delicate microorganisms that are invisible by
a brightfield optics and stain only with great difficulty. It is particularly useful in demonstrating
spirochetes from suspicious syphilitic chancres for Treponema pallidum
- Spirochetes will appear as motile, bright ―corkscrews‖ against a black background

Bacterial Examination at Fixed State (Staining)

- Process of artificially coloring the microorganisms with dyes. To make cells more visible and to
demonstrate differences between cells
o Steps in staining
 Preparation of smear
 Fixation
 Application of staining solution

Simple Staining
- The coloration of bacteria by application of a single solution of stain to a fixed smear
- The fixed film or smear is usually flooded with a dye solution for a specified period of time, after
which the solution is washed off with water
- Methylene blue, safranin, crystal violet

Differential Staining
- Elicit differences between bacterial cells or parts of a bacterial cell. It is more elaborate that the
simple staining technique in that the cells may be exposed to more than one dye solution or
staining reagent

Gram Stain
- One of the most important and widely used staining techniques in bacteriology
- Purpose
o A true STAT test in microbiology
o Judge adequacy of a specimen
o Recognition of specific morphologies
o Indicate need for additional tests
o Expand clinical diagnostic picture
- Limitations
o Only partial bacterial identification
o Some organisms do not stain
o ―no organisms seen‖ does not rule out infection
o Normal flora can mask pathogens
o Human error
o Organism do not stain as expected

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 - Biochemical Basis of Gram Stain – based on the composition of the bacterial cell wall
Gram-Positive Gram- Negative
Thick peptidoglycan layer with teichoic acid Thin peptidoglycan layer covered with proteins,
and lipoteichoic acid. phospholipids and lipopolysaccharides

Decolorizer cause increased permeability of

Teichoic acid cross-links prevent lipid-rich cell wall and primary stain (crystal
decolorization in the gram stain violet) washes out

Gram’s stain Procedure

Reagents Gram + Gram -

Color after stain application
Primary stain Crystal violet Violet Violet
Mordant Gram‘s iodine Violet Violet
Decolorizer (most Acetone: Alcohol ro Violet Colorless
critical step) 95% ethanol
Secondary stain Safranin Violet Red

Bacteria group according to its Gram’s Reaction:

- All cocci are gram positive EXCEPT: Neisseria, Veillonella, Branhamella
- All bacilli are gram negative EXCEPT: Bacillus, Mycobacterium, Clostridium, Corynebacterium,
Listeria, Erysipelothrix, Lactobacillus, Rothia
- Spirals Hard to stain but once stained it is gram negative
- Mycoplasma/ Ureaplasma: Gram negative due to the absence of cell wall
- Mycobacterium: Gram positive; Difficult to stain due to high concentration of mycolic acid in the
cell wall but once stained it will resist decolonization.

Gram Reaction and Oxygen requirement of bacterial group

Gram Positive Cocci
Aerobic Micrococcus, Staphylococcus, Streptococcus
Anaerobic Peptococcus, peptostreptococcus, sarcina
Gram Negative cocci
Aerobic Branhamella, Neisseria
Anaerobic Veillonella
Gram-Positive bacilli
Aerobic Bacillus, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria,
Mycobacterium, Nocardia
Anaerobic Actinomyces, Clostridium, Propionibacterium
Gram Negative bacilli
Acinetobacter, Aeromonas, Alcaligenes, Bordetella, Brucella,
Enterobacteriaceae, Fracisella, Legionella, Pasteurella, Pseudomonas,
Vibrio
Fusobacterium, Bacteroides

Acid Fast Stain

- Separates species of Mycobacterium from other bacteria

Procedure:

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 Technique Color of Stained Organism
Zeihl Neelsen Kinyoun Acid Fast Non-Acid fast
(hot method) (cold method)
Best for Sputum Best for tissues
Primary Stain Carbol Fuchsin Red Red
Mordant Physical (Steam) Chemical Red Red
(Tergitol)
Decolorizer Acid Alcohol Red Colorless
(0.05 or 0.1 N HCl Acid in 70%
Ethanol)
Secondary Stain Methylene blue Malachite Green Red Blue/Green

Modification

Technique Modified Process Differentiated

Pappenheim Decolorizer (alcohol and M. Tuberculosis: Red
Rosolic Acid) M. Smegmatis: Blue
Baumgarten Decolorizer M. tuberculosis: Blue
(Alcohol and Nitric acid M. Leprae

Activity 2: In a tabulated form, describe the following staining techniques as to their specific purpose,
principle and expected result
1. Auramine Fluorochrome 11. Heat and Acetic acid
2. Pappenheim‘s Stain 12. Gray‘s Method
3. Baumgarten‘s stain 13. Leiffson‘s method
4. Acridine Orange stain 14. Loeffler‘s method
5. Toluidine blue and Methylene blue stains 15. Fulton and Schaeffer‘s method
6. Silver stains 16. Dorner‘s method
7. Hiss Method 17. Neisser‘ stain
8. Tyler‘s method 19. Loeffler‘s Alkaline methylene blue (LAMB)
9. Background staining or Relief staining or Negative staining
10. Tyler‘s method

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LABORATORY
TIME ALLOTMENT: 2 HOURS

Note: This activity will not be performed. Expected answers are all theoretical. Procedures are provided
for reference only.

Name: _______________________________________
Group No. ____________________________________

Activity No. 4

MICROSCOPIC STUDY OF BACTERIA

Introduction
The second process for bacterial analysis is the microscopic method. Material may be examined
for microorganisms either in the living and unstained state, or after stains have been used, to color the
microorganisms to bring it out more clearly. In microscopy, contrast may be provided by either adjusting
the iris diaphragm or by staining. Stains adhere to bacteria materials depending on its electrical charge
of pH

Objectives:
At the end of the laboratory session, you should be able to
1. prepare a bacterial smear correctly
2. understand the principles of differential staining
3. correctly perform the basic staining techniques used in bacteriology
4. correctly interpret the staining reactions of different bacteria

Bacterial Smear Preparation

Watch: https://www.youtube.com/watch?v=on5-5oQNNqo

The preparation of a smear is required for many laboratory procedures, including the Gram-
stain. The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample
from being washed off during a staining procedure. A smear can be prepared from a solid or broth
medium or directly from clinical specimens.

Materials
Binocular Microscope
Glass slides
Bunsen burner/alcohol lamp
Slant/Broth culture of bacteria
Inoculating loop
Distilled water or NSS

Procedure
1. Wipe a glass slide with clean lint free cloth or filter paper. Pass over the flame of the Bunsen
burner/alcohol lamp to remove any grease. Cool the slide laid flat on clean surface.
2. Divide the glass slide using a wax pencil into two. Place a drop of NSS or sterile distilled water on
both sides of the slide.
3. Using an inoculating loop, flame sterilize and fish out a colony from a culture slant/dip the loop on
culture broth and emulsify the loop onto the drop of NSS/distilled water. Flame sterilize the loop
after emulsification. Repeat the same using other bacterial culture.

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4. Aseptically carry out all cultures with utmost caution. Wear proper laboratory protection in carrying
out different culture stock. Ask the assistance of the laboratory technologist/professor/instructor.
5. Air dry bacterial smears. Heat fix prepared bacterial smears by passing the slides over the flame.
6. Carefully scan your reference materials/book and/or the internet for a clear and labeled photo of the
steps of the preparation of the bacterial smear and the preparation of a hanging drop slide and
attach it below this instruction. You may also draw if you choose so.

Simple Stain Technique

Watch: https://www.youtube.com/watch?v=n5fXIpJUgD4

Simple Staining is the use of a single stain to color a bacterial organism. Most common dyes
used for simple staining are: Methylene Blue, Basic fuchsin and Crystal violet.

Principle: Bacteria are slightly negatively charged, and the dyes used for bacterial staining are
positively (basic dyes) charged causing an attraction between the cell and the dye. Acidic dyes will not
stain bacteria because of electrostatic repelling forces.

By simple staining we can observe: Cell arrangement and cell morphology.

Materials
Slant/Broth cultures of Bacillus subtilis and Staphylococcus aureus
Aqueous Carbol Fuchsin/Methylene Blue/Safranin
Glass Slides
Alcohol lamp/Bunsen burner
Inoculating loop
Binocular microscope
NSS/Distilled Water

Procedure
1. Prepare two bacterial smears of B. subtilis and S. aureus.
2. Heat fix and allow to cool.
3. Take a smear of each B. subtilis and S. aureus and flood the smears with aqueous carbol
fuchsin but use only sufficient amount of the stain to cover the smear, not the entire glass slide.
4. Allow the stain to remain for one minute.
5. Wash the slides with tap water and blot dry.
6. Take the other two smear and repeat the above procedure using aqueous methylene blue stain.
7. Examine stained preparation under oil immersion objective.
8. Look for a photo of a bacteria that was made visible using simple stains. Attach it below this
procedure and label it correctly.

Gram Staining Technique

Watch: https://www.youtube.com/watch?v=sxa46xKfIOY

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Materials
Slant/broth cultures of B. subtilis, E.coli, S. aureus and C. albicans
Gram staining set (Crystal Violet, Gram‘s iodine, decolorizer, Counterstain - Safranin)
Glass slides
Binocular microscope

Procedure
1. Prepare bacterial smears of the following organisms assigned to your group.
2. Heat fix prepared bacterial smear and allow to cool
3. Flood slide with crystal violet and allow it to remain on the surface without drying for 10 – 30
seconds. Rinse the slide with tap water, shaking off all excess.
4. Flood the slide with Gram‘s iodine to increase affinity of crystal violet and allow to remain on the
surface without drying for twice as long as the crystal violet. Rinse with tap water, shake off all
excess.
5. Flood the slide with decolorizer for 10 seconds and rinse off immediately with tap water. Repeat this
procedure until the blue dye no longer runs off the slide with the decolorizer. Thicker smear requires
more prolonged decolorizing. Rinse with tap water and shake off excess.
6. Flood the slide with counterstain (safranin) and allow it to remain on the surface without drying for
30 seconds. Rinse with tap water and gently blot the slide dry with paper towel or bibulous paper or
air dry. For delicate smears, such as CSF and other body fluids, air drying is the best method
7. Examine microscopically under an oil immersion objective
8. Look for the following photos in your reference books/materials and/or the internet and attach them
to this activity with clear and readable labels
a. Gram staining procedure
b. S. aureus gram stained
c. B. subtilis gram stained
d. E. coli gram stained
e. C. albicans gram stained

Acid Fast staining Technique

Watch: https://www.youtube.com/watch?v=Dy6dYstZpZY

A. Ziehl-Neelsen Method (Hot Technique)

Materials:
Autoclaved cultures of Mycobacterium tuberculosis
Acid fast staining kit (Ziehl-Neelsen method)
Binocular microscope

Procedure:
1. Fix smears on heated surface (60oC for at least 10 minutes).
2. Flood smears with carbolfuchsin (primary stain) and heat to almost boiling by performing the
procedure on an electrically heated platform or by passing the flame of a Bunsen burner
underneath the slides on a metal rack. The stain on the slides should steam. Allow the slides to sit

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 for 5 minutes after heating; do not allow them to dry out. Wash the slides in distilled water (Note: tap
water may contain acid-fast bacilli) drain off excess water.
3. Flood the slides with 3% HCl and 95% ethanol (decolorizer) for approximately 1 minute. Check to
see that no more red color runs off the surface when the slide is tipped. Add a bit more decolorizer
for very thick or those that continue to ―bleed‖ red dye. Wash thoroughly with water and remove the
excess.
4. Flood slides with methylene blue (counterstain) and allow to remain on surface of slides for 1
minute. Wash with distilled water and stand slides upright on paper towels to air dry. Do not blot
dry.
5. Examine microscopically, screening at 400x magnification and confirm all suspicious (i.e., red)
organisms at 1, 000x magnification using an oil-immersion lens.
6. Scan your reference materials/books/online sources and look for a clear and labelled illustration of
the procedure for the Ziehl-Neelsen staining method and attach it below this procedure together
with a clear microscopic photo of M. tuberculosis stained with the acid-fast technique.

B. Kinyoun’s Method (Cold Technique)

Materials:
Acid-fast staining kit (Kinyoun method)

Procedure:
1. Get a prepared slide of autoclaved Mtb culture or a heat fixed sputum smear.
2. Flood the smear with Kinyoun‘s carbol fuchsin. Allow the smear to stain for 3 – 5 minutes without
heating.
3. Rinse the slide with distilled water.
4. Flood the slide with 3% acid alcohol, and decolorize the smear until no more red color drains from
the slide.
5. Rinse the slide with distilled water. Drain excess water.
6. Flood the slide with malachite green and allow the stain for 20 – 30 seconds.
7. Rinse with water. Allow the smear to air dry.
8. Examine under oil immersion objective.

Questions for Research

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted
1. What are the different theories on Gram Stain Reaction?
2. Tabulate the differences of Gram-positive and Gram-negative bacteria.
3. Why should a young culture rather than old culture be used for Gram staining?
4. What are the different decolorizers used for Gram staining? How is a standard Gram stain
decolorizer prepared?

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5. What other microscopy techniques are applied for the study of Mycobacteria?
6. What is the standard reporting of AFB based on existing international guidelines?
7. How is acid alcohol being prepared?
8. What is the difference of a ―hot‖ and ―cold‖ method of acid-fast staining?
9. What is the standard reading and reporting sputum specimens?
10. How is sputum specimens screened for culture?

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WEEK 3

TIME ALLOTMENT: 9 HOURS

LECTURE: 5
LABORATORY: 4

TOPICS: lecture
1. Sterilization and Disinfection
a. Physical heat
b. Filtration
c. Chemicals
2. Cultural study of bacteria
a. Culture media
b. Inoculation techniques
c. Cultural characteristics

Laboratory
Activity 5: Media Preparation
Activity 6: Methods of Inoculation
Activity 7: interpretation of cultures

Learning objectives
At the end of the learning session, the student should be able to
1. Enumerate the different types and uses of chemical disinfectants correctly
2. Apply the basic principles of physical sterilization in the laboratory confidently
3. Prepare different culture media correctly
4. Inoculate different culture media
5. Interpret the growth of bacteria in a culture media

MICROBIAL GROWTH CONTROL

Time allotment: 2.5 hours

LESSON 1: INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Activity 1: Define the following terms and give examples if possible

1. Sterilization 12. Fungicide
2. Disinfection 13. Algicidal agents
3. Pasteurization 14. Virucidal agents
4. Disinfectants 15. Microbistatic agents
5. Antiseptics 16. Bacteriostatic agents
6. Sanitation 17. Lyophilization
7. Microbicidal agents 18. Sepsis
8. Bactericidal agents 19. Asepsis
9. Sporicidal agents 20. Aseptic technique
10. Antisepsis 21. Antiseptic technique
11. Sterile technique

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Physical methods to inhibit Microbial growth
1. Heat
2. Pressure
3. Desiccation
4. Radiation
5. Sonic disruption
6. Filtration

HEAT
- Most practical, efficient, and inexpensive method for inanimate objects and materials that can
withstand high temperature
- Kills by coagulating proteins
- Factors:
o Heat
o Time
- * The higher the temperature, the shorter the time required to kill the organisms
- Thermal death point (TDP) – the lowest temperature that will kill all organisms in a
standardized pure culture within a specified period
- Thermal death time (TDT) - the length of time necessary to sterilize a pure culture at a
specified temperature

Dry Heat – kills by oxidation

- Hot air oven
o 160C to 165C for 2 hours
o 170c to 180C for 1 hour
o For metals, glasswares, some powders, oils and waxes
- Incineration
o Burning
o For contaminated disposable materials
o Flaming – for wire loops, wire needles, forceps, mouth of tubed media and rim of petri
dishes

Moist heat -Coagulation accompanied by hydrolysis

- Autoclave
o Uses steam under pressure
o 1210C at 15 psi for 15 – 30 minutes
o Pressure – sensitive autoclave tapes and solutions containing bacterial spores can be
used for quality control
(https://www.tedpella.com/adhesive_html/Autoclave-Steam-Indicator-Tapes.htm.aspx)

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 - Boiling
o 100C, 15 – 30 minutes
o Kills vegetative forms but not spores and viruses
- Fractional Sterilization
o Tyndallization
o Steam for 30 minutes for 3 consecutive days
o Arnold sterilizer
- Inspissation
o 75 – 80C for 2 hours for 3 successive days
- Pasteurization – for milk and wine

COLD
- Does not kill microorganisms
- Slows down their metabolism
- Refrigeration – slows down growth
- Slow freezing
o Not used because ice crystals are formed
- Rapid freezing
o Uses nitrogen
o For preservation
o Places bacteria under suspended animation

DESSICATION
- Foods may be preserved by drying
- However, some microbes remain viable even after drying
- Example: N. gonorrheae and Mycobacterium tuberculosis

RADIATION
- For prevention of food spoilage, sterilization of heat sensitive equipment, preparation of vaccine
- Sunlight
o Includes IFR, visible light, UVR
o Kills only those exposed to direct sunlight

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 - UVR
o Uses UV lamp or germicidal lamp
o May be placed in rooms, cabinets containing instruments, cloth equipment, liquid and
other inanimate objects
o Reduces number of microorganisms in the air
o Disadvantages
 DNA replication is inhibited
 forms thymine dimers
 can damage the cornea and skin
 Do not penetrate cloth, glass and metals
- X-rays
o With higher energy and higher penetrating power than UV
o Produces hydroxyl radicals by the hydrolysis of water
o breaks covalent bonds on the DNA
o Spores are resistant due to its low water content
- Beta Rays
- Gamma rays
o From cobalt 60
o Can be used to process chicken and red meat
o For food processing
o May kill Salmonella and Campylobacter in chickens
 * Chicken that is irradiated is marked with the
green international symbol for radiation
(https://www.foodnavigator.com/Article/2012/02/07/Food-irradiation)

MECHANICAL METHODS – disintegrate bacteria

- Sonic vibration
o Uses sound waves
o For delicate equipment
- Trituration
o grinding
- Agitation
o shaking

FILTRATION
- Used to separate cells, larger viruses, bacteria and certain other microorganisms from the liquid
or gases in which they are suspended
- Sintered glass, plastic films, unglazed porcelain, asbestos, diatomaceous earth, cellulose
membrane filters
- HEPA – high efficiency particulate air
o To protect workers

OSMOTIC PRESSURE
- by plasmolysis
- Example: immersion of meat in salt solution
Fruits and vegetables in sugar solution

Variables of Disinfection
1. Concentration
2. Time
3. Temperature

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 4. pH
N = 1/CT
Where:
N = number of surviving bacteria
C = concentration
T = time

- increased concentration and increased time = decreased survivors

Chemical methods to inhibit microbial growth (http://www.cdc.gov/hicpac/Disinfection_Sterilization)

Alcohol
- "alcohol" refers to two water-soluble chemical compounds—ethyl alcohol and isopropyl
alcohol—
- bactericidal, tuberculocidal, fungicidal, and virucidal
- do not destroy bacterial spores.
- cidal activity drops when diluted below 50% concentration,
- optimum bactericidal concentration is 60%–90% solutions in water (volume/volume)
- mode of action
o denaturation of proteins.
o absolute ethyl alcohol, a dehydrating agent, is less bactericidal than mixtures of alcohol
and water because proteins are denatured more quickly in the presence of water .

Chlorine and Chlorine Compounds

- Hypochlorites,
o available as liquid (e.g., sodium hypochlorite) or solid (e.g., calcium hypochlorite)
o They have a broad spectrum of antimicrobial activity
o do not leave toxic residues
o unaffected by water hardness
o inexpensive
o fast acting
o remove dried or fixed organisms and biofilms from surfaces
o low incidence of serious toxicity
- mode of action
o oxidation of sulfhydryl enzymes and amino acids
o ring chlorination of amino acids
o loss of intracellular contents
o decreased uptake of nutrients
o inhibition of protein synthesis
o decreased oxygen uptake
o oxidation of respiratory components
o decreased adenosine triphosphate production;
o breaks in DNA; and depressed DNA synthesis

Formaldehyde
- used as a disinfectant and sterilant in both its liquid and gaseous states.
- water-based solution called formalin, which is 37% formaldehyde by weight.
- bactericide, tuberculocide, fungicide, virucide and sporicide
- Disadvantages
o Carcinogen – limit exposures

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 o Prolonged exposure can lead to contact dermatitis
o Fumes are irritating to the eye and respiratory system
- Mode of action
o By alkylating the amino and sulfhydryl groups of proteins and ring nitrogen atoms of
purine bases

Glutaraldehyde
- a high-level disinfectant and chemical sterilant
- Acidic glutaraldehyde are not sporicidal
- Should be made alkaline to become sporicidal
o Should be used within 14 days because polymerization blocks aldehyde functional group
which is responsible for its biociddal activity
- Glutaraldehyde is used most commonly as a high-level disinfectant for medical equipment
- Mode of action
o results from its alkylation of sulfhydryl, hydroxyl, carboxyl, and amino groups of
microorganisms, which alters RNA, DNA, and protein synthesis

Hydrogen Peroxide
- bactericidal, virucidal, sporicidal, and fungicidal properties
- mode of action
o produce destructive hydroxyl free radicals that can attack membrane lipids, DNA, and
other essential cell components.
o Catalase, produced by aerobic organisms and facultative anaerobes, is overwhelmed by
the concentrations used for disinfection

Iodophores
- Iodophors can be used both as antiseptics and disinfectants
- a combination of iodine and a solubilizing agent or carrier
- the resulting complex provides a sustained-release reservoir of iodine and releases small
amounts of free iodine in aqueous solution.
- Example: povidone-iodine, a compound of polyvinylpyrrolidone with iodine.
- retain the germicidal efficacy of iodine but are generally are non-staining and relatively free of
toxicity and irritancy
- Mode of action
o Iodine can penetrate the cell wall of microorganisms quickly, and the lethal effects are
believed to result from disruption of protein and nucleic acid structure and synthesis.

Ortho-phthalaldehyde (OPA)
- Ortho-phthalaldehyde is a high-level disinfectant
- It contains 0.55% 1,2-benzenedicarboxaldehyde (OPA).
- OPA solution is a clear, pale-blue liquid with a pH of 7.5
- advantages
o excellent stability over a wide pH range (pH 3–9)
o not a known irritant to the eyes and nasal passages
o does not require exposure monitoring,
o has a barely perceptible odor, and requires no activation
- Disadvantages
o it stains proteins gray (including unprotected skin) and thus must be handled with
caution
- Mode of action
o interact with amino acids, proteins, and microorganisms

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 o However, OPA is a less potent cross-linking agent than glutaraldehyde. This is
compensated for by the lipophilic aromatic nature of OPA
o appears to kill spores by blocking the spore germination process

Peracetic Acid
- AKA peroxyacetic acid
- Has rapid action against all microorganisms.
- Advantages
o harmful decomposition products
o enhances removal of organic material
o leaves no residue.
o remains effective in the presence of organic matter
o sporicidal even at low temperatures
- disadvantages
o can corrode copper, brass, bronze, plain steel, and galvanized iron
o can be reduced by additives and pH modifications.
o It is considered unstable, particularly when diluted
 for example, a 1% solution loses half its strength through hydrolysis in 6 days,
whereas 40% peracetic acid loses 1%–2% of its active ingredients per month
- Mode of action
o it denatures proteins, disrupts the cell wall permeability, and oxidizes sulfhydryl and
sulfur bonds in proteins, enzymes, and other metabolites
- Uses
o To chemically sterilized medical, surgical and dental equipments

Phenolics
- Fist germicide used by Lister
- Derivatives of phenol
- Phenolics are absorbed by porous materials, and the residual disinfectant can irritate tissue.
- depigmentation of the skin was reported to be caused by phenolic germicidal detergents
- Mode of action
o In high concentrations,
 acts as a gross protoplasmic poison,
 penetrates and disrupts the cell wall
 precipitates cell proteins.
o Low concentrations,
 inactivation of essential enzyme systems and leakage of essential metabolites
from the cell wall

Metals as Microbicides
- silver - used for prophylaxis of conjunctivitis of the newborn, topical therapy for burn wounds,
and bonding to indwelling catheters
- Zeolite ceramic coatings with Zn and Ag - Inactivation of bacteria on stainless steel
- silver, iron, and copper - used for environmental control, disinfection of water, or reusable
medical devices
- copper-8-quinolinolate - fungicide against Aspergillus, copper-silver ionization for Legionella
disinfection
- organic mercurials - as an antiseptic (e.g., mercurochrome) and preservative/disinfectant (e.g.,
thimerosal)

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Detergents/Soaps
- surfactants interact with the lipid in the cell membrane and with the surrounding water
- increases the surface tension
- Example: Quaternary ammonium (Quats or Zephiran

Ethylene oxide
- for sterilization of heat-sensitive equipment
- Most effective cold sterilization technique

Crystal Violet (Gentian Violet)

- skin antiseptic
- binding of + charged dye molecule to the – charged PO4 groups of nucleic acid

Malachite Green
- In LJ medium, it kills other bacteria except M. tuberculosis

ASSIGNMENT: TBA

ASSESSMENT: TBA

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LABORATORY
TIME ALLOTMENT: 2 HOURS

Name: _______________________________________
Group No. ____________________________________

Activity 5

MEDIA PREPARATION
Introduction

Microorganisms particularly bacteria are grown in the laboratory using various types of culture
media. The medium depends on the type of bacteria to be isolated or identified. Different nutrients
maybe added to the medium (high in protein or sugar). Others include pH indicators for differentiation
based on the biochemical reaction of the target bacteria. Sodium chloride, pH buffers and other
supplements can be added for specific bacterial growth requirements. Optimum growth could only be
achieved using combined proper use of culture media, growth temperature and nutrient
supplementation.

Objectives:

At the end of the laboratory session, the student should be able to

1. Correctly identify the different basic components and types of culture media.
2. prepare aseptically different culture media available for the inoculation, biochemical testing and
susceptibility testing of bacteria.

Materials:
Dehydrated Cultured Media Powder
Triple beam balance (paper cups, etc.)
Distilled water
Erlenmeyer flask
Hot plate with magnetic stirrer/alcohol/Bunsen burner set-up
Culture tubes
Petri dishes
Stirring rod
Autoclave
Hot air sterilizer

Classification of Culture Media:

- See lecture notes

Procedure
Watch: https://www.youtube.com/watch?v=KHg_PyjQPwk

Basic Steps in the Preparation of Culture Media:

The latest recommendation in selection of culture media is to make use of granulated
dehydrated preparation. Generally, manufacturer’s recommendation should be followed to achieve
optimum performance of the product.

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1. The different culture media to be prepared will be assigned per group. The needed materials,
equipments and additional instructions prior to preparation will be discussed including precautions.
Disinfect all surfaces and wear necessary personal protective equipments to allow simulated
sterility of the bench area for culture media preparation and prevent or if not minimize
contamination. Sterilization of glasswares is essential.

2. Weigh the desired quantity of granulated/powdered culture medium using a triple beam balance
depending on the volume of medium to be prepared. Dissolve in appropriate volume of distilled
water into an Erlenmeyer flask.
NOTE: Media containing agar or gelatin should be heated in boiling water bath or hot plate with
magnetic stirrer or manually by constantly stirring using a sterile stirring rod. Dissolution is complete
when the medium is clear. Do not over boil preparation.

3. Sterilize appropriately according to what type and form of media is being prepared. Liquid or broth
medium should be dispensed in appropriate volume into tubes prior to sterilization. NOTE: Other
culture media does not require heating and/or sterilization. See specific manufacturer’s
recommendation for better media performance.

4. After sterilization, allow to cool between 45 to 55oC before they are dispensed into plates to avoid
condensation of water on the lid of the petri dish. Swirl preparation prior to dispensing to ensure
even mixing. Ideally 12 – 15 mL of the medium is poured into the petri dish.

For tubed solid medium, it is recommended that the medium be dispensed first before sterilization.
Otherwise, the mouth of the tubes is flamed before medium is poured unto it.

Agar slants and/or butt/slant and butt are prepared by mounting the tubes on a slant rack and allowed
to solidify.

5. Solidified media should be checked for sterility by incubating a representative sample overnight and
examine for bacterial growth. This is especially important for mixtures containing ingredients which
have not been autoclaved such as blood agar plates.

6. Prepared culture medium should be stored in the refrigerator upside down. Wrapping of plastic or
foil is necessary for light sensitive culture media. Other culture media and/or broth are kept at room
temperature.

7. Needed culture media should be brought out from the refrigerator and stand at room temperature to
remove moist prior to use.

8. Dispose contaminated culture media as appropriate based on existing guidelines.

Illustrations
1. Carefully scan reference materials/books/internet and look for clear representative photos of the
following culture media
a. TSB h. Citrate
b. Thiglycollate i. MSA
c. MAC j. BAM
d. NB k. MR-VP
e. NA l. TSIA
f. LIA j. EMB

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 g. SIM k MHA
2. Under each photo, caption it following the instructions below

PHOTO
HERE
Physical state: __________ (solid, liquid, semi-solid)
Composition: ___________(synthetic or non-synthetic)
Use: __________________(refer to lecture notes on classification)
Preparation: ____________ (tubed (slant, slant and butt, butt) plated)

3. A maximum of 4 photos per page is recommended.

Questions for Research:

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. Complete the table

Transcription Use Sample bacteria Indicator Inhibiting

grown (for agent (for
selective selective
media only) media only
APW
BCYE
BEA
BGA
BHIB
BSA
CCFA
CTA
EMB
EYA
GNB
HEA
KIA
KVLB
LIA
L-J
MAC
MHA

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M-L
MR-VP
MSA
MTM
NA
NB
NYC
PEA
SF
SSA
SSA (for g+ cocci)
TCBS
TM
TSB
TSIA
XLD

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LESSON 3: CULTURE AND CULTURE MEDIA

TIME ALLOTMENT: 2.5 HOURS

The study of microorganisms relies on their ability to grow and survive outside the host. This is
made possible by utilizing effective and appropriate culture medium for growth, transport and storage.
Isolation and identification of microorganisms in the areas of industrial microbiology, water and food
analyses require specialized culture media.

Purpose
- Essential for diagnostic purposes when infectious disease in involved
- Develop methods for interrupting their spread and controlling their growth
- In research, to study the metabolic processes
- Understand patterns of microbial metabolism in the laboratory

Culture
- Are growth of microorganism on culture medium

Culture medium
- A material containing the necessary nutritional and environment requirements for bacterial
growth
- It is a liquid, semi-solid or solid preparation utilized to observe growth pattern of microorganism
as well as for transport and storage

Types of Culture
1. Pure culture
a. One genus
b. Isolation techniques: streak plate, pour plate, selective media (with antibiotics) and
animal inoculation
2. Mixed culture
a. More than 2 genus and species
3. Stock culture
a. For academic and industrial purposes

Classification of Culture media

1. According to physical state or consistency

Liquid Semi-solid
So solidifying or hardening 0.5% - 1.0% agar
agent Clot like consistency
Broth, infusion, milk, Brain heart Motility medium, transport
infusion medium
Allows growth of aerobes, Agar is a sulfated polymer
anaerobes and facultative made of D-galactose, 3,6-
anaerobes anhydro-L-galactose, and D-
glucuronic acid and usually
derived from red algae
Solid
Contains 2-3% agar
Provides a firm surface on
which organisms can form
colonies
Agar will melt if the temperature
reaches 80 – 90C and solidify
at 40 – 50C

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 2. According to the manner of dispensing or distribution
Plated Tubed
Dispensed in a petri dish Butt or deep
Slant
Butt and slant

3. According to composition
a. Synthetic or defined medium
All substances are known to the uses
It is used for research purposes
b. Non-synthetic or complex medium
Composed of some unknown substances (peptones, meat and yeast extracts)
Very useful for the isolation of bacteria
Ex. Nutrient broth, TSB, MacConkey agar
c. Tissue culture medium
To isolate obligate intracellular bacteria (Rickettsia, Chlamydia)
Ex. W138, HELA 229 cells, Mc Coy cells

4. According to use
a. Simple/supportive/general isolation/general purpose/basal culture medium
Supports growth of non-fastidious organisms
Ex. Nutrient agar, nutrient broth, trypticase soy agar, TSB,
b. Enriched culture medium
Medium Use Comments
Blood agar For most fastidious bacteria Tryptic soy agar with 5%
sheep blood added
Allows differentiation of
hemolysis/hemolytic pattern
Chocolate agar Enriched medium for Supplies X and V factors.
Haemophilus and Neisseria Incubate in increased CO2

REMEMBER!
Preferred blood for the preparation of BAP
1st – sheep 2nd – Horse 3rd – Rabbit

Human blood is not preferred because it contains nonspecific

inhibitors
- Citrate: inhibit grown of β-hemolytic streptococcus
- Dextrose: alter type of hemolysis

Blood is added at 40 – 50 C

c. Enrichment culture medium

Increase the number of pathogens that are outnumbered by non-pathogens.
Extends the lag phase of non-pathogens while decreasing the lag phase of
pathogens
Used to propagate the growth of certain group of organisms
They contain specific nutrients
Examples:
1. Gram negative broth (GN)

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 2. Selenite broth
3. Tetrathionate broth
4. Alkaline peptone water
5. Buffered charcoal yeast extract agar
6. Thioglycolate
d. Differential culture medium
Distinguishes group of organisms based on cultural characteristics
It allows visualization of metabolic differences between group of species of
bacteria
Ex. MacConkey, BAP, Eosin Methylene blue (EMB), Hektoen Enteric Agar (HEA)
1. MacConkey – lactose fermenters (pink), and non-lactose fermenters (colorless)
2. BAP – hemolytic patterns (streptococci)

5. Selective culture medium

a. Selects the growth of a particular organism at the same time inhibiting the growth of
other organisms
b. It is incorporated with antibiotics, dyes of chemicals to inhibit the growth of other
organisms while promoting the growth of the desired organism
c. Ex. HEA, Mac, XLD, BSA, MSA (7.5% salt)
Gentamycin blood agar – Streptococci
Bacitracin Chocolate agar – Hemophilus
Blood agar plate w/ ampicillin – Aeromonas
Phenyl ethyl alcohol – Gram (+) cocci
Columbia CNA with blood – gram (+) bacteria
Gram negative (GN) broth – salmonella spp and Shigella spp
Thayer Martin Agar (TM) – Neisseria gonorrheae
d. Inhibitory substances
Crystal/Gentian violet
Basic fuchsin inhibitory to gram (+)
Bile salt
Potassium tellurite inhibitory to gram (-)
Sodium azide
Alcohol inhibitory to swarming phenomenon
Chloral hydrate

Bacteria Selective Culture Medium

Neisseria Thayer Martin Medium
Martin Lewis Medium
New York City Medium
Staphylococcus Mannitol Salt Agar
Phenyl Ethyl Alcohol Agar
Vogel and Johnson Agar
Chapman Stone Agar
Group A Streptococcus Crystal violet Blood agar
Polymyxin B, Neomycin Fusidic acid media (PNF)
Group B Streptococcus Lim, Carrot broth
Todd and Hewitt broth with antibiotics
Granada Agar
Vibrio Thiosulfate Citrate Bile Salt Sucrose
Monsur Tellurite Taurocholate Gelatin Agar

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Brucella Castañeda medium
Wisconsin medium
Bordetella Bordet-Gengou medium
Jones Kendrick Charcoal blood agar
Regan-Lowe medium
Campylobacter Skirrow‘s medium
Yersenia Scheimann‘s selective medium
Clostridium Cooked meat medium
Thioglycolate broth
Schaedler‘s medium
Corynebacterium Cystine Tellurite medium
Tinsdale medium
Loeffler‘s medium

6. Special media
a. To isolate bacteria with specific growth requirements
b. Ex. Lowenstein-Jensen (L-J) medium, Thiosulfate Citrate Bile Salt Sucrose (TCBS)
7. Transport Medium
a. Used when there is an anticipated delay in bringing the specimen into the laboratory.it
can hold the specimen within 30m minutes
Pike‘s Media – S. pyogenes
JEMBEC – Neisseria
Alkaline Salt Transport Medium – V. cholera
Glycerol Saline Transport Media – Salmonella typhi
Mishulow‘s Medium – Bordetella
8. Culture Medium for Sensitivity or Susceptibility testing
a. Used to demonstrate the antibiotic resistance or sensitivity or an organism to different
antibiotics
Mueller Hinton Agar (MHA) – for fastidious organisms
1. pH – 7.2 – 7.4
2. depth – at least 4 mm
Middlebrook 7H 10, 7H 11 – for Mycobacterium
Wilkin-Chalgren Agar – for anaerobic bacteria
9. Biochemical medium
a. Used to demonstrate biochemical activities of bacteria that is useful in their identification
b. Ex. TSA, Citrate, IMViC, LIA

Ways to Facilitate Anaerobic Cultivation

1. Use of special culture medium incorporated with thioglycolate and cysteine (reducing agent)
2. Boiling of culture medium to remove (drive off) oxygen
3. Use of anaerobic chamber system with a vacuum pump and nitrogen gas to remove residual
oxygen
4. Use of Gaspak jar containing hydrogen and palladium catalysts to convert oxygen to water
5. For small volume, plastic bag or pouch containing calcium carbonate and catalyst can be used

 Methylene blue become colorless in the absence of oxygen

 Palladium pellets remove oxygen from the chamber by combining with hydrogen
to form water

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Lesson 4: CULTURAL CHARACTERISTICS
- One of the major features of bacteria is their appearance following growth on various media
o Color
o Abundance of growth
o Color or the culture

Evaluation of Bacterial Growth Growth in Tubed Media

- Size. Measure the diameter of the colony in

millimeters.
- Form: Punctiform, Filamentous, Rhizoid, Circular,
Irregular, Spindle like
- Elevation: Flat, Convex, Umbonate, Raised,
Pulvinate, Umbilicate
- Margin: Entire, Undulant, Lobate, Erose ,
Filamentous
- Color: White, Yellow, Black, Buff, Cream
- Density: Opaque, Translucent, Transparency
- Consistency: Butyrous, Viscid, Mebranous, Brittle

(https://laboratoryinfo.com/colony-morphology-of-bacteria/)

Agar plate colonies

- Size
o Puntiform: < 1 mm
o Small: 1-2 mm
o Medium: 3-4 mm
o Large: >5 mm
* Pseudomonas and Proteus spread across the entire agar plate
- Margin or Edge – evenly circular or with irregularities
o Rounded projections
o Irregular notches
o Threadlike or root-like projections
- Elevation
o Colonies may either be flat or raised
- Chromogenesis
o Colonies may be pigmented/colored or not
o Not all bacterial species have this distinctive feature
o Some bacteria retained the pigment within the cell while other bacteria colored the
medium
o Pigment production is best observed from growth on solid media
o May be diffusible to non-diffusible
- Optical features
o May be opaque, translucent or opalescent

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Evaluation of Bacterial Growth

(https://rsscience.com/how-to-see-bacteria-on-your-hand-bacteria-handprint/)

Growth on Agar Slant

- Amount – scanty, moderate or abundant
- Margin or edge – similar to agar plat
- Consistency of mass growth – butyrous or butter-like, viscous or stringy, dry and brittle
- Chromogenesis – similar to agar plate colonies

Growth in Nutrient broth

- Amount – similar to agar slant
- Distribution in broth
o Evenly turbid
o Confined to surface of broth as film (pellicle) or accumulated as sediment
o Granular or viscous
- Odor – putrid, fruity or aromatic, negligible, grape-like, gunpowder, fecal

Growth in gelatin stabs

- Growth along line of inoculation
o Confined to zone of inoculation streak
o Varying degrees of spreading away from the streak
- Liquefaction of gelatin
o May start evenly from top of various degrees of liquefied (funnel-like) may occur

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Other Parameters of Evaluation
- Hemolysis
o α – greening of blood agar (incomplete hemolysis)
o α‘ – small zone of alpha surrounded by a zone of beta hemolysis after refrigeration
o β – clear colorless are on blood agar (complete hemolysis), organism produces a toxin
that destroys red blood cells
o γ – no hemolysis on blood agar

Methods of Measuring bacterial growth

1. cell count – by microscopy, electronic particle counter or colony count
2. cell mass – by weighing, measuring nitrogen content, turbidimetry
3. cell activity – be relating biochemical activity

Methods of Quantifying Bacterial growth

METHODS
Microscopic count
- a measured volume of a bacterial
suspension is dispensed inside a defined
area on a microscope slide
Plate Count
- Most commonly used method
- It measureS the number of viable cells
(non-aggregate cells)
- 30 – 300 colonies
Membrane or Molecular filter
- Polycarbonate membrane filter
Turbidimetric method (cell mass)
- 10M to 100M cells/mL
Dry weight determination (cell mass)
Biochemical activity
APPLICATION
- enumeration of bacteria in milk and
vaccine
- breed count method (milk)
- Petroff-Hauser counter – prokaryotes
- Hemocytometer – prokaryotes and
eukaryotes
- Enumeration of bacteria in milk, food,
water and soil
- Enumeration of bacteria in food and water
(including lakes and streams)
- Microbial assays in broth or aqueous
suspensions
- For filamentous organism (fungi)
- Enumeration of bacteria in milk

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LABORATORY
TIME ALLOTMENT: 1.5 HOURS

Name: ___________________________________
Group No. ________________________________

Activity 6

METHODS OF INOCULATION

Introduction
.
A mixed culture contains two or more bacterial species that are known and can be easily
separated based on cultural or biochemical characteristics. Culturing techniques provide a means for
maintaining adequate nutrition for the organisms so they can continue to survive. As organisms grow in
a culture they consume the available nutrients and periodically need to be transferred to fresh media to
continue to grow. Certain culturing techniques not only provide the organisms with a fresh supply of
nutrients but also allow for the separation of bacterial cells to obtain isolated colonies.

These culturing procedures are known as isolation techniques. Streak plates allow for the
growth of isolated colonies on the surface of the agar. An isolated colony is a colony that is not touching
any other colonies and is assumed to be a pure culture.

Objectives:

At the end of the laboratory period, the student should be able to

1. confidently perform the different inoculation techniques used in bacteriology
2. identify correctly which inoculation to technique will be used on a specific culture media
3. strictly follow aseptic techniques during inoculation of bacteria

Materials:
Mixed culture TSIA
Sterile nutrient broth Inoculating loop
Nutrient agar plate Inoculating needle
LIA slant Alcohol lamp/Bunsen burner

Procedure:

Aseptic Technique (UK Standards for Microbiology Investigations, 2017)

Watch: https://www.youtube.com/watch?v=bRadiLXkqoU

When handling specimens or cultures, the use of an aseptic technique is crucial to avoid
contamination and to protect the worker from infection.
The following points should be observed when culturing specimens or performing
subcultures:

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 1. caps and lids from containers should not be placed on the workbench, but retained in the
hand while the sample is being processed, taking care not to contaminate the hand or
cap. Caps and lids should be replaced as soon as possible
2. lids from agar media should be placed on the bench to face upwards and after the plates
are inoculated, the lids should be replaced immediately to avoid any contamination
3. if the work is being carried out on the open bench, a disposable jar should be in close
proximity to the operator in order to discard the loops
4. keeping samples away from the face when opening culture containers. This can be
achieved by wearing the appropriate PPE when handling cultures
5. aerosol production should be minimised by:
a. opening caps of clinical specimens slowly in a microbiological safety cabinet as
the contents of containers are sometimes under pressure
b. avoiding vigorous swirling or shaking of the sample prior to opening by mixing the
sample gently
c. avoiding expelling the last drop from a pipette o removing excess fluid from a
swab put in a suspension (to be inoculated on an agar plate) by turning the swab
against the inside of the container
6. when forceps or scissors are used for handling specimens, they should be autoclaved
and sterilised before use. Use disposable forceps or scissors if available, and dispose
into a sharps bin after use

INOCULATION OF TUBED CULTURE MEDIUM

Watch: https://www.youtube.com/watch?v=nMoM8Ku5-8A

A. Broth medium from a swab

a. Collect a swab specimen. Your instructor will assign which specimen to collect.
b. Hold the nutrient broth with your left hand
c. Remove the cover. Flame-sterilize the mouth of the tube
d. Drop the cotton swab with the collected specimen into the culture tube.
e. Break-off of cut off the swab stick from the swab
f. Flame the mouth of the tube and replace the cover
g. Incubate at 37C for 24 hours

B. Broth Medium from a plate

a. Select an isolated colony from the dish culture
b. Flame the mouth of the tube
c. Tip the tube and insert the needle. Touch the inner surface of the glass tube, just
above the point where the surface of the medium makes an acute angle, and rub
the sides of the tube
d. Flame-sterilize the mouth of the tube and recap
e. Flame the needle
f. Incubate the tube at 37C for 24 hours

C. Inoculation of a slanted medium

a. Hold the tube lightly with the left hand and the loop with the right hand
b. With the little finger on the right hand, remove the cap of the tube
c. Flame-sterilize the mouth of the test tube and proceed with the inoculation
d. Starting from the butt end of the slant, draw the loop or needle over the surface
tracing a zigzag course from side to side towards the end of the slant
e. Flame-sterilize the mouth of the tube before recapping
f. Incubate at 37C for 18 – 24 hours

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D. Inoculation of Butt-Slant medium
a. Repeat ―a to c‖ from the previous procedure
b. Starting from the center of the surface of the media, thrust an inoculating needle
down to almost the bottom of the tube. Withdraw the inoculating needle along the
same route of inoculation
c. Starting from the point of exit, draw the needle over the surface, tracing a zigzag
course from side to side towards the end of the slant
d. Flame-sterilize the test tube and recap.
e. Incubate at 37C for 18 – 24 hours

E. Inoculation of a semi-solid medium and solid butt medium

a. Perform aseptic technique
b. With a previously sterilized inoculating needle, stab the media until almost to the
bottom and then withdraw.
c. Flame-sterilize the mouth of the tube and then recap
d. Incubate at 37C for 18 – 24 hours

INOCULATION OF PLATED MEDIA

Watch: https://www.youtube.com/watch?v=WYvTj6pIhbo

A. The uninterrupted streak method

a. Hold the petri dish with left hand, with the cover side up. With the thumb and little
finger, raise the lid and proceed with the inoculation
b. Start inoculation at the further side of the plate tracing a zigzag pattern from side
to side until you reach the middle and the widest part of the plate
c. Rotate the plate 180 degrees. Continue too inoculate the second half of the plate
in the same manner

B. The Interrupted Streak method

a. Divide the agar plates into four quadrants

b. With a loopful of the broth culture, inoculate one quadrant in a zigzag manner
starting near the periphery towards the center
c. Flame-sterilize the loop, pass it with the organism across the section just
previously streaked and inoculate the next quadrant
d. Do the same for the third and fourth quadrant

C. Radial Streak method

a. Place a loopful of the broth culture of the organisms near the edge of the plain
sugar plate.
b. From this source of organism, make radial straight-line streaking towards the
other sided at about 10-degree angles. Start from one side of the plate until the
whole plate has been inoculated. The loop is lifted every time the other end of the
plate is reached and streaking is again started at the source of the organism.

D. Overlap Streak Method

a. Start inoculation at the further side of the plate tracing a zigzag course from side
to side until you finish inoculating approximately one third of the plate.

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 b. Rotate the plate 90-degrees counterclockwise. Continue the inoculation from the
further side of the plate again making sure that the loop touches portion of the
plate previously streaked. Continue inoculation until you have covered another
plate third of the plate. Repeat the procedure until the plate is completely
inoculated.
c. Flame sterilizes the loop. Incubate the plate 37c for 24 hours.

E. Pour Plate
Watch: https://www.youtube.com/watch?v=Ppe_bgnPFHU

This method is extensively used in bacteriological studies of milk and water and isolation
of possible bacterial pathogens in either specimen. It is used in isolation of pathogens
mixed with other organisms in a mixed culture. Occasionally, it is used in blood culture
work and in the isolation of Salmonella from the feces.
a. To three test tube, add 9ml of sterile saline.
b. To the first test tube, add 1ml of the broth culture and mix thoroughly.
c. Transfer 1ml of the contents of the first tube to the second tube containing 9ml of
sterile saline.
d. Transfer 1ml of the contents of the second test tube to the third test tube
containing 9ml of saline.
e. From each tube using a separate sterile pipette, transfer 1ml into a sterile
corresponding petri dish.
f. Add about 8-12ml of melted and cooled agar about 45c to each of the three petri
dishes.
g. Rotate the petri dish a few times on a surface to allow the agar to solidify.
h. Incubate at 35c for 24 hours.

ILLUSTRATIONS
1. Draw the different methods of inoculation.

QUESTIONS FOR RESEARCH

1. What is isolation streaking? How is this done?
2. What are the different types of inoculating loops and needles?
3. What are the different standard loop measurements?
4. Define the following terms:
a. Inoculum
b. Colony
c. Stock Culture
5. What specimen preparation is needed for large volumes of specimen and contaminated
specimen?
6. Discuss the method of streaking for colony count.
7. What are the different ways of providing incubation conditions to cultures?

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Week 4
Time Allotment: 9 hours
Lecture: 6 hours
Laboratory: 3 hours

Topic: lecture
1. Specimen Management
2. Quality Assurance in The Microbiology Lab
3. Antimicrobial agents
a. Definition
b. Ways of Function
c. Classification
d. Drug Resistance
4. Antimicrobial Susceptibility Testing (Automated, Diffusion, Dilution)

Laboratory
Activity 7: Specimen collection
Activity 8: Antimicrobial Susceptibility Testing

Learning Objectives
At the end of the learning session, the student should be able to
1. Collect appropriate specimen for bacterial culture and analysis
2. Process the collected specimen correctly
3. Correctly state the mode of action of the different antimicrobials
4. Identify the drugs used for antimicrobial susceptibility testing

Lesson 1: Specimen Management

Time allotment: 2 hours

SPECIMEN COLLECTION AND HANDLING

Specimen collection and transportation are critical considerations, because any results
the laboratory generates is limited by the quality of the specimen and its condition on arrival in
the laboratory
Careful skin preparation before procedures such as blood cultures and spinal taps
decreases the chance that organisms normally present on the skin will contaminate the
specimen

General Considerations
1. Specimens should be collected during the acute (early) phase of an illness (or within 2-3
days for viral infection)
2. If possible, specimens should be collected before antibiotics are administered
3. Swabs are generally poor specimens (except nares and throat specimens) if tissue and
needle aspirates can be obtained
a. For anaerobes, aspirates are preferred to swabs
4. The specimen collected should be a representation of the diseased area
5. The quantity of the specimen should be sufficient enough for diagnostic testing

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Specimen Transport
1. Ideally, specimens should be transported to the laboratory within 30 minutes of collection
a. For anaerobic bacteria, transport should not take more than 10 minutes
b. For CSF samples, it should be transported within 15 minutes
2. All specimen containers should be leak-proof
3. All specimens should be transported within sealable, leak-proof, plastic bag; specimen
bags should be marked with biohazard label
4. Use of appropriate special preservatives or holding media for transport of specimen
delayed for more than 30 minutes is important in ensuring organism viability
a. Ex. Changes in temperature – Nesseria meningitidis
Changes in pH – Shigella spp

Specimen Preservation
1. Preservatives
a. Boric acid – maintains the appropriate colony counts (urine)
b. Polyvinyl alcohol (PVA) maintains the integrity of trophozoites and cysts
(stool)
c. Buffered formalin
2. Transport of holding media
a. Maintains the viability of microorganisms present in a specimen without
supporting the growth of any of the organisms
b. Charcoal sometimes is added to these media to absorb fatty acids present in the
specimen that could kill fastidious organisms (Neisseria gonorrheae and
Bordetella pertussis)
c. Examples: Stuart‘s medium, Amie‘s medium, Cary Blair, Transgrow, JEMBEC

Anticoagulants
- Used to prevent clotting of blood, bone marrow and synovial fluid
- 0.025% (w/v) Sodium Polyanethol Sulfonate (SPS) – this concentration of SPS is used
because Neissseria spp and some anaerobic bacteria are sensitive to higher
concentrations
- Heparin – used for viral cultures, it may inhibit the growth of g(+) bacteria and yeast

Specimen Storage
- If specimen cannot be processed immediately after receiving it in the laboratory, they
must be stored at
o Refrigerator temperature = 4C
 Urine, stool, viral specimens, sputa and swabs
 Specimens suspected of containing anaerobic bacteria should never be
stored in the refrigerator
o Body temperature = 37C
 CSF should always be stored at this temperature
o Ambient (room) temperature = 22C
o Freezer Temperature = -20C or -70C
 Serum for serological studies – frozen up to 1 week at -20C
 Tissues or specimen for long storage – (-70C)

Specimen Labelling
- Patient‘s name, Identifying number (hospital number), birth date and source
- Patient‘s information must be provided on the specimen label

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 - Identification of a mislabeled specimen or requisition should not be done over the
telephone

SPECIMEN PROCESSING
- When a specimen is received with multiple requests but the amount of the specimen is
insufficient, the clinician should prioritize the testing
- When multiple specimens arrive at the same time, priority should be given to those that
are most critical – CSF, tissue, blood and sterile fluids
- Urine, throat, sputa, stool or would drainage specimens can be saved later
- AFB, viral and fungal specimens – batch processing

1. Gross examination
a. Stool should be examined for evidence of barium (chalky white color), bloody,
cloudy or clotted
2. Direct Microscopic examination
a. The quality of the specimen can be assessed
Sputa can be rejected if it represents saliva and not lower respiratory tract
secretions by quantitation of white blood cells or squamous epithelial cells
= < 10 epithelial cells and > 25 pus cells
b. The microbiologist and clinician can be given an early indication of what may be
wrong with the patient (4+ WBC‘s in the exudate)
c. The work of the specimen can be guided by comparing what grows in culture to
what was seen on smear
Three different morphotypes (cellular types) are seen on gram stain but
only 2 grows out on culture, the third organism may be an anaerobic
bacterium

3 types of stains for direct examination

1. Gram stain
a. Helps visualize rod, cocci, WBC, RBC or squamous epithelial cells
2. Acid Fast stains
a. Ziehl-Neelsen
b. Kinyoun
c. Auramine-Rhodamine
3. Fungal Stains
a. KOH
b. PAS (Periodic Acid Schiff)
c. Calcoflour white

SPECIMEN PREPARATION
1. Homogenization – grinding of tissue
2. Concentration – by centrifugation of filtration of large volume of sterile fluids (peritoneal
or pleural)
3. Decontamination – for Legionella or Mycobacteria

*swab specimens – are often vortexed (mixed) in 0.5 – 1.0 mL of saline or broth for 10
– 20 seconds to dislodge material from fibers

Inoculation of Solid Media

1. Quantitatively by a dilution technique/quantitative loop
a. Urine cultures and tissues from burn victims

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 b. Plates inoculated for quantitation are usually streaked with 1:100 or 1:1000 loop
2. Semi-quantitative using an ordinary inoculating loop
a. Plates are usually struck out in 4 quadrants
b. Streaking for isolation – the microorganisms present in the specimen are
successfully diluted out as each quadrant is steaked until finally each morphocyte
is present as a single colony
Manner of reporting (grade)
 4+ - many, heavy growth; if growth is out to the fourth
quadrant
 3+ - moderate growth; if growth is out to the third
quadrant
 2+ - few or light growth; if growth is out to the second
quadrant
 1+ - rare, if growth is in the first quadrant

Incubation conditions
- 35 – 37C – for most bacteria, AFB and viruses
- 28C – for fungi

1. Aerobes – ambient air with contains 21% O2 + 0.03 % CO2

2. Anaerobes – 0% O2, 5-10% CO2 _ 5-10%H2 + 80 – 90% N2 (anaerobe jar, bag or
chambers)
3. Capnophiles – 2-10% O2 + 8 – 10% CO2

Processing of Commonly submitted specimens to a microbiology laboratory

1. Abscess (lesion, wound, pustule, ulcer)

a. Superficial abscess
Wipe area with sterile saline solution or 70% alcohol
Aerobic swab moistened with Stuart‘s or amie‘s medium
Swab along leading edge of wound
b. Deep abscess
Wipe area with sterile saline solution or 70% alcohol
Aspirate material from wall of excise tissue

2. Blood or bone marrow

a. Disinfect venipuncture site with 70% alcohol and betadine
b. Draw blood at time of febrile episode
c. Draw 2 sets from right and left arm
d. For blood, do not draw more than 3 sets in a 24-hour period
e. Culture bottles – aerobic and anaerobic culture bottles (vacutainer tubes with
SPS)
f. Considerations if antimicrobials have been administered
Neutralize antimicrobials like if patient is on penicillin, ass penicillinase to
the medium. However, penicillinase is not effective for methicillin,
cloxacillin, nafcillin, which are penicillinase resistant penicillin
g. Culture media
For aerobes: TSB, BHI, Columbia
For anaerobes: chopped meat or pre-reduced broth
h. Ratio of blood to culture medium
Adult: 1:10

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 Neonates: 5:10
i. Incubate for 7 days
Subculture in 24 h for preliminary result, 3 days, 5 days and 7 days for
final result.
BAP, CAP and MAC is used

3. Body fluids – amniotic, abdominal, ascites/peritoneal, bile, joints/synovial)

a. Disinfect skin before aspirating the sample/needle aspiration
b. It may need to concentrate the specimen by centrifugation or filtration (stain and
culture sediment)
c. Inoculate the sample as soon as received

4. Bone
a. Disinfect skin before surgical procedure
b. The sample should be taken from affected area for biopsy
c. The specimen may require homogenization

5. CSF
a. Disinfect skin prior to aspiration
b. Consider rapid testing like gram stain and cryptococcal antigen test
c. Storage: 6 hours at 37C, except for viruses, which can be held at 4C for up to 3
days
d. Gram stain can be performed by cytocentrifugation
e. Specimen is placed in 3 to 4 tubes
Tube procedure Preservation
1 Chemistry and Serology Freezer
2 Microbiology Room temperature/incubator
3 Cell count Refrigerator
f. Preliminary tests: centrifuge; use sediment for
Smears – gram stain, india ink
Culture – Haemophilus influenzae, Neisseria meningitidis, Streptococcus
pneumoniae
g. Culture media: BAP, CAP, MAC
h. Enrichment broth: BHI

6. Ear
a. Inner ear
Clear ear canal with mild soap solution before myringotomy (puncture of
the eardrum)
Aspirate material behind the eardrum with syringe if eardrum is intact
Use swab to collect material from ruptured ear drum
b. Outer ear
Wipe away crust with sterile saline
Aerobic swab moistened wit Stuart‘s or amie‘s medium may be used
Firmly rotate swab in outer canal to collect the sample

7. Eye
a. Conjunctiva
Obtain sample from both eyes

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 Aerobic swab moistened wit Stuart‘s or amie‘s medium may be used or a
swab pre-moistened with sterile saline
b. Corneal scraping
The clinician instills local anesthetic before specimen collection
Bedside inoculation of the following media should be done: BAP, CAP,
SDA, 7H10, FT

8. Foreign bodies
a. IUD
Disinfect skin before remove of the IUD
b. IV, catheters, pins, prosthetic valves
Do not culture foley catheters
Specimens should be rolled back and forth across the agar using sterile
forceps 4x
 colonies are associated with clinical significance

9. GI tract
a. Gastric aspirate
The specimen should be collected early in the morning before the patient
eats or gets out of bed
Most gastric aspirates are on infants or for acid fast bacilli (AFB)
Sample must be neutralized within one hour of collection
b. Gastric biopsy
Rapid urease test or breath test for Helicobacter pylori
c. Rectal swab
Insert swab – 2-5cm past the anal sphincter
Direct exam – methylene blue for fecal leukocytes
d. Stool culture
Collect 3 specimens every other day (outpatients)
Collect 3 specimens everyday (inpatients)
Wait 7-10 days if patients have received antiparasitic compounds
Direct exam: methylene blue for fecal leukocytes

10. Genital tract

a. Bartholin cyst
Disinfect skin before collection
Specimen is aspirated
b. Cervix
Remove mucus before collection of specimens
Do not use lubricant on speculum; swab deeply into cervical canal
Swab must be moistened with Stuart‘s or Amie‘s medium
c. Cul-de-sac
Sample aspiration done by gynecologists
d. Endometrium
Surgical biopsy or transcervical aspirate via sheathed catheter
e. Urethra (female)
Remove exudates from urethral opening
Collect discharge by massaging urethra against pubic symphysis or insert
flexible swab 2-4 cm into urethra and rotate swab for 2 seconds
Swab must be moistened with Stuart‘s or Amie‘s medium
f. Vagina

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 Remove exudates before specimen collection
Swab must be moistened with Stuart‘s or Amie‘s medium
Swab the secretions and mucus membrane of vagina
g. Prostate
Clean glands with soap and water
Swab must be moistened with Stuart‘s or Amie‘s medium
Collect the secretions on swab or in tube
h. Urethra (male)
Insert flexible swab 2-4 cm into urethra and rotate for 2 seconds or collect
discharge on JEMBEC transport system
Swab moistened with Stuart‘s or Amie‘s medium
11. Hair, Nails, or skin scrapings (for fungal culture)
a. Nails or skin: wipe with 70% alcohol
b. Hair: collect hairs with intact shaft
c. Nails: send clippings of effected area
d. Skin: scrape skin at leading edge of lesion

12. Respiratory tract

a. Lower (Bronchial Alveolar lavage. Bronchial brush, bronchial wash)
Anerobic culture is appropriate only if sheathed (protected) catheter was
used
b. Sputum, tracheal aspirate (suction)
Sputum: have patient collect from deep cough; induced sputa on pediatric
or uncooperative patient may be watery because of saline nebulization
It should be examined immediately or be kept in the refrigerator for 1-3
hours. Do not do anaerobic cultures on expectorated sputum for oral
anaerobes might contaminate the culture
Transport device: sterile leak proof container
Transport time: less than 2 hours
Incubation temp: 35 – 37 C
Incubation period: 48 hours

- Suitability Criteria for Sputum Culture

Group Leukocytes Epithelial cells
6 >25 <5
5 >25 <10
4 >25 10 – 25
3 >25 >25
2 10 – 25 >25
1 <10 >25
Cell numbers per x 100 (LPF)
*only sputum samples in group 4 – 6 should be cultured
Or: observe the Bartlett‘s Criteria where sputum for culture should have PMN cells of
more than 25/LPF and epithelial cells of less than 10/LPF

c. Upper RT
Nasopharynx
 Swab is moistened with Stuart‘s or Amie‘s media
 Insert flexible swab through the nose into posterior nasopharynx
and rotate for 5 seconds

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  Specimen of choice for Bordetella pertussis

- Commonly isolated organisms form nasopharyngeal swab

H. influenzae Enriched CAP or BAP with Staphylococcus streak across
the inoculum
N. meningitidis Enriched CAP or TMA
F. pertussis Charcoal cephalexin medium

Pharynx (Throat)
 Swab moistened with Stuart‘s or Amie‘s medium
 Swab posterior pharynx and tonsils
 Routine culture for Group A Streptococcus (S. pyogenes) only

- Commonly isolated organisms from a throat swab

Alpha Streptococcus Most abundant normal flora; potential contaminant
Group A Streptococcus Most common pathogen; include anaerobic
conditions for beta Streptococcus (some are not
hemolytic unless conditions are anaerobic)

Culture on Todd-Hewitt broth for fluorescence

microscopy of beta Streptococcus

13. Tissue
a. Disinfect the skin
b. Do not dry out the specimen
c. Moisten with sterile, distilled water if not bloody
d. May need to homogenize

14. Urine
a. Clean voided midstream (CVS) of urination
Female
 Clean area with soap and water
 Hold labia apart and begin voiding
Male: clean glands with soap and water, retract foreskin
b. Straight catheter (in and out)
Clean urethra with soap and water
Insert catheter into bladder and allow first 15 mL to pass, then collect the
remainder
c. Indwelling catheter
Aspirate 5 – 10 mL of urine with needle and syringe
d. Supra-pubic aspirate
Needle aspiration above the symphysis pubis through abdominal wall into
the full bladder

- Indication of UTI: urinary tract infection is exhibited if the gram stain of an uncentrifuged
urine has 1 or more organisms per OIF with WBC or a colony count of 100,000 (10 6)

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 organisms per mL. It should also be noted that the presence of many squamous cells
indicates vaginal or urethral contamination

TRANSPORT TIME

By Type of Specimen
Specimen Time Recommended
Respiratory 1h
Gastrointestinal 1h
Blood 1h
CSF Immediately
Other body fluids Immediately
Urine 1h
Exudates and Transudates 30 min

By Bacteriological Examination
Type of Examination Time Recommended
Respiratory 30 min
Gastrointestinal 1h
Blood 1h
CSF 1h
Other body fluids 1h
Urine 1h
Exudates and Transudates 1h

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LABORATORY
TIME ALLOTMENT: 1.5 hours

Note: this activity will not be performed. Procedures are written for reference.

Name: ____________________________________________
Group No. _________________________________________

Activity No. 7

SPECIMEN COLLECTION AND TRANSPORT

Introduction
NOTE: The following guidelines were based on the published Standard Operating Procedures for Microbiology of the
World Health Organization (WHO).

The laboratory diagnosis of an infectious disease begins with the collection of a clinical
specimen for examination or processing in the laboratory (the right one, collected at the right
time, transported in the right way to the right laboratory). Proper collection of an appropriate
clinical specimen is the first step in obtaining an accurate laboratory diagnosis of an infectious
disease. Guidelines for the collection and transportation of specimens should be made available
to clinicians in a lucidly written format. The guidelines must emphasize two important aspects:
1. Collection of the specimen before the administration of antimicrobial agents.
2. Prevention of contamination of the specimen with externally present organism or
normal flora of the body.

General Rules for Collection and Transportation of Specimens:

1. Apply strict aseptic techniques throughout the procedure.
2. Wash hands before and after collection.
3. Collect the specimen at the appropriate phase of disease.
4. Make certain that the specimen is representative of the infectious process (e.g. sputum
is the specimen for pneumonia and not saliva) and is adequate in quantity for the
desired tests to be performed.
5. Collect or place the specimen aseptically in a sterile and/or appropriate container.
6. Ensure that the outside of the specimen container is clean and uncontaminated.
7. Close the container tightly so that its contents do not leak during transportation.
8. Label and date the container appropriately and complete the requisition form.
9. Arrange for immediate transportation of the specimen to the laboratory.

SPECIMEN COLLECTION

The clinical state of the patient will not necessarily be reflected by the result of laboratory
investigation despite correct laboratory performance unless the specimen is in optimal condition
required for the analysis. Some of the important specimens and their proper collection and
transportation methods are described here so as to ensure quality.

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A. BLOOD

Whole blood is required for bacteriological examination. Serum separated from blood is
used for serological techniques. Skin antisepsis is extremely important at the time of collection
of the sample. Tincture of iodine (1-2%), povidone iodine (10%) and chlorhexidine (0.5% in 70%
alcohol) are ideal agents. However, some individuals may be hypersensitive to iodine present in
some of these. While collecting blood for culture, the following points must be remembered:
1. Collect blood during the early stages of disease since the number of bacteria in blood is
higher in the acute and early stages of disease.
2. Collect blood during paroxysm of fever since the number of bacteria is higher at high
temperatures in patients with fever.
3. In the absence of antibiotic administration, 99% culture positivity can be seen with three
blood cultures.
4. Small children usually have higher number of bacteria in their blood as compared to
adults and hence less quantity of blood needs to be collected from them.
Volume of blood to be collected at different ages
Age Volume in 2 bottles
< 2 years 2 mL
2 – 5 years 8 mL
6 – 10 years 12 mL
>10 years 20 mL

B. CEREBROSPINAL FLUID (CSF)

Examination of CSF is an essential step in the diagnosis of any patient with evidence of
meningeal irritation or affected cerebrum. Almost 3-10 ml of CSF is collected and part of it is
used for biochemical, immunological and microscopic examination and remaining for
bacteriological or fungal examination. The following important precautions need to be taken for
CSF collection and transportation:

Appearance and interpretations of CSF

Clear and colourless Normal
Clear with Tyndall effect
High protein content
(sparkling appearance against incident light)
Clear yellowish Old haemolysis
Clear red Fresh haemolysis
Turbid blood-stained Haemorrhage
Turbid white High cell or protein content
Turbid clot (after overnight storage) Fibrin clots

G. SPUTUM

Sputum is processed in the laboratory for etiological investigation of bacterial and fungal
infections of the lower respiratory tract. It is of utmost importance in the diagnosis of pulmonary
tuberculosis.
1. Select a good wide-mouthed sputum container, which is preferably disposable, made of
clear thin plastic, unbreakable and leak proof material.

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 2. Give the patient a sputum container with the laboratory serial number written on it. Show
the patient how to open and close the container and explain the importance of not
rubbing off the number written on the side of the container.
3. Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and spit in
the sputum container by bringing it closer to the mouth.
4. Make sure the sputum sample is of good quality. A good sputum sample is thick,
purulent and sufficient in amount (2-3 ml).
Give the patient an additional container with laboratory serial number written on it for an early
morning specimen. Explain to the patient to rinse his/her mouth with plain water before bringing
up the sputum.

D. URINE

Under normal circumstances urine is sterile. The lower part of the urethra and the
genitalia are normally colonized by bacteria, many of which may also cause urinary tract
infection. Since urine is a good growth medium for all sorts of bacteria, proper and aseptic
collection assumes greater importance for this specimen.
For microbiological examination urine must be collected as a "clean catch-mid-stream"
specimen.
Urine specimens should be transported to the laboratory within one hour for
bacteriological examination, because of the continuous growth of bacteria in vitro thus altering
the actual concentration of organisms.

H. STOOL

Fecal specimens for the etiological diagnosis of acute infectious diarrheas should be
collected in the early stage of illness and prior to treatment with antimicrobials. A stool specimen
rather than a rectal swab is preferred.
1. The feces specimen should not be contaminated with urine.
2. Do not collect the specimen from bed pan.
3. Collect the specimen during the early phase of the disease and as far as possible before
the administration of antimicrobial agents.
4. 1 to 2 gm quantity is sufficient.
5. If possible, submit more than one specimen on different days.
6. The fresh stool specimen must be received within 1-2 hours of passage.
7. Store at 2-8oC.
8. Modified Cary and Blair medium is recommended as a good transport medium. It is a
very stable medium and can be stored for use in screw – capped containers. It is a semi-
solid transport medium. At least two swabs should be inoculated. Most pathogens will
survive for up to 48 hours at room temperature. Specimens are unacceptable if the
medium is held for more than one week or if there is detectable drying of the specimen.

Alternative transport media are Venkataraman-Ramakrishnan medium (V-R fluid) or alkaline

peptone water. VR fluid should be prepared in 30 ml (1 oz) screw capped bottles (MacCartney
bottles). It preserves vibrios for more than six weeks and has also proved to be a very
convenient medium for transportation as it can be kept at room temperature after collection of
the specimen.

F. THROAT SWAB

1. Depress the tongue with a tongue blade.

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 2. Swab the inflammed area of the throat, pharynx or tonsils with a sterile swab taking care
to collect the pus or piece of membrane.
3. Transport in sterile transport tube.

G. BONE MARROW

Bone marrow is collected by a doctor who is well trained in this procedure

1. Decontaminate the skin overlying the site from where specimen is to be collected with
70% alcohol followed by 2% tincture of iodine.
2. Aspirate 1 ml or more of bone marrow by sterile percutaneous aspiration.
3. Collect in a sterile screw-cap tube.
4. Send to laboratory immediately.

H. RECTAL SWAB

1. Insert swab at least 2.5 cm beyond the anal sphincter so that it enters the rectum.
2. Rotate it once before withdrawing.
3. Transport in Cary and Blair or other transport medium.

TRANSPORTATION OF SPECIMENS

Specimens to be sent to other laboratories require special attention for safe packing of
the material. Guidelines are usually issued by national authorities and the same should be
strictly followed. For hand-carried transportation over a short distance, the specimen should be
placed upright in appropriate racks. For long distance transportation, it should be placed in three
containers

1. A primary container which has the specimen and is leakproof with a screw-cap.
2. A secondary container which is durable, waterproof and made of metal or plastic
with a screw-cap. It should have enough absorptive material to absorb the
contents of the primary container should the latter break or leak. On its outside,
the details of the specimen should be pasted.
3. A tertiary container is usually made of wood or cardbox. It should be capable of
withstanding the shocks and trauma of transportation. Dry ice can be kept
between this and the secondary container along with sufficient absorbents and
provision for the escape of carbon dioxide to prevent a pressure build-up inside.

In general, most specimens should be processed in the laboratory within 1 to 2 hours

after collection. In practice, a 2-to 4-hour time limit is probably more practical during a normal
working day. The laboratory must be organized to permit processing of the specimens as soon
as they arrive, and the collection of most specimens should be limited to the working hours of
the laboratory. However, some arrangements must be made to allow for the initial handling of
the few specimens that have to be collected outside of the laboratory‘s working hours.
A continuous effort must be made in order to ensure proper collection and transportation
of clinical specimens. Full cooperation of nursing staff and others concerned with specimen
collection is required and can be achieved once they are made aware of the principles involved
and the significance of what they are being asked to do.

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Illustrations:
1. Carefully scan reference materials/books/internet and look for clear and labeled photos
of the following. Study them carefully and attach them to this activity.
a. Throat Swab
b. Nasal Swab
c. Vaginal Swab
d. Ear Swab
e. Lumbar Tap
f. Paracentesis
g. Thoracentesis
h. Pericardiocentesis
i. Venipuncture
j. Amniocentesis
2. Two photos are recommended per page
3. On a separate page, download a labeled photo of the complete steps for phlebotomy.

Questions for Research:

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. What are the different criteria for rejection of specimens submitted in the microbiology
section?
2. During phlebotomy, what are the considerations to be made before, during and after the
procedure?

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LESSON 2: QUALITY ASSURANCE IN THE LAB
TIME ALLOTMENT: 1 HOUR

ACTIVITY
What are the different factors involved in quality assurance in the microbiology laboratory?
Discuss thoroughly.

LESSON 3: INHIBITING THE GROWTH OF MICROORGANISMS IN VIVO

TIME ALLOTMENT: 2 HOURS

Antimicrobial chemotherapy
- Treatment of diseases through chemical compounds
- 17th century
o Drugs have been used for the treatment of infectious diseases
o Example:
 Quinine for malaria
 Emetine for amoebiasis

Paul Ehrlich
- The science of chemotherapy began with Paul Ehrlich
- He is the father of chemotherapy
- Discovered salvarsan or arsphenamine or 606
- contributions
o He formulated the principles of selective toxicity
o Recognized the specific relationships between microbial pathogens and drugs
o Development of drug and drug resistance
o The role of combined therapy

Antibiotics
- Chemotherapeutics of microbial origin
- Sources of antibiotics
o From fungi or bacteria
o Chemically prepared

Characteristics of antibiotics
- Selective toxicity
o Implies that a drug is harmful to a parasite without being harmful to the host
- Kill or inhibit the growth of pathogens
- Cause no allergic reaction in the host
- Be stable when stored in solid or liquid form
- Remain in specific issues in the body long enough to be effective
- Kill the pathogen before they mutate and become resistant to it

Classification of Antibacterial Agents

Broad V. narrow spectrum antibiotics

- Narrow spectrum antibiotics
o Kills only a limited group of organisms

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 o G+ or G-
o Ex. Vancomycin, nalidixic acid, colistin
- Broad Spectrum antibiotics
o For both G+ and G- bacteria
o Ex. Ampicillin, chloramphenicol and tetracycline
Competitive inhibitors
- Inhibit growth of microorganism by competing with an enzyme needed to produce an
essential metabolite
- Bacteriostatic – inhibit growth
- Examples: sulfa drugs

Mechanism of Action
1. Inhibition of cell wall synthesis
2. Inhibition of cell membrane function
3. Inhibition of protein synthesis (inhibition of translation and transcription of genetic
material)
4. Inhibition of nucleic acid synthesis (either DNA or RNA synthesis)
5. Inhibition of enzyme activity

Inhibition of cell wall synthesis

- Beta-lactam antimicrobial agents
o Antibiotics with the beta-lactam ring
o Binds enzymes involved in cell wall synthesis
o PBP‘s (penicillin binding proteins) – site in the cell wall of bacteria where the
beta-lactam ring binds to
o Examples:
 Penicillins – penicillin, ampicillin, piperacillin
 Cephalosporins – cefalozin, cefuroxime, ceftriaxone
 Monobactams – aztreonams
 Carbapenems – imipenem, meropenem
- Glycopeptides
o Vancomycin – binds to precursors of cell wall synthesis
 Usually ineffective against G- bacteria
o Bacitracin – inhibits the recycling of certain metabolites required for maintaining
the peptidoglycan layer
 It is toxic and thus for topical use only

Inhibitors of Cell Membrane Function

- Polymyxin B and colistin
o Disrupt cell membrane
o Leads to leakage of macromolecules and ions essential for cell survival
o More active against G- bacteria

Inhibitors of Protein Synthesis

- Aminoglycosides
o Binds to protein receptors on the organism‘s 30S ribosomal unit
o Ex. Gentamycin, tobramycin, amikacin, netilmicin, streptomycin and kanamycin
o For G+ and G- bacteria
o Not for anaerobes
- Macrolide-lincosamide-Streptogamin (MLS) group

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 o Binds to receptors on the bacterial 50S ribosomal subunit
o Ex. Erythromycin, azithromycin, clarithromycin, clindamycin
- Oxazolidinones
o Synthetic drug
o Not expected to be affected by drug resistance mechanisms
o Ex. Linezolid
- Chloramphenicol
o Inhibits addition of new amino acids by binding to the 50S ribosomal subunit
o For G- and G+ bacteria
- Tetracyclins
o Binds to the 30S ribosomal subunit
o Inhibits tRNA –amino acid complex from binding to the ribosome
o For G- and G+ bacteria

Inhibitors of DNA and RNA synthesis

- Fluoroquinolones
o AKA quinolones
o Derived from nalidixic acid
o Binds and interferes with DNA gyrase for bacterial DNA supercoiling
o Ex. Ciprofloxacin and ofloxacin
o For G- and G+ bacteria
- Metronidazole
o Direct interaction between the activated drug and DNA that results to the
breakage of the DNA strand
o Activation happens at anaerobic conditions
o For anaerobic G- bacteria
- Rifampin
o Binds to the enzyme DNA –dependent RNA polymerase and inhibits synthesis of
RNA
o For G+ bacteria

Inhibitors of other metabolic processes

- Sulfonamides
o Binds to dihydropteroate synthase to disrupt the folic acid pathway
o Folic acid PW is important for the precursors needed for DNA synthesis
o For G+ and G- bacteria
- Trimetoprim
o Targets dihydrofolate reductase in the folic acid PW
o For G+ and G- bacteria
- Nitrofurantoin
o May have several targets involved in bacterial protein and enzyme synthesis
o May directly damage DNA
o For G+ and G- bacteria causing UTI

DRUG RESISTANCE

“Superbugs”
- Bacteria that become resistant to one or more antimicrobial agents
- Multi-drug-resistant - pathogens that are resistant to several different antimicrobial
agents

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 1. MRSA (Methicillin-resistant Staphylococcus aureus), MRSE (methicillin-resistant S.
epidermidis)
- Resistant to all anti-staphylococcal drugs except vancomycin, synercid and zyvox

2. VISA (vancomycin-intermediate S. aureus)

- Organism has developed resistance to the usual dosage of vancomycin, thus higher
dosages are needed.

3. VRE (vancomycin-resistant Enterococcus Spp)

- Resistant to most anti-enterococcal drugs including vancomycin
- Enterococcus causes nosocomial UTI

4. MDRTB (multi-drug-resistant Mycobacterium tuberculosis)

- Resistant to all antitubercular drugs and combinations of these drugs

5. Multi-drug-resistant strains of pseudomonas spp., Salmonella spp., Shigella spp., and

Neisseria Gonorrhoeae

6. Beta-lactamase producing strains of Streptococcus pneumoniae and Haemophilus

influenzae

Mechanisms by which bacteria become resistant to antimicrobial agents

LESSON 4: ANTIMICROBIAL SUSCEPTIBILTY TESTING

TIME ALLOTMENT: 1.5 hours

TEST TUBE DILUTION/BROTH DILUTION METHOD

- Involves challenging the organism of interest with
antimicrobial agents in a broth environment (Mueller-
Hinton Broth)
- Specific amount of antibiotic is prepared in a
decreasing concentration in broth by serial dilution

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 techniques, and standard number of the test organism is inoculated
- Standard inoculum size: 5x105 CFU/mL
- Absence of turbidity of broth signifies inhibition of bacterial growth by the antibiotics
tested.
- Can be used to determine MIC and MLC concentrations
o Microdilution
 Broth volume: 0.05 – 0.1 mL
 Schaeder‘s broth, West Wilkins broth, BHI
o Macrodilution
 Broth volume: 1.0 mL or greater
- The lowest concentration of the antibiotic without bacterial growth after 16 to 24 hours of
incubation is the minimum inhibitory concentration (MIC)
- The lowest concentration of the antibiotic without bacterial growth when sub-cultured to
a fresh medium is the minimum lethal concentration (MLC)
- MH broth with 2% NaCl – to improve detection of oxacillin-resistant staphylococci

ANTIMICROBIAL GRADIENT METHOD

- the antimicrobial gradient diffusion method uses the principle of establishment of an
antimicrobial concentration gradient in an agar medium as a means of determining
susceptibility
- The Etest (bioMérieux AB BIODISK)
o commercial version
o It employs thin plastic test strips that are impregnated on the underside with a
dried antibiotic concentration gradient
o marked on the upper surface with a concentration
scale
- 5 or 6 strips may be placed in a radial fashion on the
surface of an appropriate 150-mm agar plate that has
been inoculated with a standardized organism
- After overnight incubation, the tests are read by viewing
the strips from the top of the plate.
- The MIC is determined by the intersection of the lower part
of the ellipse shaped growth inhibition area with the test
strip.
- the gradient diffusion method has intrinsic flexibility by being able to test the drugs the
laboratory chooses.
- This method is best suited to situations in which an MIC for only 1 or 2 drugs is needed
or when a fastidious organism requiring enriched medium or special incubation
atmosphere is to be tested (eg, penicillin and ceftriaxone with pneumococci)
- Generally, Etest results have correlated well with MICs generated by broth or agar
dilution methods.
- However, there are some systematic biases toward higher or lower MICs determined by
the Etest when testing certain organism-antimicrobial agent combinations.
- This can represent a potential shortcoming when standard MIC interpretive criteria
derived from broth dilution testing are applied to Etest MICs that may not be identical

AGAR DILUTION METHOD

- The antimicrobial concentrations and organisms to be tested are brought together on an
agar-based medium rather than in a broth
- One or more bacterial isolates are tested per plate, MIC can also be determined
- Standard inoculum size: 1x104 CFU/mL

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 - Shelf life of agar dilution plate is only one week for most antimicrobial agents
- After incubation, the plates are examined for growth
- Reference method for anaerobes: Brucella agar with laked blood and vitamin K
(Wadsworth method)

DISC DIFFUSION METHOD (KIRBY-BAUER TEST)

- The disk diffusion susceptibility method is simple and practical and has been well-
standardized.
- The test is performed by applying a bacterial inoculum of approximately 1–
2×108CFU/mL to the surface of a large (150 mm diameter) Mueller-Hinton agar plate
- Up to 12 commercially-prepared, fixed concentration, paper antibiotic disks are placed
on the inoculated agar surface.
- Plates are incubated for 16–24 h at 35°C prior to
determination of results.
- The zones of growth inhibition around each of the
antibiotic disks are measured to the nearest
millimeter.
- The diameter of the zone is related to the
susceptibility of the isolate and to the diffusion rate of
the drug through the agar medium.
- The zone diameters of each drug are interpreted
using the criteria published by the Clinical and
Laboratory Standards Institute (CLSI, formerly the
National Committee for Clinical Laboratory Standards or NCCLS) or those included in
the US Food and Drug Administration (FDA)-approved product inserts for the disks.
- The results of the disk diffusion test are ―qualitative,‖ in that a category of susceptibility
(ie, susceptible, intermediate, or resistant) is derived from the test rather than an MIC.
- advantages
o test simplicity
o does not require any special equipment,
o the provision of categorical results easily interpreted by all clinicians,
o flexibility in selection of disks for testing.
- disadvantages
o lack of mechanization or automation of the test.
o not all fastidious or slow growing bacteria can be accurately tested by this
method
 the disk test has been standardized for testing streptococci, Haemophilus
influenzae, and N. meningitidis through use of specialized media,
incubation conditions, and specific zone size interpretive criteria

Turbidity Standard
- the use of a standard inoculum size is as important as culture purity and is accomplished
by comparison of the turbidity of the organism suspension with a turbidity standard
- 0.5% McFarland Turbidity Standard
o 99.5 mL of 1% H2SO4 + 0.5 mL of 1.175 % of barium chloride
o Is equivalent to 1.5 x 108 colony forming units per mL
- Pure cultures are grown or are directly prepared from agar plates to match the turbidity
of the 0.5 McFarland standard

Preparation of Pure Culture for Susceptibility testing

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 - Pure inocula (cultures) are obtained by selecting 4-5 colonies of the same morphology,
inoculate them into broth medium and incubate for 3-5 hours to achieve a turbid
suspension
- Alternatively, 4-5 colonies 16 – 24 hours of age may be selected from an agar plate and
suspended in broth or 0.85% NSS to achieve a turbid suspension
- Matching turbidity using the unaided eye is facilitated by holding the bacterial
suspension and McFarland tubes side by side and viewing them against a black-lined
background.
- If the bacterial suspension initially does not match the standard‘s turbidity, the
suspension may be diluted, or supplemented with more organisms, as needed.

Important Considerations
1. Within 15 minutes of inoculation, the antimicrobial agent disks are applied and the plates
are inverted for incubation to avoid contamination of moisture on the agar surface that
can interfere with the interpretation of the test results
2. When disks containing known concentration of antimicrobial agent are placed on the
surface of a freshly inoculated plate, the agent immediately begins to diffuse and
establish a concentration gradient around the paper disk.
3. Upon incubation, the bacteria grow on the surface of the plate except where the
antibiotic concentration in the gradient around each disk is sufficiently high to inhibit
growth
4. The diameter of the zone of inhibition around the disk is measured in millimeters using a
caliper
5. A wide zone surrounding a disk signifies more susceptibility of the organism to the
antibiotic
6. Zone width is related to antibiotic concentration, diffusion rate through agar and solubility
7. Susceptibility is indicated by the presence of zone of inhibition around the antibiotic disk
8. The zone of inhibition is inversely related to MIC – the larger the zone of inhibition, the
lower the MIC
9. MHA containing 5% sheep‘s blood is used to testing streptococci and other fastidious
organisms

Factors Affecting Zone of Inhibition

1. Thickness of the susceptibility agar plate (4-6 mm)
a. If the agar plate is too thick, zone sizes would be smaller, if the agar is too thin,
zone sizes would be larger
2. The amount of inoculum or test organism
3. The growth rate of the test organism
a. Air at 35 – 37C for most bacteria (16-24 hours)
b. 5-7% CO2 for N. menigitidis
c. Prolonged incubation may result in false resistance interpretation
4. pH of the medium (7.2 – 7.4)
a. decrease pH affects the activity of antimicrobial agents
b. aminoglycosides and macrolides may lose potency, penicillin may have increase
activity
5. number of disk per plate
a. 12 disks per plate
b. Placement of more than 12 disks result in overlapping zones (erroneous results)
6. Concentration of divalent cations (calcium and magnesium)
a. May affect testing of aminoglycosides and tetracyclins against P. aeruginosa

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Causes of False Resistance
1. Use of unduly heavy inocula of cultures or undiluted specimen materials
2. The examination of test plates after zones have become overgrown
3. The use of disk with inadequate drug concentration due to prolonged storage, failure to
refrigerate as from disk container opened frequently
4. Use of wrong organism

SERUM BACTERICIDAL TEST

- Determines the activity of one or more antimicrobial agents present in serum against an
organism that has been recovered from the patient

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LABORATORY
TIME ALLOTMENT: 1.5 HOURS

Note: This activity will not be performed. Procedures are written for reference only and all
results are theoretical.

Name: _______________________________________
Group No. ____________________________________

Activity no. 8

ANTIMICROBIAL SUSCEPTIBILITY TESTING

(KIRBY-BAUER DISC DIFFUSION METHOD)

Introduction
Antimicrobial susceptibility testing (AST) determines the concentration of an
antimicrobial that inhibits microbial growth, for both microbicidal and microbiostatic agents
(Brown et al., 2016; Sanguinetti and Posteraro, 2018; Humphries et al., 2019; van Belkum et al.,
2019). The importance of accurate AST in at least guiding antibiotic use in the clinic cannot be
underestimated (Doern et al., 1994; Kumar et al., 2009; Weiss et al., 2012; Holmes et al., 2016).
During the development of novel antimicrobials, AST is vitally important; (i) to determine
the preclinical activity of drug candidates and allow the identification of lead compounds, (ii) to
facilitate the determination of the likelihood of resistance development, (iii) to provide estimates
of likely in vivo and critically, clinical efficacy when testing compounds in biological matrices
replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum,
urine, biofilms, etc. (Breteler et al., 2011; Macia et al., 2014; Bottger et al., 2017; Ersoy et al.,
2017; Nizet, 2017; Savini et al., 2017; Starr and Wimley, 2017; Haney et al., 2019).
(https://www.frontiersin.org/articles/10.3389/fcimb.2020.00326/full)

Objectives:
At the end of the laboratory session, the student should be able to
1. perform correctly antibiogram testing among select bacteria by disc diffusion method.
2. interpret the results accurately of antimicrobial susceptibility testing
3. apply confidently the test for diagnostic purposes.

Required Materials:

Sterile cotton swab

Sterile forceps
Mueller Hinton Agar Plates
18-hour broth culture of the following microorganism (as available):
a. S. aureus
b. E. coli
c. P. aeruginosa

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 Commercially prepared antibiotics

Procedure:

A. 0.5 McFarland Standard Preparation

McFarland Standards are used to standardize the approximate number of bacteria in a liquid
suspension by comparing the turbidity of the test suspension with the McFarland Standard.
A McFarland Standard is a chemical solution of barium chloride (BaCl 2) and sulfuric acid
(H2SO4); the reaction between these two chemicals results in the production of a fine
precipitate, barium sulfate. When shaken well, the turbidity of a McFarland Standard is
visually comparable to a bacterial suspension of known concentration as indicated in the
table.

NOTE: McFarland Standards should be stored in standing position at 4°C to 8°C and
protected from light during 12 weeks.

B. Kirby-Bauer Disc Diffusion Method

1. Standardize the inoculums by comparing the 0.5 McFarland Standard (BaSO4). If the
test organism is more turbid, dilute with nutrient broth; if more dilute, incubate further.
2. With a sterile swab, carefully streak the entire surface of the Mueller Hinton agar with the
broth cultures of the organism.
3. Label the plate with microorganism used.
4. Place equidistant from each other and on circular fashion, one disc each of antibiotics.
5. Press the discs firmly on the agar.
6. Invert the plates and incubate at 35oC for 16 – 24 hours.
7. After incubation, measure with a vernier caliper or ruler the diameter of the zone of
inhibition of each antibiotic and express results in millimeters.
8. Interpret the results by comparing the zone size with the Zone diameter Interpretive
Standard based on new NCCLS (now CLSI) guidelines.

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 Antimicrobial Susceptibility Flowchart:

Streaking Method (Entire)

Measuring Zone of Inhibition

Organism Zone of Inhibition Interpretation

1.
2.
3.

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McFarland Standard Preparation (cont.)

Composition of McFarland turbidity standards

Background lines for viewing turbidity of a suspension in

comparison to a turbidity standard

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 Comparison of the 0.5 McFarland turbidity standard with inoculum suspension

Questions for Research:

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. What is the principle involved in disk diffusion technique?

2. What are the different methods of antimicrobial susceptibility testing?
3. Define the following:
a. Resistant
b. Susceptible
c. Intermediate susceptibility
4. What are the different factors influencing antimicrobial susceptibility testing?
5. What are the different errors in results and interpretation of antimicrobial susceptibility
testing?

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WEEK 5
TIME ALLOTMENT: 9 HOURS
LECTURE:
LABORATORY:

Topics: Lecture

1. Principles of Disease and Epidemiology

a. Normal Flora (Normal Flora-Host relationship)
b. Epidemiologic Triangle (Etiologic Agent, Environment & Host)
c. Factors for Disease Occurrence
d. Bacterial Infection (Classification; Signs & Symptoms
e. Development of Disease
f. Disease Growth

Laboratory
Activity no. 9: Microbial mechanisms of pathogenicity

Learning Objectives
At the end of the learning session, the student should be able to
1. Correctly identify the normal resident microbiota in different areas of the body that may
interfere with the isolation of pathogenic bacteria
2. Understand the epidemiologic triangle
3. Differentiate correctly the terms used in epidemiology
4. Identify ways to break the chain of infection

Lesson 1: Normal Flora (normal Flora Host Relationship)

Ecology
- The systematic study of the interrelationships that exist between organisms and their
environment
Microbial Ecology
- The study of the numerous interrelationships between microorganisms and the world
around them
- Involves:
- How microbes interact with other microbes
- How microbes interact with other organisms other than microbes
- How microbes interact with the non-living world around them

Symbiotic relationships
- Symbiosis
o Defined as the living together or close association of two dissimilar organisms
o Usually, two different species
- Symbionts/symbiotes
o Any microorganism that spends a portion or all of its life associated with another
organism of a different species

Categories of symbiosis
- Ectosymbiosis
o One organism remains outside the other
- Endosymbiosis
o One organism is present within the other

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Three types of symbiotic relationship
- Commensalism
o Relationship in which one organism, the commensal, benefits while the other, the
host is neither harmed nor helped
o Both the host and the commensal ―eat at the same table
o E.g. Entamoeba coli and man
- Mutualism
o A symbiotic relationship that is beneficial t both symbionts
o The mutualist and the host are metabolically dependent on each other
o E.g. Lichens – association between ascomycetes (fungus) and certain genera of
either green algae or cyanobacteria
- Parasitism
o Branch of biology which deals the study of the phenomena of dependence of one
living organism on another temporarily or permanently for the purpose of
procuring food and shelter
o Parasite – the organism that lives in the body of another organism for survival
o Host – an organism that harbors a parasite nd provides physical protection and
nourishment to the specific parasite
o Categories of Parasites
 Ectoparasite
 Attached to the skin or temporarily invades the superficial tissues
of the host‘s body
 Endoparasite
 A parasite established within the body of its host and produces an
infection
Other Relationships
- Neutralism
o A symbiotic relationship in which neither the symbiont is affected by the
relationship
o A situation in which different microorganisms occupy the same ecological niche
but have absolutely no effect on each other
- Synergist (synergistic infections)
o Two or more microorganisms ―team up‖ to produce a disease that neither could
cause by itself
 ANUG – Acute Necrotizing Ulcerative Gingivitis or Vincent‘s disease or
trench mouth
 Caused by Fusobacterium, Actinomyces, Prevotella spp
 Bacterial vaginosis
 Mobiluncus and Gardnerella spp

Indigenous Microflora of Humans

- Also referred to as normal flora
- Includes all microbes that reside on or within that person
- A fetus has no indigenous microflora, during and after delivery, a newborn is exposed to
many microorganisms from its mother food, air and virtually everything that touches the
infant

Categories of Microorganisms
- Resident

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 o Normally grow on or in the skin
- Transient
o Those organisms that are temporarily present

Skin

Factors that affect the microflora of the skin

- The skin is subjected to periodic drying
o Lack of moisture drives many resident microbiota into a dormant state
o Scalp, ears, axillary areas, genitourinary and anal regions, perineum, palms have
moisture that is sufficient to support a resident microbiota
o Lack of moisture drives many resident microbiota into a dormant state
o Scalp, ears, axillary areas, genitourinary and anal regions, perineum, palms have
moisture that is sufficient to support a resident microbiota
- The skin has a slightly acidic pH (4-6)
o Due to the organic acids produced by normal staphylococci and secretions from
skin oil and sweat glands
o The acidic pH discourages colonization by many microorganisms
- Sweat contains a high concentration of NaCl
o It makes the skin hyperosmotic and osmotically stresses most microorganisms
- The skin can secrete certain inhibitory substances
o It may be bactericidal and/or bacteriostatic
o Sweat glands – release lysozymes (muramidase) – an enzyme that lyses S.
epidermidis and other gram + bacteria by hydrolyzing the B – 1,4 glycosidic
bonds inn the bacterial cell wall peptidoglycan
o Oil glands – secrete complex lipids that may be partially degraded by enzymes
from Propionibacterium acnes that can change it to oleic acid that have strong
antimircorbial activity against gram – bacteria and some fungi

Microbes on the superficial skin

- Staphylococcus epidermidis
- Aerobic corynebacteria

Organisms that normally occur in the scalp

- Pitysporum ovale
- Pitysporum orbicularis

Microbes found in skin glands

- Propionibacterium acnes – normally harmless but is associated with acne vulgaris and
comedones

Transient flora found around orifices

- Staphylococcus aureus – nostrils and perianal region
- Clostridium perfringens – colonizes the perineum and thighs especially those patients
who suffer from diabetes

Nose and Nasopharynx

- Nose
o S. aureus
o S. epidermidis
o Diphtheroids

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 - Nasopharynx
o Lies above the soft palate
o Streptococcus pneumoniae – non-encapsulated
o Neisseria meningitidis – non-encapsulated
o Haemophilus influenzae

Oropharynx
- Lies between the soft palate and the upper edge of the epiglottis
o S. aureus
o S. epidermidis
o Alpha hemolytic streptococci (S. oralis, S. gordinii, S. salivarius)
o Diphtheroids
o Branhamella catarrhalis

Palatine and pharyngeal tonsils

- Micrococcus
- Porphyromonas, Prevotella, and Fusobacterium

Respiratory Tract
- The upper and the lower respiratory tract (trachea, bronchii, bronchioles, alveoli) do not
have a normal microbiota
- Reasons
o The continous stream of mucus generated by the ciliated epithelial cells
o The phagocytic action of the alveolar macrophages

Oral Cavity

Mouth
- Organisms should resist the mechanical removal by adhering to surfaces like the gums
and teeth
- Mouth is colonized within hours after a human is born
- Initial microbiota
o Streptococcus
o Neisseria
o Actinomyces
o Veillonella
o Lactobacillus
- As teeth grows
o Streptococcus parasanguis
o S. mutans
o S. salivarius – attaches to the buccal and gingival epithelial surfaces and
colonizes the saliva

Eye
- Commensals of the Conjunctiva
o S. epidermidis
o S. aureus
o Aerobic corynebacteria (diphtheroids)

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 o S. pneumoniae
- Cultures from the eyelids
o Branhamella catarrhalis
o Escherichia
o Klebsiella
o Proteus
o Enterobacter
o Neisseria
o Bacillus

External Ear

- Coagulase negative Staphylococci

- Corynebacterium
- Less frequently seen Fungi
o Bacillus - Aspergillus
o Microccocus - Alternaria
o Neisseria - Penicillum
- Occasionally seen - Candida
o Proteus - Saccahromyces
o Escherichia
o Pseudomonas

Stomach
- Most bacteria are killed due to the very acidic pH (2 – 3) of the gastric contents
- Stomach contains less than 10 viable bacteria per mL of gastric fluid
- Streptococcus, Staphylococcus, Lactobacillus, Peptostreptococcus and Candida spp.

Small Intestines
- Duodenum
o Gram + cocci and rods
o Contains few microorganisms because of the combined influence of the
stomach‘s acidic juices and the inhibitory action of bile and pancreatic secretions
- Jejunum
o Enterococcus faecalis
o Lactobacilli
o Diphtheroids
o Candida albicans
- Ileum
o Anaerobic gram-negative bacteria
o Family Enterobacteriaceae
o The microbiota takes on the characteristics of the colon microbiota
Large Intestines
- Has the largest microbial population of the body
- Microbiota consists primarily of anaerobic, gram -, non-sporing bacteria and gram +,
spore forming and non-sporing rods
- The vast majority of microorganisms are anaerobic
- Fungi
o Candida albicans
- Protozoans
o Trichomonas hominis

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 o Entamoeba humanni
o Endolimax nana
o Iodamoeba butschlii
o Entamoeba coli
- Initial residents
o Bifidobacterium genus – found in breastfed infants because milk contains a
disaccharide amino sugar that they require as growth factor
o Lactobacillus – found in formula-fed infants because the formula lacks the
required growth factor for bifidobacterium

Genitourinary Tract
- Upper Genitourinary tract
o Includes the kidneys, ureters and urinary bladder
o Usually free of microorganisms
- Lower GUT
o Distal urethra and external opening of the urethra
 Neisseria spp.
 Staphylococci
 Streprococci
 Enterococci
 Diphtheroids
 Mycobacteria
 Mycoplasma
 Enteric Gram – rods
Genitalia
- Vagina
o Microflora varies with the stage of sexual development
 Puberty and following menopause
 Alkaline secretions: diphtheroids, streptococci, staphylococci, and
coliforms
 Childbearing years
 Acidic secretions (pH 4 – 5): lactobacilli, α – hemolytic
streptococci, staphylococci and yeasts

Lesson 2: Microbial mechanisms of Pathogenicity

Pathogen
- A microorganism capable of causing a disease
- Able to overcome the host‘s defenses
- Could be due to toxins, enzymes, capsules, etc…
Pathology
- Scientific study of diseases (pathos – suffering; logy – study)
- Aspects of pathology
o Etiology – study of the cause of disease
o Pathogenesis – development of the disease
o Pathology – concerned with the structural and functional changes brought about
by diseases and its final effect
Disease
- Occurs when an infection results in any change from the state of health

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 - An abnormal state in which part or all of the body is not properly adjusted or is not
capable of carrying its normal function
- An infection may exist in the absence of any detectable disease
- Signs and symptoms of a disease
o Dolor
o Calor
o Rubor
o Tumor
o Functio laesa
- Symptom
o Some evidence of a disease that is experienced or percieved by the patient
o Subjective
o Ex: ache or pain. Tinnitus, blurred vision, nausea, dizziness, itching and chills
 Symptomatic disease (clinical dse) – patient experiences symptoms
 Asymptomatic disease (subclinical dse) – patient is unaware because
he does not experience symptoms
- Sign of a disease
o Objective evidence of a disease
o Ex: abnormal heart or breath sounds, blood pressure, pulse rate, abnormal lab
results, radiographs, ultrasounds, CT scans

Communicable disease
- An infectious disease that is transmissible from one person to another
- spreads from one host to another, either directly or indirectly
- illness due to the transmission of the products of an etiologic agent or reservoir to a
susceptible host directly or indirectly
- Ex: gonorrhea, chicken pox, measles, genital herpes, typhoid fever
Contagious disease
- A communicable disease that is easily transmitted
- easily spread from one person to another
o illness due to direct transmission of etiologic agent from reservoir to susceptible
host
- Ex: influenza

Infection
- Colonization by a pathogen
- A person can be infected by a certain pathogen but not have the infectious disease
caused by that pathogen due to the following reasons:
o The microbe may land on a anatomical site where it is unable to multiply
o Many pathogens must attach to specific receptor sites before they are able to
multiply and cause damage
o Antibacterial factors may be present at the site where the pathogen lands
(lysozymes in saliva, sweat and tears)
o The indigenous microflora may inhibit the growth of the foreign microbe
o The indigenous microflora may produce antibacterial factors
o The individual‘s nutritional and overall health status often influences the outcome
of the pathogen/host encounter
o The person may be immune to that particular pathogen
o Phagocytes my engulf and destroy the pathogen
Classification of Infectious diseases

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According to how they behave in the host:
- Communicable disease
- Contagious disease
- Non-communicable – is not spread from one host to another

According to Frequency Occurrence

- Incidence –frequency/ number of new occurrences of disease, injury or death, i.e.
number of transitions from well to ill, from uninjured to injured, or from alive to dead in
the study population DURING THE TIME PERIOD BEING EXAMINED
- Prevalence - number of persons in a defined population who have a specified disease
or condition AT A POINT IN TIME (usually the time a survey is done)
o Sporadic – occasionally
o Endemic - a disease that is constantly present in a population
o Epidemic – if many people in a given area acquire a certain disease in a
relatively short period of time
o Pandemic – occurs worldwide

According to Severity or Duration of a Disease

- Acute infections – has rapid onset, followed by a relatively rapid recovery
o Ex: measles, mumps, influenza
- Chronic infection – diseases that has an insidious (slow) onset and lasts a long time.
o Ex: tuberculosis, leprosy (Hansen‘s disease), syphillis
- Subacute infections – disease having a sudden onset and then develop into a long
lasting disease
o Ex: bacterial endocarditis
- Latent infection – it may go from being symptomatic to asymptomatic and then some
time later go back to being symptmatic
o Ex: Cold sores, genital herpes, shingles, syphillis

According to Extent of Host Involvement

- Localized infection
o Remain in one site or may spread
o Ex: pimples, boils, abscesses
- Systemic infection
o AKA generalized infection
o When the infection has spread throughout the body
o Happens when pathogens are not contained at the original site of infection
o Ways in which pathogens are spread
 Lymph
 Blood
 Phagocytes
- Focal – can arise from infections in the teeth, tonsils or sinuses
o Bacteremia – bacteria in the blood
o Septicemia – bacteria in the blood actually multiplies
o Toxemia – toxins in the blood
o Viremia – viruses in the blood

According to Extent of Infection

- Primary infection – first infection that often allows another organism to appear on the
scene
o Viral respiratory infection – destroys cilia of the respiratory tract

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 - Secondary infection – caused by organisms following an initial or primary infection
o Bacterial pneumonia

Lesson 3: The Natural History of Disease

Activity1: Draw the Epidemiologic Triad and label how they are interconnected with each other.
Do not download photos form the internet.

The Etiologic Agent

- The causative agent of a disease
- Genetic/Intrinsic
- Acquired/Extrinsic
o Physical – radiation, increase or decrease in temperature
o Chemical – lead, alcohol, mercury, chloroform
o Nutritional – malnourishment
o Infectious – bacterial, viral, fungal, parasitic
Environment
- Involves the mode of transmission
- Contact transmission
o Direct contact transmission – through physical contact between its source and a
susceptible host. No intermediate object is involved (touching, kissing, sexual
intercourse)
o Indirect contact transmission – transmitted by means of non-living objects
(fomites)
o Droplet transmission – microbes are spread in droplet nuclei that travel only short
distances
- Vehicular transmission
o Waterborne transmission – spread by water contaminated with untreated or
poorly treated sewage
o Foodborne transmission – transmitted by food that re incompletely cooked,
poorly refrigerated, or prepared under unsatisfactory conditions
o Airborne transmission – spread of agents of infection by droplet nuclei in dust
that travel more than one meter from the reservoir to the host
- Vectors (living things that carry pathogens from one host to another)
o Mechanical transmission – passive transport of the pathogens on the insect‘s
feet or other body parts
o Biological transmission – active transmission wherein the pathogen multiplies
inside the arthropod
Host
- A susceptible host
- responsible for the degree to which the individual can adapt to the stressors produced by
the agent
- influenced by:
- genotype
o nutritional status
o immune system
o social behavior

Activity 2: Draw the chain of infection and show ways by which the chain can be broken. Do not
download photos

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THE PATHOGEN
- the agent causes the disease
o Viruses
o Bacteria
o Protozoa
o Parasite
o Protein

RESERVOIR
- site of natural environmental location in which the pathogen is normally found living the
from which infection of the host can occur
- source - the location from which the pathogen is immediately transmitted to the host
directly or indirectly
o animate sources – humans and animals
o inanimate sources – water, soil or food
 period of infectivity – the time during which the source is infectious or
disseminating the pathogen
- Carrier – an infected individual who is a potential source of infection for others
o 4 types of carriers
 Active carrier – an individual who has an overt clinical case of a disease
 Convalescent carrier – an individual who has recovered from the
infectious disease but continues to harbor large numbers of pathogen
 Healthy carrier – an individual who harbors the pathogen but is not ill
 An incubatory carrier – an individual who is incubating the pathogen in
large numbers but is not yet ill
 Casual, acute or transient carriers – harbor the pathogen for only a brief
period of time (hours, days or weeks)
 Chronic carriers – harbor the pathogen for long periods (months, years or
life)

TRANSMISSION TO THE HOST

- Airborne transmission
- Contact transmission
- Vehicle transmission
- Vector borne transmission

- External – pathogen is carried on the body surface of the pathogen

o Ex: flies carrying Shigella on their feet from the fecal source
- Internal – carried within the vector
o Harborage transmission – the pathogen does not undergo morphological or
physiological changes within the vector
 Ex: Yersenia pestis by rat fleas
o Biologic transmission – pathogen undergoes morphological or physiological
change within the vector
 Ex: Malarial parasites
PORTAL OF ENTRY
- Mucous membranes – respiratory, GIT and GUT, conjunctiva
- Skin – cuts, abrasions, hair follicles, sweat gland ducts

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 - Parenteral – microorganisms are deposited directly into the tissues beneath the skin or
into the mucous membranes when these barriers are penetrated or injured. Punctures,
injections, bites, cuts, wounds, surgery, splitting due to swelling or drying

SUSCEPTIBILITY OF THE HOST

- Depends on
o Pathogenicity of the organism
o Non-specific and specific defense mechanism of the host

PORTAL OF EXIT
- Active escape – takes place when a pathogen actively moves to a portal of exit and
leaves the host
- Passive escape – occurs when a pathogen or its progeny leaves the host in feces, urine,
droplets, saliva or desquamated cells

- FACTORS
o Portal of entry
 Examples
 Salmonella species – typhoid – swallowed
 Staphylococcus aureus – pneumonia – respiratory route
o Furuncles – skin penetration
o Food poisoning – gastrointestinal
o Virulence – intensity of the pathogenicity
 Ability to multiply in vitro
 Example: Neisseria vs MTB
o Number of pathogen – in general, more pathogens would mean more chances of
infection
o Resistance of the host

STAGES OF A DISEASE
1. Period of Incubation
a. The time interval between the actual infection and the first appearance of any
signs and symptoms
b. It is dependent on the virulence of the microorganism and the resistance of the
host
 Factors that affect the length of incubation
 Overall health and nutritional status of the host
 The immune status of the host (immunocompetent or
immunocompromised)
 The virulence of the pathogen
 The number of pathogens that enter the body
2. Prodromal period
a. A relatively short period that follows the period of incubation
b. Characterized by early, mild symptoms of disease, such as general aches and
malaise
c. Patient feels ―out of sorts‖
3. Period of illness/Clinical period/Fastigium/Acme
a. During this period, the disease is most active

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 b. Host exhibits overt signs and symptoms such as fever, chills, sore throat, muscle
pain, photophobia etc
c. communicable diseases are most easily transmitted at this time
4. Period of decline (defervescence)
a. Signs and symptoms of the disease subsides
b. The fever decreases, and the feeling of malaise diminish
c. Patient is vulnerable to secondary infections
5. Period of Convalescence
a. The person regains strength and the body returns to its pre-disease state
b. Recovery has occurred
c. Longer for viral respiratory diseases

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LABORATORY
TIME ALLOTMENT

Name: _________________________________________
Group NO. ______________________________________

Activity No. 9

MICROBIAL MECHANISMS OF PATHOGENICITY

Introduction

Virulence is the degree or intensity of pathogenicity. There are three characteristics that
determines virulence: invasiveness which is the ability of the organism to spread to adjacent
tissues, infectivity which is defined as the ability of the organism to establish a focal point of
infection and the degree that the pathogen causes morbid symptoms is known as the
pathogenic potential. Toxigenicity is the ability of the bacteria to produce toxins.
Toxins are chemical substances that can damage the host and produce disease.
Toxigenicity plays an important role in the ability of microorganisms to cause diseases. Toxins
produce fever dur to an endogenous pyrogen that acts on the hypothalamus and triggers IL-1
which a fever producing cytokine. The release of bradykinin causes the blood vessels to dilate
leading to hypotension and shock.

Materials:

Reference materials: Books, Internet

Procedure

Some organisms are better able to cause a disease. For example, 10 Shigellla cells can
cause shigelloses while 100 – 1000 cells of salmonella is needed to cause salmonellosis.
Streptococcus pyogenes can cause a multitude of diseases including tonsilitis and scarlet fever.
To induce an infectious disease, a pathogen must be able to initially be transported to the
host, adhere to, colonize, or invade the host, multiply (grow) or complete its life cycle on or in
the host, initially evade host defense mechanism and possess the mechanical, chemical or
molecular ability to damage the host

1. Fill up the table below with the description of the steps of the pathogenesis of infectious
diseases

Steps Description
Entry

- Active entry

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 - Passive entry

Attachment

Multiplication

Invasion or Spread

Evasion of Host
Defenses

Damage to host tissue

2. Virulence factors are attributes that enable pathogens to attach, escape destruction and
cause disease. They are phenotypic characteristics and thus are dictated by the
organism‘s genotype.

A. Fill up the table below the with the description of the following
adherence factors of bacteria and an example of a bacteria that
has it.
Adherence Factors Description
Filamentous
hemagglutinin

Fimbriae

Glycocalyx or capsule

Lectin

Mucus gel

Pili

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Receptors

S layer

Slime

Teichoic acid and

Lipoteichoic acid

Ligand

B. Microbial products involved in pathogen dissemination in a

mammalian host
Product Organism Involved Mechanism of Action
Coagulase

Collagenase

Deoxyribonuclease
(with Ca and Mg)

Elastase and
Alkaline protease

Exotoxin B
(cysteine Protease)

Hemolysins

Hyaluronidase

H2O2 and NH3

Immunoglobulin A
protease

Lecithinase

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Leukocidins

Porins

Protein A

Streptokinase
(Fibrinolysis,
Staphylokinase)

C. Tabulate the difference between Exotoxin and Endotoxin (you may

add more rows as needed. The table given is only a format for you
to follow)

Point of difference Exotoxin Endotoxin

Questions for Research

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. Differentiate lethal dose from infective dose

2. Differentiate Infection from intoxication
3. What are the different classifications of exotoxins? Give examples of bacteria that
produce them.
4. Describe ways by which the following bacterial products can be detected in the lab using
microbiological techniques
a. DNAse
b. Catalase
c. Coagulase
d. Streptokinase
5. Describe concisely how the following toxins work.
a. Diphtheria toxin g. toxic shock toxin
b. Tetanospasmin h. alpha toxin
c. Botulinum toxin i. Erythrogenic toxin/pyrogenic toxin
d. Enterotoxin
e. Pertussis toxin
f. Anthrax toxin

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WEEK 6
TIME ALLOTMENT:
LECTURE:
LABORATORY:

TOPIC: LECTURE: IMMUNOLOGY

1. Innate Immunity
(Physiologic Barriers & Non-Adaptive Mechanisms)
2. Specific Host Defense
3. Types of Immunity
4. Practical Application (Antisera& Vaccines)

LABORATORY:
Activity no. 10: Immunology

Learning Objectives

At the end of the learning session, the student should be able to

1. Describe completely the body‘s different lines of defenses.
2. Understand the complement system and how it is triggered
3. Differentiate active and passive immunity correctly
4. Discriminate between humoral and cellular immunity

IMMUNOLOGY
Immunology

- The branch of microbiology that deals with the study of body defenses against invading
microorganisms

Immune Response

- Involve complex interactions among many different types of body cells and cellular
secretions

Resistance

- The ability to ward off disease thru the body defenses

2 types of Resistance
1. Non-specific resistance – refers to all body defenses that protect the body from any
kind of pathogens
 resistance that is not acquired through contact with an antigen
 innate
2. Specific resistance (immunity) - refers to defenses (antibodies) against specific
microorganisms

Susceptibility

- Vulnerability or lack of resistance

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Lines of Body Defenses
Function

1. Some defenses are designed to keep out some microorganisms

2. Other defenses remove the microorganism if they do get in
3. Other combats them if they remain inside

Non-Specific Resistance
1. First line of body defense: skin and mucous membranes
2. Second line of body defense: phagocytosis and complement system

First Line of Body Defense

Mechanical Factors/Physical barriers
1. Skin
 Closely packed cells, continuous layering and presence of keratin, provides a
formidable barrier to the entrance of microorganisms
 few microbes are capable of penetrating the skin
 due to the presence of dry, acidic pH and fatty acids, alcohols
 sloughing removes attached bacteria
 SALT
 cool temperature
2. Mucous membrane
 Line the gastrointestinal, urinary and reproductive tracts
 Inhibit the entrance of many microorganisms but they offer less protection than
the skin
 in the respiratory tract and GIT
 mucus traps microbes
 microorganisms are constantly driven upwards by cilia
 rapid shedding of cells
 MALT
 enzymes are also secreted in the GIT
3. Lacrimal Apparatus
 Protects the eyes
 A group of structures that manufactures and drains tears
 Contains enzymes (lysozymes)
 Flushing action of tears
4. Salivary glands
 Produce saliva that help to dilute the number of microorganisms and wash them
from the surface of the teeth and the mucous membrane of the mouth to prevent
colonization by microbes
5. Ciliary escalator
 Covers the cell of the mucous membrane or the lower respiratory tract

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  It propels inhaled dust and microorganism, that have become trapped in mucus,
upward toward the throat
6. Epiglottis
 Covers the larynx during swallowing and prevent microorganisms from entering
the lower respiratory tract

7. Urethra
 Cleansed by the flow of urine to prevent microbial colonization in the urinary tract
8. Vaginal Secretions
 Move microorganisms out of the female body

Chemical Factors
1. Sebum
 Composed of fatty acids which inhibit the growth of certain pathogenic bacteria
and fungi
2. Lysosyme/Muramidase
 Present in perspiration which helps maintain body temperature
 Eliminates certain waste and flush microorganisms from the surface of the skin
3. Gastric juice
 Mixture of HCl, enzymes and mucus that produce a very high acidity of gastric
juice
 The acidity destroys bacteria and most bacterial toxins
4. Transferrins
 Iron-binding protein in blood that inhibits bacterial growth by reducing the amount
of available iron

Second line of Defense

1. Reticuloendothelial System
 involves mononuclear phagocytic cells
 flushing action of lymph fluid
2. Phagocytosis
 Is the ingestion of a microorganism or nay particulate matter by a cell
(phagocyte)
 Mechanism of Phagocytosis
 Chemotaxis
o The chemical attraction of phagocytes to microorganisms
 Adherence
o Is the attachment of the phagocytes‘ plasma membranes to the
surface of the microorganisms of their foreign materials
o It is mediated by opsonins through the process of opsonization
o Opsonization – coating of a microorganism wsith certain plasma
proteins (opsonin) that promote the attachment of the
microorganisms to the phagocytes
 Ingestion

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 o Occurs when the plasma membrane of the phagocytes extends
projections called pseudopods that engulf that microorganisms
 Digestion
o Takes place when phagosomes (membrane containing the
ingestion antigen) fuses with the lysosome of the phagocytes with
subsequent release of the proteolytic enzymes with digest the
antigen
 White blood cells or Derivatives of WBC
 White blood cells - Increase in number during bacterial infections
(leukocytosis)
o Neutrophils
 Highly phagocytic and motile
 Active in the initial stage of an infection
o Basophil
 Release substance known as histamine
 Histamine – important in inflammation and allergic
responses
o Eosinophil
 Produce toxic proteins against certain parasites such as
helminthes
o Monocytes
 Not actively phagocytic in blood, but when they enter the
body tissues and mature,they become macrophage
3. Inflammation
 A defensive response against microbial infection, physical and/or chemical
agents
 release of monokines, IL – 1 and TNF
 results to vasodilation causing edema at the infection site with the accumulation
of plasma
 fibrin limits the spread of microbes
 integrins and selectins causes leukocytes to attach to blood vessels and promote
their movement across the blood vessels going to the irritant (chemotaxis)
 chemokines also induce chemotaxis
 5 signs of inflammation
 Rubor – redness
 Calor – heat
 Dolor – pain
 Tumor – swelling
 Functio laesa – loss of function
 Functions of inflammation
 To destroy infectious agents and remove its by-products from the body
 To limit the effects of the injurious agent and its by-products by confining
or walling it off

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  To repair or replace tissue damaged by the injurious agent or its by-
products
 Stages of inflammation
 Vasodilation and increased membrane permeability
 Phagocyte migration
o Margination
o Diapedesis
 Repair
4. Fever
 A systemic response of the body to microbial invasion, which is due to the
release of IL-1 (endogenous pyrogen)
 IL-1 induces the hypothalamus to release prostaglandin that sets the
hypothalamus‘ thermostat at a higher temperature, thereby causing fever
 During fever, blood vessels constrict, rate of metabolism increases and shivering
occurs, all of which raise the body temperature
 Significance of fever
 IL-1 steps up the production of T-lymphocytes
 High temperature intensifies the effect of interferon
 Inhibits growth of some microorganisms by decreasing the amount of iron
available to them
 Helps body tissues to repair quickly since the increase in body
temperature speeds up the body‘s reaction

5. The complement system

 A defensive system of serum proteins that participate in lysis of foreign cells,
inflammation, and phagocytosis
 Two Pathways of Activation
 Classical pathway – activated by an immune complex
 Alternative pathway – activated through direct interaction with a
bacterium
 Consequences of complement activation
 Opsonization
o Coating of antigen to facilitate phagocytosis
 Inflammation
o Complement casues basophils and mast cells to release
histamine and other substances that stimulates the inflammatory
reaction
 Cytolysis
o Due to the destruction of plasma membrane causing the cellular
contents to break out
o The activated components of the complement proteins, with C9
proteins possibley playing a key role, attacks the invading cell‘s
membrane and produce circular lesion called transmembrane
channels, this leads to loss of ions and eventual cytolysis

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 6. Interferons
 Classes of similar antiviral proteins produced by certain animal cells after viral
stimulation
 Produced by virus infected cells
 It interfered with viral multiplication
 Produced by fibroblasts in connective tissues, by lymphocytes and by
other leukocytes
 Interferons diffuse to uninfected neighboring cells to manufacture mRNA
for the synthesis of antiviral proteins (AVP)
 AVP are proteins which functions as enzymes that disrups various stages
of viral multiplication

Third Line of Defense

Immune response

- Specific host defense mechanism that represents the third line of defense against
pathogens

Immunity

- Involves a specific defensive response when the host is invaded by foreign organism or
other foreign substances
- Results from the production of specialized lymphocytes and antibodies

Lymphocytes

- Not phagocytic but play a key role in specific immunity

Antigens

- Organisms or substances that provike a response from the immune system

Antibodies

- Special glycoproteins produced by the lymphocytes to recognize, bind with, inactivate

and destroy specific microorganisms

Acquired Immunity

- Refers to the protection and organism develops against certain types of microbes of
foreign substances
- Types
o Acquired active immunity
 When a person is exposed to microorganisms/foreign substances and the
immune system responds by producing specialized lymphocytes and
specialized proteins called antibodies

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 o Acquired passive immunity
 Antibodies are transferred from one organism to another. The recipient
does not actively synthesize the antibody but has them provided by an
outside source

Naturally Acquired Immunity

- Obtained when a person is exposed to ann antigen in the course of daily life and the
immune system responds by producing antibodies and specialized lymphocytes
- Types
o Naturally acquired Active Immunity
 Obtained when a person is exposed to antigens in the course of daily life
o Naturally acquired passive immunity
 Involves natural transfer of antibodies from the mother to infant

Artificially Acquired Immunity

- Involves transfer of performed antibodies

- Types
o Artificially acquired active immunity
 Results from vaccination or immunization
 Vaccines – specially prepared antigens
 Live
 Attenuated
 Toxoid
 Recombinant
o Artificially acquired passive immunity
 Involves introduction of antibodies into t he body
 Antibodies come from an animal or person who is already immune to the
disease

Serum

- Where antibodies can be found in an immune animal or person

Antiserum

- Generic term from blood-derived fluids containing antibodies

Five types of antibodies

Characteristic IgG IgM IgA IgD IgE

s
location Blood, lymph, Blood, lymph, B- Secretions B-cell surface, Bound to mast
intestine cell surface (tears, saliva, blood, lymph cells and
mucus, basophils
intestine, milk), throughout the

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 blood, lymph body, blood
Placental yes no no no No
transfer
Complement Yes yes no no no
activation
Function Enhance Especially Localized Not known but Allergic
phagocytosis, effective against protection in presence on c- reactions;
neutralized microoganisms mucosal cells may possibly
toxins and and surfaces indicate expulsion or
viruses, and agglutinating function in lysis of
protects fetus antigens; first initiation of protozoan
and newborn antibody immune parasites
produced in response
response to
initial infection

Immune System
Organs of the immune system
1. Lymph nodes
2. Bone marrow
3. Spleen
4. Liver

2 main components of the immune system

1. Humoral immune response
 Antibodies – proteins secreted by plasma cells (differentiated B-cells) as a
reaction to antigen stimulation
 The system involves antibodies produced by B-cells in response to a
specific antigen
 Antibodies primarily defend against bacteria, viruses and toxins in blood
plasma and lymph
 Antigens – foreign substances that provide antibody formation
2. Cellular immune Response
 The system depends on T cells and does not involve antibody production
 Cellular immunity is primarily a response to intracellular parasites, transplanted
tissues and cancer cells
 T cells contain antigen receptors attached to their surfaces which allow T
cells to recognize and react to an antigen
 Most effective against bacteria and antigen located within phagocytes or
infected host cells and against fungi, protozoan and helminthes
 It also responds to transplanted tissue by mounting an immune response
to reject it
 Types of T cells

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  T helper cells (T4) cells – necessary for B cell activation and T cell
dependent antigen
 Delayed hypersensitivity T cells (TD cells) – provide protection against
infectious agents and cause inflammation associated with tissue
transplant rejection
 T suppressor cells (Ts) – regulate immune response and help maintain
tolerance
 T cytotoxic cells (Tc) – destroys target cells on contact
 Other cells involved in cell - mediated immunity
 Killer cells – attack antibody-coated target cells
 Natural killer cells – attack and destroy target cells

Lymphokines – protein secretions of TD cells and other non-lymphoid cells, which mediate
immune response
Summary of some cytokine and T cell lymphokine

Lymphokine/cytokine Function
Macrophage Attracts macrophages to infection site
chemotactic factor
Macrophage migration Prevents macrophages from leaving the infection site
inhibition factor
Macrophage activation Activates macrophage to improve phagocytic activity
factor
Interleukin 1 Promotes multiplication and activation of b-cells and T cells; triggers fever response
in body
Interleukin 2 Stimulates proliferation and differentiation of T cells and natural killer cells
Lymphotoxin Destroys non-lymphocytic target cells in vitro
Interferon Inhibits viral replication
Transfer factor Intensifies the action of sensitized T cells

Development of an Immune Response

Primary immune response

- Elicited by the entry of new antigen into the body

- The antibody will encounter an Antigen presenting cell (APC)
o Endothelial and NK cells
o Macrophages – engulf Ag by endocytosis then attach fragments of the Ag on
their surface
o Dendritic cells – most potent of the APC‘s. have branched projection
 2 classes of DC‘s
 Myeloid-related dendritic cell (mDC)
o Langerhan‘s cell
 Lymphoid-related dendritic cell (plasmacytoid dendritic cell; pDC)
o B cells

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  These cells can send two signals (antigen signal and co-stimulatory
signal) to Th cells to get activated
 Other cells send only one signal (antigen signal)
- Importance of APC’s
o APC‘s present antigens to Naïve Th cells
o Th cells activate T cells and B cells
 Naïve T cells – mature T cells released by the thymus which have not
encountered an antigen yet
 Naïve B cells – mature B cells that have been released by the bone
marrow and which have not yet encountered an antigen yet
o Th cells also stimulate the production of the following cells
 Tc cells – effector cells of cellular immunity
 Memory T cells – carry image of the Ag and effects secondary immune
response
 Suppressor T cells – inhibit the activity of B cells and other T cells to
ensure than an immune response does not get out of hand
- Events in a primary immune response
o An APC presents a new antigen to a naïve Th cell which primes it
o A naïve B cell that has the same Ag on its surface encounters the primed Th cell
o The primed Th cell releases the appropriate cytokines that activates the B cell
o The B cell proliferates and after many generations, the progenies differentiate
into plasma cells and memory B cells
 Plasma cells – produce Ab‘s
 Memory B cells – effects secondary immune response

Secondary Immune Response

- Elicited by re-exposure to an Ag that has previously triggered a primary immune

response
- Has short induction phase (1-2 days) and more rapid build up
- Due to memory B and T cells
- So effective that pathogenic microorganisms are eliminated before the person manifests
any symptoms

Abnormal Immune Responses

1. Allergic reactions – overwhelming reaction to an Ag
2. Anaphylactic shock – fatal allergic reactions
3. Autoimmune response – the immune system fails to distinguish self from non-self and
the effector cells attack the body‘s own tissues and cells

ACTIVITIES: TBA

ASSIGNMENTS: TBA

ASSESSMENT: TBA

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LABORATORY
TIME ALLOTMENT: 2 hours

Activity No. 10

INTRODUCTION TO IMMUNOLOGY and VACCINATION

Introduction

Immunology is the study of the immune system and is a very important branch of the
medical and biological sciences. The immune system protects us from infection through various
lines of defense. If the immune system is not functioning as it should, it can result in disease,
such as autoimmunity, allergy and cancer. It is also now becoming clear that immune responses
contribute to the development of many common disorders not traditionally viewed as
immunologic, including metabolic, cardiovascular, and neurodegenerative conditions such as
Alzheimer‘s.

From Edward Jenner‘s pioneering work in the 18 th Century that would ultimately lead to
vaccination in its modern form, to the many scientific breakthroughs in the 19 th and
20th centuries that would lead to, amongst other things, safe organ transplantation, the
identification of blood groups, and the now ubiquitous use of monoclonal antibodies throughout
science and healthcare, immunology has changed the face of modern medicine.

Immunological research continues to extend horizons in our understanding of how to

treat significant health issues, with ongoing research efforts in immunotherapy, autoimmune
diseases, and vaccines for emerging pathogens, such as Ebola and COViD19. Advancing our
understanding of basic immunology is essential for clinical and commercial application and has
facilitated the discovery of new diagnostics and treatments to manage a wide array of diseases.
In addition to the above, coupled with advancing technology, immunological research has
provided critically important research techniques and tools, such as flow cytometry and antibody
technology. (British Society for Immunology)

Objectives
At the end of the laboratory session, the student should be able to
1. Discern innate from acquired immunity completely
2. Correctly comprehend the body‘s defenses against infection
3. Describe how phagocytosis is achieved by phagocytes

Materials
Reference books

Procedure:

Phagocytosis
1. Look for a clear and labeled photo of phagocytosis from your reference
materials/books/internet and study it carefully.
2. Attach the photo below this procedure

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Antibody structure
1. Carefully scan reference materials/book and the internet for a clear and labeled
photo of a typical antibody structure and study it in detail noting its parts and its
function in immunology.
2. Attach the photo of the typical antibody structure below this procedure

Vaccination
1. Look for a clear and labeled photo on the route of administration of the following
vaccines using your reference materials
a. BCG vaccine
b. Polio vaccine
c. DPT, Hepatitis and tetanus toxoid vaccine
d. Measles

Questions for Research

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified
o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. Complete the table regarding leukocytes

Types of Granulocyte or Normal value (%) Function

Leukocytes Agranulocyte in peripheral
blood

Neutrophils

Basophils

Eosinophils

Monocytes

Lymphocytes

2. Complete the table regarding antibodies

IgG IgM IgA IgD IgE

Form

Molecular

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weight
(Daltons)
% of Total
immunoglobulin
Concentration
in serum
Number of
antigenic
binding sites
Functions

3. Complete the table on the Expanded Program of Immunization of the WHO

Vaccine Minimum Numbe Dos Minimu Site of Route of

(abbreviation Age r of e m administratio administratio
s should be Requiremen Doses interval n n
transcribed) t of doses
BCG
DPT
OPV
Hepatitis B
Measles

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Week8
Topic: Gram-positive Cocci
Time allotment: 8 hours

Lesson 1: Staphylococcus

Learning objectives
At the end of the learning session, the student should be able to
1. Discuss the general characteristics of Staphylococci
2. Identify the different virulence factors of Staphylococci
3. Differentiate Staphylococcus aureus from other Staphylococci species

Introduction:
The staphylococci are catalase-producing, gram-positive cocci. On stained smears, they exhibit
spherical cells (0.5 to 1.5 μm) that appear singly, in pairs, and in clusters. The genus name,
Staphylococcus, is derived from the Greek term staphle, meaning ―bunches of grapes.‖
Although the Gram stain can be characteristic of staphylococci, microscopy alone cannot
differentiate staphylococci from other gram-positive cocci. Staphylococci are members of the
newly formed family Staphylococcaceae.

Micrococcaceae family includes two genus namely: Staphylococcus and Micrococcus

Staphylococcus
General Characteristics

 Staphylococci are catalase-producing, gram-positive cocci.

 On stained smears, they exhibit spherical cells- 0.5 to 1.5 um, that appear singly, in
pairs, and in clusters.
 The genus name, Staphylococcus is derived from the Greek term ―staphale‖ meaning
―bunch of grapes‖
 They are nonmotile and non-spore-forming and aerobic or facultatively anaerobic except
for S. saccharolyticus which is an obligate anaerobe
 Colonies produced after 18 to 24 hours of incubation are medium sized and appear
cream-colored, white or rarely light gold, and ―buttery-looking‖
 Some species are Beta-hemolytic
 They are responsible for several suppurative types of infections

Clinically Significant Species

Staphylococcus aureus

 It is the most clinically significant species of staphylococci.

 It is responsible for numerous infections ranging from relatively mild to life threatening.

 Virulence Factors
o Enterotoxins- are heat-stable endotoxin that cause various symptoms, including
diarrhea and vomiting

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 o Toxic Shock Syndrome Toxin-1- causes nearly all cases of menstruating-
associated Toxic Shock Syndrome
o Exfoliative toxin- It is also known as epidermolytic toxin. It causes the epidermal
layer of the skin to slough off and is known to cause staphylococcal Scalded Skin
Syndrome.
o Cytolytic toxin
 Alpha hemolysins-in addition to lysing erythrocytes, it also damage
platelets and macrophages and cause severe tissue damage
 Beta hemolysins- acts on the sphingomyelin in the plasma membrane of
erythrocytes
o Enzymes
 Staphylocoagulase- causes bacterial cells to agglutinate in plasma
 Hyaluronidase- hydrolyzes hyaluronic acid present in the intracellular
ground substance that makes up connective tissues, permitting the
spread of bacteria during infection.
 Lipases- acts on lipids present on the surface of the skin, particularly fats
and oil secreted by the sebaceous glands
o Protein A- It has the ability to bind the Fc portion of immunoglobulin IgG

 Epidemiology
o The primary reservoir for staphylococcus is the human naris, with colonization
also occurring in the axillae, vagina, pharynx, and other skin surfaces.
o Transmission of S. aureus may occur by direct contact with unwashed,
contaminated hands and by contact with inanimate objects

 Infections caused by Staphylococcus aureus

S. aureus infections can be suppurative or toxin mediated.

o Skin and Wound Infections- They are suppurative in nature. Typically, the abcess
is filled with pus and surrounded by necrotic tissues and damaged leukocytes.
 Folliculitis- is a relatively mild inflammation of a hair follicle or oil gland;
the infected area is raised and red.
 Furuncles- It is an extension of folliculitis, are large, raised, superficial
abscesses.
 Carbuncles- occur when larger, more invasive lesions develop from
multiple furuncles.
 Bullous impetigo- causes staphylococcal pustules that are large and
surrounded by a small zone of erythema
o Scalded Skin Syndrome- It is a bullous exfoliative dermatitis that occurs primarily
in newborns and previously healthy young children
o Toxic Shock Syndrome- It is a rare but potentially fatal, multisystem disease
characterized by a sudden onset of fever, chills, vomiting, diarrhea, muscle
aches, and rash, which can quickly progress to hypotension schock.

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 o Toxic Epidermal Necrolysis-It is most commonly drug induced, but some cases
have been linked to infections and vaccines
o Food Poisoning- Enterotoxin A and B have been associated with gastrointestinal
disturbances.

Staphylococcus epidermidis

 Infections caused by S. epidermidis are predominantly hospital acquired.

o Predisposing Factors: Instrumentation procedures such as catheterization,
medical implantation and immunosuppressive therapy
 It is a common cause of healthcare-acquired UTIs

Staphylococcus saprophyticus

 It has been associated with UTIs in young women

Staphylococcus lugdunensis

 It can cause both community-associated and hospital acquired infections

Other Coagulase-Negative Staphylococci: S. warneri, S. capitis, S. simulans, S. hominis, and S.

scheliferi

 Microscopic Examination
o Numerous gram-positive cocci, along with polymorphonuclear cells in purulent
exudates, joint fluids, aspirated secretions, and other body fluids are seen when
these sites are infected with staphylococci
o A culture should be done regardless of the results of microscopic examination
because the genus or the species cannot be appropriately identified by
microscopic morphology alone

 Isolation and Identification

o Staphylococci grow easily on routine laboratory culture media, particularly sheep
blood agar.
o A selective medium such as Mannitol Salt Agar, Columbia colistin-nalidixic acid
Agar, or Phenylethyl alcohol Agar can be used for heavily contaminated
specimens
o CHROMagar Staph aureus is a proprietary selective and differential medium for
isolation and identification of S. aureus.

 Cultural Characteristics
o Staphylococci produce round, smooth, white, creamy colonies on SBA after 18 to
24 hours of incubation at 35oC to 37oC.
o S. aureus can produce haemolytic zones around the colonies and may rarely
exhibit pigment production with extended incubation

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 o S. epidermidis colonies are usually small – to medium sized, nonhemolytic gray
to white colonies
o S. saprophyticus forms slightly larger colonies with about 50% of the strains
producing yellow pigment
o S. haemolyticus produces medium-sized colonies, with moderate or weak
hemolysis and variable pigmentation

 Identification Methods
o Staphylococci have been traditionally differentiated from micrococci on the basis
of ocidation-fermentation (O/F) reactions produced in O/F glucose medium.
 Staphylococci ferment glucose whereas micrococci fail to produce acid
under anaerobic conditions
o A modified oxidase test can be used to rapidly differentiate staphylococci from
micrococci.
 Most staphylococci are negative whereas micrococci are positive
o S. aureus is often identified by the coagulase test,
 Clumping factor, formerly referred to as cell-bound coagulase, causes
agglutination in human, rabbit, or pig plasma and is considered an
important marker for S. aureus
o Test for pyrrolidonyl arylamidase activity can be used to differentiate S. aureus
(negative) from S. lugdunensis, S. intermedius and S. schleiferi (positive)

Lesson 2: Streptococcus and Enterococcus

Learning objectives
At the end of the learning session, the student should be able to
1. Discuss the general characteristics of Streptococci
2. Identify the different virulence factors of Streptococcus
3. Differentiate Streptococcus from Enterococcus species
Introduction:
Streptococcus and Enterococcus spp. belong to the family Streptococcaceae. Members of both
genera are catalase-negative, gram-positive cocci that are usually arranged in pairs or chains. A
negative catalase test result differentiates streptococci and enterococci from staphylococci.
Weak false-positive catalase reactions can be seen when growth is taken from media containing
blood, owing to the peroxidase activity of hemoglobin. Compared with other gram-positive cocci,
the cells of enterococci and some streptococci appear more elongated than spherical. The
streptococcal cells are more likely to appear in chains when grown in broth cultures

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Streptococcaceae- includes Streptococcus and Enterococcus spp
Streptococcus and Enterococcus spp
General Characteristics

 Members of both genera are catalase negative, gram-positive cocci that are usually
arranged in pairs or chains.
 A negative catalase test result differentiates streptococci and enterococci from
staphylococci.
 Compared with other gram positive cocci, the cells of enterococci and some enterococci
appear more elongated than spherical.
 Most members of the genera Streptococcus and Enterococcus behave like facultative
anaerobes
 Some species are capnophilic, requiring increased concentration of carbon dioxide.
 Growth is poor on nutrient media such as trypticase soy broth

Clinically Significant Streptococci

Streptococcus pyogenes
 Antigenic Structure
o The group is antigen is unique, placing the organism in Lancefield group A.
o M protein is attached to the peptidoglycan of the cell wall and extends to the cell
surface
o The M protein is essential for virulence S. pyogenes colonizes the throat and skin
on humans making these sites primary sources of transmission
 Virulence Factors
o The best defined virulence factor in S. pyogenes is M protein encoded by the
genes emm
 M protein molecule causes streptococcal cell to resist phagocytosis and
plays a role in adherence of the bacterial cell to mucosal cells.
o M1 serotype is the most common serotype seen in pharyngitis
o Fibronectin-binding protein (Protein F) and Lipotechoic acid- are adhesion
molecules that mediate attachment to host epithelial cells.
o Hyaluronic acid capsule- it prevents opsonized phagocytosis by neutrophils or
macrophages. It also allows the bacterium to mask its antigens and remain
unrecognized by its host
o Streptolysin O-responsible for hemolysis on Sheep Blood Agar plates incubated
anaerobically
 O refers to this hemolysin being oxygen labile
o Streptolysin S- lyses leukocytes and is non-immunogenic
 Oxygen stable
o Hyaluronidase- spreading factor is an enzyme that solubilizes the ground
substance of mammalian connective tissues.
o Streptococcal pyrogenic endotoxins/erythrogenic toxins- red spreading rash

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  Clinical Infections
o Bacterial pharyngitis
 It is the most common clinical manifestation of Group A Streptococcus
 Strep throat is most often seen in children between 5 and 15 years of
age.
o Pyodermal infections
 Skin or pyodermal infections include impetigo, cellulitis, erysipelas, wound
infection, or arthritis
 Impetigo- a localized skin disease, begins as small vesicles that progress
to weeping lesions
 Erysipelas- rare infection of the skin and subcutaneous tissues observed
frequently in elderly patients
 Cellulitis- can develop following deeper invasion by streptococci.
o Necrotizing Fasciitis
 An invasive infection characterized by rapidly progressing inflammation
and necrosis of the skin, subcutaneous fat, and fascia.
o Streptococcal Toxic Shock Syndrome
o Poststreptococcal Sequelae
 Two serious complications or sequelae of GAS disease are rheumatic
fever and acute glomerulonephritis

 Laboratory Diagnosis
o Sheep Blood Agar plate is inoculated and streaked for isolation.
o Several selective media, such as SBA containing sulfamethoxazole (SMZ) or
colistin and polymixin B found in some formulations of Streptococcus selective
agar.
o Key tests are bacitracin susceptibility or pyrrolidonyl-alpha-naphtylamide (PYR)
hydrolysis.
o When the origin of an isolate is not the throat and serologic identification is not
used, additional tests should be part of the early identification scheme: hippurate
hydrolysis, CAMP test, bile esculin test, and growth in 6.5% sodium chloride
(NaCl) broth.

Streptococcus agalactiae
 Antigenic structure
o All strains of S. agalactiae have the group B-specific antigen, an acid-stable
polysaccharide located in the cell wall.
o S. agalactiae is the only species that expresses group B antigen
o There are 9 recognized capsular polysaccharide serotypes.
 Serotypes Ia, IIb and II contain a terminal residue of sialic acid

 Virulence Factors
o The capsule prevents phagocytosis but is ineffective after opsonisation.

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  Sialic acid appears to be the most significant component of the capsule
and a critical virulence determinant

 Clinical Infections
o Causes invasive disease in the newborn. Two clinical symptoms are associated
with neonatal GBS disease:
 Early onset infection - <7 days old
 Late onset infection- at least 7 days old to about 3 months old

 Laboratory Diagnosis
o Grows on Sheep Blood Agar as grayish white mucoid colonies surrounded by a
small zone of Beta hemolysis
o Detection of GBS in pregnant women is accomplished by collecting vaginal and
rectal material with swabs between 35 and 37 weeks.
 Samples should be inoculated into selective broth, such as Todd-Hewitt
broth containing 10 ug/mL colistin and 15 ug/mL nalidixic acid.

Streptococcus pneumoniae

 Also known as pneumococcus

 Virulence Factors
o Capsular polysaccharide
o Hemolysin
o Immunoglobulin A protease
o Neuraminidase
o Hyaluronidase

 Clinical Infections
o It is an important human pathogen that causes pneumonia, sinusitis, otitis media,
bacteremia, and meningitis.
 Pneumonia may be complicated by a pleural effusion that is usually
sterile (empyema).
 Meningitis usually follows other S. pneumonia infections, such as otitis
media or pneumonia.
 Bacteremia often occurs during the course of a serious infection
o Atypical haemolytic uremic syndrome in children

 Laboratory Diagnosis
o The cells characteristically seen on Gram stain appear as gram-positive cocci in
pairs (diplococcic)
o Brain-heart infusion agar, trypticase soy agar with 5% sheep RBCs, or chocolate
agar are necessary for good growth.
o Optochin susceptibility- S. pneumoniae is susceptible
o Bile solubility test- S. pneumoniae is bile soluble

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Viridans Streptococci

 They are constituents of the normal microbiota of the upper respiratory tract

Enterococcus

 They were previously classified as group D streptococci.

 Natural inhabitants of the intestinal tracts of human and animals
 The commonly identified species in clinical specimens are E. faecalis and E. faecium.
 Most enterococci are nonhemolytic or alpha haemolytic, although some straims show
Beta hemolysis.
 Enterococci sometimes exhibit pseudocatalase reaction.

 Virulence Factors
o They have a survival advantage over other organisms in that they can grow in
extreme conditions and are resistant to multiple antimicrobial agents.
o Extracellular surface adhesion proteins, extracellular serine protease, and
gelatinase of E. faecalis are thought to play a role in the colonization of the
species and adherence to heart valves and renal epithelial cells.
o Cytolysin-shows similarity to bacteriocins produced by gram-positive bacteria and
is expressed by quorum-sensing mechanism.

 Clinical Infections
o Enterococci are frequent causes of nosocomial infections
o Urinary tract infections are the most common followed by bacteremia

 Laboratory Diagnosis
o Trypticase soy or brain-heart infusion agar supplemented with 5% sheep blood is
routinely used to culture enterococci.
o If the clinical specimen is obtained from a contaminated site or is likely to contain
gram-negative organisms, selective media containing bile esculin azide, colistin
nalidixic acid, phenylethyl alcohol, chromogenic substrates, or cephalexin-
aztreonam-arabinose agar should be used
o Enterococcus spp. are identified based on their:
 ability to produce acid in carbohydrate broth
 ability to hydrolyze arginine
 tolerance of 0.04% tellurite
 utilization of pyruvate
 ability to produce acid from methyl-α-D-glucopyranoside

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Note: The following activity shall not be performed. All answers will be based on theoretical
results. Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Activity No. 11
GRAM-POSITIVE COCCI
Introduction:
The gram-positive cocci are a very heterogenous group. Historically, the genus Staphylococcus
was included with the genus Micrococcus in the family Micrococcaceae. However, molecular
phylogenetic and chemical analysis has indicated that these two genera are not closely related.
The Staphylococcus spp. has now been combined with the Bacillaceae, Planococcaceae, and
Listeriaceae into the order Bacillales. There are approximately 39 species and 21 subspecies
within the genus Staphylococcus.
Objectives
At the end of the of the laboratory session, the student should be able to
To isolate and identify Gram-positive cocci into Genus specie level using available primary
inoculating media and biochemical tests.
To purify and store identified microorganisms for future use and/or subsequent additional
biochemical tests for subspecies identification.
Materials
Two (2) TSB (one with 6.5% NaCl)
Fluid thioglycollate broth
Sterile cotton swab
Tongue depressor
Alcohol lamp
Primary inoculating media (BAP, CAP, MSA)
Nutrient agar slant
Inoculating loop
3% Hydrogen Peroxide
Rabbit‘s plasma
Glass slide
Gram Stain Set
Microscope
Oil immersion
Antibiotic discs (Bacitracin, Optochin, SXT, etc.)

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Procedure:
1. Collect a nasal swab and place the swab into a TSB with 6.5% NaCl. Collect two
throat swabs and separately place one swab onto a FT broth and plain TSB. Incubate at
35oC for 18 to 24 hours and observe for turbidity.
2. Clear broth cultures must be re-incubated for another 24 hours. If the broth appears
clear, discard the broth and clean glasswares.
3. For turbid cultures, subculture onto primary inoculating media indicated. Incubate at
35oC for 18 to 24 hours. Observe for the colonial characteristics (e.g., hemolytic pattern
on BAP, etc.) and document on chart provided.
4. Do a colonial Gram stain, if the smear shows Gram-positive cocci perform catalase
test as appropriate. Catalase positive is presumptive of Staphylococci species and
Micrococcus species while a negative catalase test is presumptive of Streptococci
species.
5. Subculture onto nutrient agar slant as appropriate significant colonies for identification.
6. Perform additional biochemical tests as appropriate (see algorithm below).

Biochemical Tests:
1. Catalase Test
a. Place a drop of 3% hydrogen peroxide on a slide and emulsify a well isolated
colony.
b. Observe for the evolution of gas indicated by bubble formation or
effervescence.
2. Coagulase Test
a. Slide Method
i. Place a drop of sterile distilled water on clean and dry glass slide and
emulsify a well isolated colony.
ii. Add a loopful of fresh rabbit‘s plasma (EDTA, citrate or oxalated). Mix
thoroughly and then observe for visible white clumps. If positive,
confirm using the tube method.
b. Tube Method
i. Inoculate a 0.5 mL of rabbit‘s plasma (EDTA, citrated or oxalated) with
a well isolated colony.
ii. Incubate at 35oC and observe for the coagulum or clot formation after
4 hours. If negative, incubate at room temperature for another 20
hours.
Alternative Procedure: Tube Coagulase Test
1. Dilute 1:5 rabbit‘s plasma with physiologic saline or NSS.
2. Dispense onto sterile 12x75 tubes. Add 0.5 mL of a 24 hour suspension of the
bacterial colony.
3. Place in a water at 37oC and observe for coagulum formation at 15 to 20
minutes interval.
3. 6.5% NaCl Tolerance Test (Salt Tolerance Test)
a. Inoculate a well isolated bacterial colony onto a trypticase soy broth
containing 6.5% NaCl.
b. Incubate at 35oC for 18 to 24 hours.
c. Interpretation: Turbidity indicates growth of organism that can tolerate high
concentration of salt – POSITIVE.
4. Novobiocin Test
a. Inoculate a standardized suspension of the isolated organism on blood agar
plate. Impregnate a novobiocin disk.
b. Incubate at 35oC for 18 to 24 hours.
c. A zone of inhibition indicates that the organism is SENSITIVE. No zone of
inhibition – RESISTANT.
5. Bacitracin Test
 a. Inoculate a standardized suspension of the isolated organism on blood agar plate.
Impregnate a bacitacin disk (0.04 U) or Taxo A.
b. Incubate at 35oC for 18 to 24 hours.
c. Interpretation: Sensitive: A zone of inhibition is observed.
Resistant: No zone of inhibition.
6. Optochin Test
a. Inoculate organism on BAM using the overlap streak method.
b. Place the optochin disk or Taxo P on the site of inoculation.
c. Interpretation: Sensitive: Zone of inhibition greater that 15 mm.
Resistant: No zone of inhibition.
7. Superoxol Test (for Neisseria spp.)
a. Place a drop of superoxol (30% hydrogen peroxide) reagent on clean, dry slide.
b. Emulsify a well isolated colony onto the drop of reagent.
c. Vigorous production of bubbles is indicative of a positive result.
8. Carbohydrate Fermentation Test (for Neisseria spp.)
a. Inoculate the test organism on five peptone medium containing 1% GLU, MAL,
LAC, FRUC, SUC. Add the phenol red indicator.
b. Incubate for 18-24 hours at 37oC.
c. Interpretation: Positive: Red to yellow change in color.

Additional Tests: (as necessary)

Sensitivity Test: Polymyxin B, SXT, etc.
Ornithine
Alkaline Phosphatase
β-glucosidase, galactosidase, etc.
Voges-Proskauer
PYR
Urease
Hippurate Hydrolysis
Bile Esculin Hydrolysis

Results and Observations:

Plain TSB: _____________ TSB with 6.5% NaCl: ____________ FT: ______________
Gram Stain Reaction: _____________________________________________________
Blood Agar Plate: ________________________________________________________
______________________________________________________________________
_____________________________________________________________________
Chocolate Agar Plate: ____________________________________________________
______________________________________________________________________
______________________________________________________________________
Mannitol Salt Agar: _______________________________________________________
______________________________________________________________________
_________________________________________________________________
Catalase Test: ______________________ Coagulase Test
a. Slide Method: _____________________
b. Tube Method: ____________________

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Other/s:
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

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Algorithm of Identification:

Specimens: Pus, transudate & exudates, urine, throat swab, nasal swab, urethral swab, wound
swab, etc.

Culture Broths: Fluid Thioglycollate (FT), Typticase Soy Broth (TSB), Nutrient Broth (NB)

Illustrations:
1. Draw and label the typical microscopic feature of the following:
a. Staphylococcus aureus
b. Streptococcus pyogenes
c. Streptococcus agalactiae

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 d. Streptococcus pneumonia
Questions for Research:
1. What are the different tests used to differentiate Micrococcus species from Staphylococcus
species?
2. Describe the general characteristics of the Genus Staphylococcus.
3. What are the different selective media used for isolating Staphylococcus species? Describe
the colonial characteristics of S. aureus, S. epidermidis and S. saprophyticus on the different
selective media.
4. What is the principle of the following tests below?
a. Catalase Test
b. Coagulase Test
c. Bile Esculin Hydrolysis
d. CAMP Test
e. Bile Solubility Test
f. Salt Tolerance Test
g. Hippurate Hydrolysis
h. Neufeld Quellung Reaction
5. Enumerate and characterize the different types of hemolysis observed on blood agar plate?
6. Differentiate streptolysin O and streptolysin S.
7. Explain the four classification schemes of Streptococci species.
8. What is ASTO or ASO Test? Discuss the principle its importance.

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Week 9: Gram-negative Cocci
Topic: Neisseria
Time allotment: 2 hours

Learning objectives
At the end of the learning session, the student should be able to
1. Describe the characteristics of the pathogenic Neisseria species and Morraxella
catarrhalis
2. Explain the importance of proper specimen collection and transport for the culture
and isolation of pathogenic Neisseria species

Introduction:
The family Neisseriaceae includes the genera Neisseria, Kingella, Eikenella, Simonsiella, and
Alysiella
Neisseriaceae contains the genus Neisseria, Kingella, Eikenella, Simonsiella, Alysiella.

Neisseria
General Characteristics

 They are aerobic, nonmotile, non–spore-forming, gram-negative diplococcic

o Neisseria elongata, Neisseria weaveri, and Neisseria bacilliformis are known
exceptions and are rod-shaped.
 All species are cytochrome oxidase- and catalase-positive except for N. elongata and N.
bacilliformis, which are catalase negative
 Many Neisseria spp. are capnophilic, requiring carbon dioxide (CO2) for growth, and
have optimal growth in a humid atmosphere.
 Neisseria gonorrhoeae (often called gonococci) and Neisseria meningitidis are the
primary human pathogens of the genus.

Pathogenic Neisseria Species

Virulence Factors
Pathogenic Neisseria spp. have several characteristics that contribute to their virulence,
including the following:

 Receptors for human transferrin

 Capsule (N. meningitidis)
 Pili (fimbriae)
 Cell membrane proteins
 Lipooligosaccharide (LOS) or endotoxin; lipid A moiety and core LOS that differentiates
it from the lipopolysaccharide found in most gram-negative bacilli and is loosely
attached to the underlying peptidoglycan
 Immunoglobulin A (IgA) protease that cleaves IgA on mucosal surfaces

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Neisseria gonorrhoeae

 Humans are the only natural host for N. gonorrhoeae, the agent of gonorrhoea
 Gonorrhea is an acute pyogenic infection of nonciliated columnar and transitional
epithelium; infection can be established at any site where these cells are found.
Gonococcal infections are primarily acquired by sexual contact and occur primarily in the
urethra, endocervix, anal canal, pharynx, and conjunctiva

 Clinical Infections
o Gonorrhea has a short incubation period of approximately 2 to 7 days.
 In men, acute urethritis occurs usually resulting in purulent
discharge and dysuria. It is the most common manifestation
 The endocervix is the most common site of infection in women.
Symptoms of infection include dysuria, cervical discharge, and
lower abdominal pain
o Newborns can acquire ophthalmia neonatorum-a gonoccal eye infection
during vaginal delivery through an infected birth canal. This can result in
blindness if not treated immediately.

 Laboratory Diagnosis
o The specimen of choice for genital infecctions in men is the urethra
and in women is the endocervix.
o Calcium alginate and cotton swab are inhibitory to N. gonorrhoeae, so
Dacron or rayon swabs are preferred.
o Direct Microscopic Examination
 Smears for direct Gram stains should be prepared from
urogenital specimens.
 A gram stain is not recommended for pharyngeal
specimens because commensal Neisseria spp. can be
present.
 The demonstration of gram-negative intracellular
diplococci from the urethral discharge of a symptomatic
male correlates at a rate of 89% with culture and is
evidence of gonococcal infection.
o Culture
 N. gonorrhoeae typically does not grow on SBA
 The medium of choice for cultivation is Chocolate agar
 Because it supports the growth of many other organisms
found as commensals in the specimens collected for the
recovery of gonococci, a selective medium is necessary.

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Neisseria meningitidis

 Similar to N. gonorrhoeae, N. meningitidis is also found only in humans.

 N. meningitidis can be found as a commensal as well as an invasive pathogen
 It is an important etiologic agent of endemic and epidemic meningitis and
meningococcemia and rarely pneumonia, purulent arthritis, or endophthalmitis.

 Clinical Infections
o When the bacteria adhere to the nasopharyngeal mucosa it leads in a
subclinical infection or mild symtoms.
o When the bacteria enters the bloodstream, 2 main diseases can occur:
 Fulminant meningococcemia or meningitis
 Waterhouse-Friderichsen syndrome- when sepsis becomes
fulminant and spreads rapidly causing disseminated
intravascular coagulation, septic shock or haemorrhage in the
adrenal glands occurs.

 Laboratory Diagnosis
o Specimen: CSF, blood, nasopharyngeal swabs and aspirates, joint
fluids, and less commonly sputum and urogenital sites.
o Direct Microscopic Examination
 On Gram-stained smears from specimens such as CSF
meningococci appear as intracellular and extracellular gram-
negative diplococcic

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 o Culture and Incubation
 Specimens for isolation of N. meningitidis should be cultured on
SBA and Chocolate agar.

Note: The following activity shall not be performed. All answers will be based on theoretical
results. Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Activity No. 12
GRAM-NEGATIVE COCCI: Neisseria spp
Introduction:
The family Neisseriaceae contains the genus Neisseria as well as Kingella, Eikenella,
Simonsiella, Alysiella, and several other genera. Currently, the genus Neisseria contains 25
species; about 12 species and biovars can be isolated from humans
Objectives
At the end of the of the laboratory session, the student should be able to
To know the general characteristics and medical importance of the Genus Neisseria.
To be able to isolate and identify Neisseria species using microscopic examination of colony
and available biochemical test.Materials
Materials:

Prepared slides of Neisseria Candle Jar Set-up

gonorrheae and Neisseria Thayer Martin Agar
meningitides Broth culture of N. gonorrheae
Microscope Sugar Fermentation Broths (Glu,
Oil immersion objective Fru, Lac, Mal, Suc)
Atlas & Books Oxidase Reagent
Chocolate Agar Plate 30% hydrogen peroxide
Blood agar plate
Procedure:
1. Streak specimen and/or subculture unknown broth cultures onto primary inoculating media.
Incubate BAP aerobically, CAP and TMA on candle jar set-up for 18 to 24 hours.
2. After 24 hours, observe colony characteristics from the different agars and Gram stain typical
Neisseria colonies. Perform superoxol test by placing 1 drop of 30% hydrogen peroxide on a
clean dry slide, pick a colony and emulsify on the reagent. Observe for rapid evolution of gas
(brisk bubble formation).

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3. Perform oxidase using either a commercial paper strip reagent of using a cotton swab and/or
filter paper. Pick a colony and onto a petri dish or slide with a filter paper strip, add oxidase
reagent and observe for purple color.
4. Subculture typical colonies onto sugar fermentation set-up and incubate for 18 to 24 hours.
Observe for the change in color of the sugar solutions (red to yellow).
5. Record all observation and interpret results and identify unknown organism accordingly.
Illustrations:
1. Draw and label the characteristic microscopic morphology of the following:
a. N. gonorrheae
b. N. meningitides

Questions for Research:

1. What are the different culture media used for the isolation of Neisseria species?
2. Describe and differentiate the types 1 and 2 from T3 and T4 colonies.
3. What is Phadebact Test for Neisseria species?
4. Tabulate the carbohydrate fermentation of the different species of the genus Neisseria.

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Week 10: Mycobacteria
Topic: Mycobacterium
Time allotment: 2 hours

Learning objectives
At the end of the learning session, the student should be able to
1. Explain the general characteristics of the genus Mycobacterium
2. Differentiate Mycobacterium tuberculosis from other members of MTC
3. Describe the distinct colonial appearance of Mycobacteria

Mycobacteria

General Characteristics
 Mycobacteria are slender, slightly curved or straight, rod-shaped organisms 0.2 to 0.6
μm × 1 to 10 μm in size.
 They are nonmotile and do not form spores.
 The cell wall has extremely high lipid content; thus, mycobacterial cells resist staining
with commonly used basic aniline dyes, such as those used in the Gram stain, at room
temperature
 Mycobacteria take up dye with increased staining time or application of heat but resist
decolorization with acid-ethanol
o This characteristic is referred to as acid fastness— hence, the term AFB—and is
a basic characteristic in distinguishing mycobacteria from most other genera

Pathogenic Species

Mycobacterium tuberculosis
 TB is one of the oldest documented communicable diseases

Primary Tuberculosis
 TB is usually a disease of the respiratory tract.
 After exposure to M. tuberculosis, whether a person develops TB is determined by his or
her cellular immune response, amount of exposure and virulence of the strain.
 In many exposed individuals, the immune system does not eliminate the bacteria. The
pathologic features of TB are the result of a hypersensitivity reaction to mycobacterial
antigen
.
Reactivation Tuberculosis
 The risk of reactivation TB is about 3.3% during the first year after a positive PPD skin
test and a total of 5% to 15% thereafter in the person‘s lifetime.
 Progression from infection to active disease varies with age and the intensity and
duration of exposure

Extrapulmonary Tuberculosis
 Cases of extrapulmonary disease are a common presentation in individuals with human
immunodeficiency virus infection, although it is most often in association with pulmonary
disease.

Identification of Mycobacterium tuberculosis

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  Colonies are typically raised with a dry, rough appearance. The colonies are
nonpigmented and classically described as being buff-colored.
 Biochemically, It is characteristically positive for niacin accumulation, reduction of nitrate
to nitrite, and production of catalase

Mycobacterium bovis
 Mycobacterium bovis produces TB primarily in cattle but also in other ruminants, as well
as in dogs, cats, swine, parrots, and humans.

Nontuberculous Mycobacteria
Slow Growing Species
 Mycobacterium avium Complex: Mycobacterium avium and Mycobacterium intracellulare
o These organisms are common environmental saprophytes and have been
recovered from soil, water, house dust, and other environmental sources.
 Mycobacterium avium subsp. Paratuberculosis
o It is the causative agent of Johne disease, an intestinal infection occurring as a
chronic diarrhea in cattle, sheep, goats, and other ruminants
 Mycobacterium kansasii
o It second to MAC as the cause of Nontuberculous Mycobacteria
o The most common manifestation is chronic pulmonary disease involving the
upper lobes, usually with evidence of cavitation and scarring.
 Mycobacterium genavense
o It is the cause of disseminated infections in patients with AIDS
 Mycobacterium haemophilum
o It occurs primarily in patients who are immunocompromised.
o Submandibular lymphadenitis, subcutaneous nodules, painful swellings, ulcers
progressing to abscesses, and draining fistulas are often the clinical
manifestations
 Mycobacterium malmoense
o M. malmoense appears as a short coccobacillus without cross bands on acid-
fast–stained smears
 Mycobacterium marinum
o Cutaneous infections in humans occur when traumatized skin comes into contact
with salt water or inadequately chlorinated fresh water containing the organism
 Mycobacterium scrofulaceum
o Causes cervical lymphadenitis in children.
 Mycobacterium simiae
o Original strains were isolated from the lymph nodes of monkeys
 Mycobacterium szulgal
o The most common manifestation is pulmonary disease similar to TB
 Mycobacterium ulcerans
o It is a rare cause of mycobacteriosis, also referred to as Buruli ulcer
 Mycobacterium xenopi
o It has been recovered from hot and cold water taps and birds

Rapidly Growing Species

 Mycobacterium chelonae–Mycobacterium abscessus Group

o The three most important rapidly growing mycobacteria causing human infections
are M. abscessus subsp. Abscessus, M. chelonae, and M. fortuitum.

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 o M. chelonae is the species of rapidly growing mycobacteria most likely isolated
from disseminated cutaneous infections in immunocompromised patients.
o Both M. fortuitum and M. chelonae have been associated with a variety of
infections of the skin, lungs, bone, central nervous system, and prosthetic heart
valves.
 Mycobacterium fortuitum Group
o The M. fortuitum group contained the species M. fortuitum, M. peregrinum.
o The M. fortuitum group is the most common rapidly growing mycobacteria
associated with localized cutaneous infections.
 Mycobacterium smegmatis Group
o The M. smegmatis group contains two species, M. smegmatis and M. goodie
o It has been implicated in rare cases of pulmonary, skin, soft tissue, and bone
infections.

Mycobacterium leprae
 Mycobacterium leprae is the causative agent of Hansen disease, an infection of the skin,
mucous membranes, and peripheral nerves

 Isolation and Identification of the Mycobacteria

o Direct Detection Methods
 Microscopy is considered a reasonably sensitive and rapid procedure for
the presumptive identification of Mycobacterium spp. in clinical
specimens.
 Acid-Fast Stains- Visualization of acid-fast bacilli in sputum or other
clinical material should be considered only presumptive evidence of
tuberculosis

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 Culture

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 Note: The following activity shall not be performed. All answers will be based on theoretical
results. Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Activity No. 13
Mycobacteria
Introduction:
The quality of work in AFB diagnostic microscopy depends on a number of factors like specimen
collection, quality of reagent, staining technique, reading of smear, reporting and recording and
training of technician. However, collecting a suitable specimen and making a good smear are
critical as quality of rest of the procedure depends upon it. Smear preparation must be
performed carefully and with attention to detail.

Objectives
At the end of the of the laboratory session, the student should be able to
Know the different methods of digestion, decontamination and concentration methods used for
the culture of Mycobacteria spp.
Know the different methods of staining and recent advancement in the microscopic study and
identification of Mycobacterial infection.
Prepare a good smear and be able to read, grade and report sputum smears based on local
and standard guidelines set by the DOH, CDC and WHO.
Materials:
Sputum specimen Alcohol lamp
Glass slides AFB stain
Coconut midrib Prepared AFB slides
Sand-alcohol set-up
Procedure:
PREPARING SPUTUM SMEARS
1. Numbering the slides

 Select new, clean, grease-free, unscratched slides which are free from fingerprints
 Using a pencil, record the laboratory register serial number and order number of the sputum
specimen on the frosted end of the slide. If plain unfrosted slides have to be used, labeling is
best done using a diamond pencil.
 Ensure that the number on each slide corresponds to the number on the specimen container.

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2. Sputum smearing
 Using the end of an applicator stick or wire loop, select and pick up the yellowish purulent
particles of sputum.
 Prepare the smear in an oval shape in the center of the slide. The smear size should be 2–3 cm
in length x 1–2 cm wide, which will allow 100–150 fields to be counted in one length.
 For good spreading of sputum, firmly press the stick perpendicular to the slide and move in
small concentric circles or coil-like patterns.
 Place the used stick into a discard container.
 Use a separate stick for each specimen.
 Alternatively, if a wire loop is used instead of a broken stick, dip the wire loop in an sand-alcohol
bottle. Remove the excess sputum from the wire loop by moving it up and down. After each
smear is completed, heat the wire loop in a flame until red-hot.
 Thorough spreading of the sputum is very important; it should be neither too thick nor too thin.
Prior to staining, hold the smear about 4-5 cm over a piece of printed paper. If letters cannot be
read, it is too thick.
3. Air drying of smear
 Allow the smear to air dry completely at room temperature.
 Do not dry smears in direct sunlight or over a flame.
4. Heat fix smear
 After the slide is completely dry, use forceps to hold the slide upwards.
 Pass the slide over the flame 2–3 times for about 2–3 seconds each time. Do not heat the slide
for too long or keep it stationary over the flame, or elsethe slide will be scorched.
STAINING WITH ZIEHL-NEELSEN CARBOL FUCHSIN SOLUTION

Principle of acid fastness

The cell wall of acid fast bacilli contains fatty acids known as mycolic acids, which makes them resistant
to the action of many chemicals. Because of this, the bacilli cannot be stained easily like in Gram‘s
stain. Strong dye concentration, application of heat, addition of phenol and longer staining time are
required to stain the bacilli. Once stained it is difficult to destain them. This property is used to
differentiate the AFB from all other materials like bacteria, cells and mucus will get decolorized by the
action of strong acid or acid-alcohol, leaving the acid fast bacilli stained with primary stain, which is
basic fuchsin incase of ZN staining or the fluorescent dye like auramine in case of fluorescent staining
method.
A good stained smear by ZN method shows strong red AFB, against weak blue background.
Contact time of carbol fuchsin is very important during staining, minimum five minutes are required for
good quality stains, however 10 minutes are preferred to get a strong AFB staining. The color and
intensity of background is important so that it does not mask the AFB. Destaining must be complete, if
required, destaining step should be repeated to ensure complete absence of red color from the
background. It is not possible to destain the AFB by using cold watery acids.
Overview of Ziehl-Neelsen staining procedure:
1. Arrange slides in serial order on staining bridge, with smear side up
2. Flood slides with filtered carbol fuchsin stain
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3. Gently heat to steam
4. Keep the staining reagent for at least 10 minutes
5. Rinse with water and drain
6. Apply decolorizing solution for 3 minutes
7. Rinse with water and drain
8. Apply methylene blue counter stain for NOT MORE THAN 1 minute
9. Rinse with water and drain
10. Air dry on a slide rack
READING THE SMEAR

 Carefully rotate the 100x objective over the smear and focus it.
 Systematically examine the smear under the 100X objective.
 Scan smears by moving across the smear in a horizontal direction.
 Stop and observe each field before moving onto the next field.
 Read at least 100 high power fields before reporting a negative result.
(Note: Fewer than 100 fields may be read if the slide is found positive for AFB.)
 Usually examining 100 fields takes about 5 minutes.
Appearance of AFB in the smear

AFB stain red or pink against a blue background with the Ziehl-Neelsen staining method. They are
usually thin and rod-shaped, but occasionally may appear as coccoid (beaded), filamentous (thread-
like), or clumped (in group) forms.
WHO/IUATLD GRADING SCALE: Reporting scale

Follow the scale when reporting smears:

 If no AFB are seen in at least 100 fields, report as negative for AFB.
 If 1–9 AFB are seen in 100 fields, report actual number of AFB seen.
 If 10–99 AFB are seen in 100 fields, report as (1+).
 If 1–10 AFB/field in at least 50 fields report as (2+).
 If more than 10 AFB/field in at least 20 fields, report as (3+).
Questions for Research:
1. What are the different methods of acid fast staining?
2. What are the different digestion and decontamination method for Mycobacterium tuberculosis?
3. What are the different culture media used for the isolation of Mycobacterium species?

Algorithm in Identification: TB Culture (Done in Reference Laboratories)

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Week 10: Gram-positive bacilli
Topic: Aerobic Non spore-formers
Time allotment: 8 hours

Learning objectives
At the end of the learning session, the student should be able to
1. Discuss the characteristics of each organism included in this group
2. Specify the desired requirements for growth of each group of organisms
3. Explain the clinical significance of Corynebacterium species other than Corynebacterium
diphtheria

Aerobic Non spore-formers

Non–Spore-Forming, Nonbranching Catalase-Positive Bacilli

Corynebacterium
 Majority of the species are found as indigenous microbiota on the skin and mucous membranes
of humans and animals
 The species are non-motile, non-encapsulated, non-spore-forming, and highly pleomorphic
rods.
 The species are glucose and maltose fermenters except C. urealyticum and C.
pseudodiphtheriticum.
 Microscopy: Slightly curved, gram-positive rods with unparallel sides and slightly wider ends that
produce a ―club-like‖ shape
 Culture: BAP-colonies have a small zone of Beta hemolysis, although some strains non-
hemolytic.
 This genus has three groups:
o A. Lypophilic corynebacteria
 The members of this group are fastidious corynebacteria that require 48 hours of
incubation before any growth can be observed
 The addition of lipids in the culture medium can enhance the bacterial growth
 Examples: Corynebacterium jeikeium and C. urealyticum
o B. Non-lipophilic corynebacteria
 The members of this group exhibit fermentative or oxidative metabolism
 Examples: C. diphtheria, C.pseudodiphtheriticum, C. pseudotuberculosis,C.
ulcerans
o Corynebacterium species that are associated with human infections and diseases.
 Examples: C. diphtheria, C. jeikenum, C. pseudotuberculosis,
C.pseudodiphtheriticum, and C. urealyticum

Corynebacterium diphtheria
 It is also known as the diphtheria bacillus or Kleb-Loffler bacillus
 It is a non-lypophilic, facultative anaerobe but grows best under aerobic conditions
 It is not part of the indigenous microbiota of the respiratory tract, and only inhabits the human
nasopharynx in a carrier state
 It is acquired through inhalations of contaminated respiratory droplets or direct contact with
infected cutaneous lesion
 It rarely enters the bloodstream

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  It is readily killed by heat and by most usual disinfectants
 It is a glucose and maltose fermenter
 Virulence Factor: Diphtheria toxin
 Preferred medium: Enriched medium with serum, cysteine, and potassium tellurite
 Microscopy:
o Its cells have rounded ends and ―club-shaped sweiings‖
o Its highly pleomorphic cells are arranged in pairs and create X, V, Y and L formations
that closely resembles Chinese letters
 Biochemical test: (-) Urease (+)nitrate reduction
 Best specimens: Nasopharyngeal and throat swabs
 Three biotypes of Corynebacterium diphteriae
o Intermedius- very small, flat, dry, and grayish-black colonies; non-hemolytic
o Mitis- small, black, and covex colonies that have a ―fried-egg‖ appearance; hemolytic
o Gravis- large, flat, and dark gray colonies that have a ―daisy-head‖ appearance; non-
hemolytic
 Diphtheria Toxin
o It is a heat-labile polypeptide
o It is produced by strains with a lysogenic beta-phage that carries the TOX gene
o It causes tissue necrosis and exudate formations over the tonsils, larynx and pharynx
o It favors an alkaline pH (7.8-8.0), an aerobic environment, and sufficient amount of iron
in the medium consumed

 Related Infections and Diseases

o 1. It is an acute, infectious disease that is characterized by the production of a systemic
toxin and a false membrane lining of the throat mucous membrane that may eventually
lead to respiratory obstruction
 Signs and symptoms: Low-grade fever; thick mucopurulent nasal discharge; and
cough
 Control measure: Immunization (DPT vaccine)
 Diphtheria antitoxin is administered to neutralize any unabsorbed exotoxin in the
patient‘s tissues
o 2. Cutaneous or skin diphtheria (Veldt sore)
 The toxin is absorbed systemically but is less severe

Corynebacterium urealyticum

 It is one of the most frequently isolated and most clinically significant Corynebacteria species
 It is a urinary pathogen, a strict aerobe, and lipophilic
 It does not ferment glucose and maltose
 Microscopy: Arranged in V-shaped forms and palisades
 Culture: BAP- Colonies are pinpoint, white, smooth, and are non-hemolytic
 Biochemical test: Rapid urease producer

Corynebacterium pseudodiptheriticum (Hoffman bacillus)

 It is an indigenous microbiota of the human nasopharynx

 It causes respiratory infection, UTI, and cutaneous wound infections in immunocompromised
patient, such as those who have AIDS

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  Microscopy: Cells are arranged in parallel rows or palisades and do not exhibit any other
characteristic ―pleomorphism‖ that is similar to other corynebacteria
 Biochemical test: (+) Urease and nitrate production

Corynebacterium jeikenum

 It is a skin microbiota that is found in inguinal, axillary, and rectal sites

 It is an obligate aerobe and a multi-antibiotic resistant bacterium
 It is isolated from immunocompromised individuals
 It is a coomon cause of diphtheroid prosthetic valve endocarditis in adults
 Microscopy: Pleopmorphic, club-shaped, and arranged in V-shaped forms
 Culture: BAP- Colonies appear large when added with 1% Tween 80
 Biochemical test: (-) Urease and nitrate reduction.
Corynebacterium ulcerans
 It is acquired through animal contact or ingestion of unpasteurized dairy products
 It is also isolated from skin ulcers and exudative pharyngitis
 It is associated with diphtheria-like sore throats since a significant isolates have shown to
produce a diphtheria-like toxin
 Culture:
o BAP- Colonies have a narrow zone of Beta hemolysis
o CTBA- Colonies have a surrounding brown halo
o Loeffler‘s serum agar- Colonies exhibit growth
 Biochemical test: (+) Urease and gelatinases, (-) nitrate reduction

Corynebacterium pseudotuberculosis
 It is an animal pathogen that humans can contract through direct contact with infected animals.
 It produces a dermonecrotic toxin that causes death of various cell types
 It can also produce diphtheria toxin
 Culture:
o CTBA- Colonies exhibit a black color and are surrounded with a brown halo
o BAP-Colonies are small and yellowish-white
 Biochemical test: (+) Urease (-) gelatinase

Laboratory Diagnosis
 Specimen: Nasopharyngeal and throat swabs, urine. blood, and wound discharge
o Nasopharyngeal and throat swabs are the best specimens for isolation of C. diptheriae
o A calcium alginate swab is preferred for collection
 1. Staining
o Gram staining: Highly pleomorphic, Gram-positive, short or slightly curved rods
o The various arrangements of C. diptheriae are due to its incomplete fission during
multiplication
o Methylene blue statining: Bacterial cells exhibit a beaded formation
o Neisser‘s and Albert‘s stains are used for staining metachromatic granule
o Neisser‘s staining method is composed mainly of either methylene blue or crystal violet
o Malachite green and toluidine are the components of Albert‘s stain.
 2. Culture
o Culture media: BAP, CAP, CTBA, Tinsdale agar, and Loeffler serum agar

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 o The primary inoculation of throat swabs to a Loeffler slant and the overnight inoculation
and subculture of any growth to CTBA may respectively improve the recovery of C.
diptheriae for both media
o CTBA and Tinsdale agar should be incubated for at least 48 hours at 35C
o The multiplication of corynebacteria occurs within the range of 15C to 40C
 Cytine tellutite blood agar (CTBA)
 It is the preferred medium for the isolation and identification of
corynebacteria
 It is a modification of Tinsdale agar
 It is both a selective and differential medium
 It contains sheep blood, bovine serum, cysteine, and potassium tellurite
 (+) Result: Colonies of corynebacteria exhibit a black or brown color after
48 hrs of incubation
 (+) Black or brown colonies surrounded by a brown halo: C. diphtheria, C.
ulcerans, and C. pseudotuberculosis
 Tinsdale agar
 It is composed of sheep blood, cysteine, potassium tellurite, and sodium
thiosulfate
 (+) Result: Colonies exhibit a black colorand are surrounded by abrown
halo
 All the biotypes of C. diphteriae have brown haloes around their colonies
 Loeffler‘s serum agar
 It is useful for observing the microscopic morphology and metachromatic
granules of C. diphteriae
 (+) Result: C. diphteriae colonies exhibit a ―poached-egg‖ appearance
 The metachromatic granules of C. diptheriae are called ―Babes-Ernst‖
bodies
 Christensen urea slant
 This is used to observe the urease production of C. uralyticum
 3. Schick test
o It is a skin test used to determine the susceptibility of a person to diphtheria
o The procedure involves the intradermal introduction of a small amount of diphtheria toxin
into the arm of the individual who is suspected to be harboring the disease
o (+) Result: Redness and Swelling around the site

Listeria

Listeria monocytogenes
 It is both a human and animal pathogen
 It is aerobic or facultatively anaerobic and non-spore forming
 It is motile with pretrichous flagella and exhibits a characteristic ―tu,bling motility‖
 It can grow in a high salt medium with up to 10% NaCl
 It has an optimal growth between 30C and 37C but can also grow at 4C
 It is recovered from the soil, dust, water, dairy products, and processed meats
 It causes miscarriage or stillbirth in humans
 Virulence factors: Listeriolysin O, catalase, supeoxide dismutase, phospholipase C, and p60

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  Mode of acquisition: Ingestion of contaminated food such as meat, chicken, dairy products, and
vegetables
 Microscopy: Coccobacillary in form and are arranged singly or in short chains that resemble
streptococci
 Culture: BAP-colonies are small, smooth, transluscent, grayish blue, and are surrounded by a
narrow zone of Beta hemolysis

Related Infections: Listeriosis

 It is a serious infection that affects neonates, pregnant women, and immunocompromised hosts
 Processed meat products should be thoroughly cooked or heated before consumption as a
primary preventive measure
 Immunocompromised individuals and pregnant women are predisposed to listeriosis after
ingesting contaminated dairy products like cheese.
 An infected pregnant woman may pass the infection to the fetus
 Macrophages and T-lymphocytes are the most important host defense against the infection

The different types of listeriosis are as follows

a. Maternal disease (Pregnancy)
 It usually occurs during the third trimester of pregnancy
 It leads to miscarriage or stillbirth
 Signs and symptom: Flu-like illness, fever, headache, and myalgia
b. Neonatal disease
 It is associated with an intrauterine infection due to the aspiration of infected amniotic
fluid
 The infected infants are at full term and appear healthy at birth
 It leads to meningitis that is usually seen by the third week of life
c. Disease of immunocompromised host
 It develops though the ingestion of contaminated dairy products and processed meat
products.

Laboratory Diagnosis
 Specimens: Blood, CSF, and swabs of lesions
 It is commonly isolated from blood and CSF
 1. Motility test
o Wet mount/hanging drop method: Exhibits a "tumbling motility‖ at room temperature
o SIM test: Has an ―umbrella-shaped‖ or inverted Christmas tree‖ pattern at room
temperature at 25C but not at 35C
 2. Culture
o Culture media: BAP, CAP, BHI, thioglycollate medium, McBride agar, and nalidixic
acid medium
o Enrichment technique: Cold enrichment (4C using broth)
o Colonies and hemolytic patterns may be confused with those of group B streptococci
o Growth occurs at a wide temperature range of 0.5C to 45C
o It requires a slightly increased amount of CO2
 3. Biochemical test
o (+) Glucose fermentation
o (+) Catalase and motility
o (+) CAMP reaction-―block type‖ hemolysis
o (+) Hippurate and bile esculin hydrolysis
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 o (+) growth in 6.5% NaCl
o (+) Voges-Proskauer and Methyl red tests
o (-) H2S production, nitrate reduction, and urease

Non–Spore-Forming, Nonbranching Catalase-Negative Bacilli

Erysipelothrix rhusiopathiae
 It is not part of the indigenous human microbiota
 It is facultatively anaerobic, non-motile, and non-sporeforming
 It is the only catalase-negative, non-spore-forming, Gram-positive, rod-shaped bacterium that
produces hydrogen sulfide.
 It is isolated from wild and domestic animals like birds and fish
 Major: reservoir: Domestic swine
 Microscopy: Thin, pleomorphic rods with the tendency to form long filaments that are arranged
in single, short chains or in a V-shaped formation
 Gelatin stab culture: Has a pattern of a ―pipe cleaner‖ or a ―test tube brush‖ at 22C
 Related infections and diseases: Endocarditis, septicemia, and erysipeloid (red-skin infection)
 Mode of transmission: Direct contact with infected excreta, blood, and flesh of animals through
skin breaks
 Predisposed individuals: Veterinarians and fish handlers

Laboratory Diagnosis
 Best specimens: Tissue biopsies or aspirates from skin lesion
 The organism is localized in the deeper layer of the subcutaneous tissue
 The outward margin of the lesion is the best site for collecting specimens
 1. Culture
o Culture media: BAP, nutrient broth, Columbia CAN agar
o Culture: BAP- Colonies are pinpoint with alpha-hemolytic zone
o The organism can grow on BAP and CAP for up to seven days
o The types of colonies that appear on its culture as follows
 a. Large and rough colonies-curled, slender, filamentous with a tendency to over-
decolorize and become Gram-negative bacilli
 b. Small and smooth colonies-transparent, glistening, and slender rods
 2. Biochemical tests
o (+) H2S production in a TSI medium
o (+) Glucose and lactose fermentation
o (-) Catalase, oxidase, esculin hydrolysis, nitrate reduction, VP, and urease

Arcanobacterium
 The species of this genus are classified as pleomorphic, non-motile, rod-shaped, and Gram-
positive
 They can cause pharyngitis and endocarditis
 Culture: BAP-Colonies have a narrow zone of Beta hemolysis; exhibit pitting of the agar with a
black opaque dot
 Biochemical test: (-) catalase
 Significant species: A. haemolyticum, A. pyoges, and A. bernardiae
 A. haemolyticum is both lipase-and lecithinase-positive and has a positive reverse CAMP
reaction due to phospholipase D

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Kurthia
 The members of this genus are obligately aerobic Gram-positive rods that are motile by
peritrichous flagella
 Culture:
o a. BAP-Colonies exhibit a large ―medusa-head‖ appearance
o b. Nutrient agar- Colonies exhibit a rhizoid growth
 Biochemical test: (+) Catalase; (-) gelatinase and oxidase

Aerobic Actinomycetes
 The mebers of this large and diverse group are composed of Gram-positive bacilli
 The members are classified as aerobes with a branching filamentous growth that extends along
the agar due to the substrate hyphae, and into the agar due to the aerial hyphae
 They cause diseases in humans and many animals
 Microscopy: Filamentous Gram-positive rods with a beaded appearance
 Culture: Cells elongate to form branching, filamentous forms while some organisms form
hyphae on the agar surface or into the agar.
Acid-fast Aerobic Actinomycetes
Nocardia
 The species of this genus are partially acid-fast, obligate aerobic, non-motile, and catalase-
positive
 Their cell wall contains peptidoglycan, meso-diaminopimelic acid (DAP), and the sugars,
arabinose and galactose
 The species grow on media that are used to recover fungi
 Microscopy: Gram-positive bacilli with long, thin, beaded, branching filaments
 Culture: Colonies exhibit wrinkled, chalk-like, and orange-tan pigmentation.
 Species: N. brasilienses, N. farcinica, N. asteroids, and N. nova
 Differential test: Resistant to lysozyme

Related Infections and Diseases

 Infection is acquired through the inhalation of the organism that are present in dust and soil
 Nocardia species can cause invasive pulmonary infections with hematogenous dissemination
throughout the body
 1. Actinomycetoma (Actinomycotic mycetoma)
o It is a chronic, localized, painless subcutaneous infection that is characterized by the
presence of sulfur granules in the affected tissue
o Causative agent; N. brasiliensis
 2. Pulmonary disease
o It is a confluent bronchopneumonia where the sputum is thick and purulent, although the
encapsulation of the abscess is absent
o The affected tissues do not have sulfur granules
o Causative agent: N. cyriacigeorgica and N. farcinica

Laboratory Diagnosis
 Specimens: Biopsy or drainage material from actinomycetoma, sputum, bronchoalveolar lavage,
lung tissue, transthoracic aspirate of a nodule or abscess, CSF and blood
 Pus and tissue specimens are the best samples in performing a wet mount and staining
 1. Staining technique
o Modified Ziehl-Nielsen or Kinyoun stain is used to observe a partially acid-fast Nocardia

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 o Gomori‘s methenamine-silver (GMS) stain is used for histopathological examination of
tissue specimens
 2. Culture
o Culture media: BAP. CAP, BHI, TMA, Lowestein-Jensen media, Saboraud Dextrose
agar (SDA) without chloramphenicol, Middlebrook (MB) media, litmus milk broth, potato
destrose agar, and tap water agar
o Some Nocardia species are Beta-hemolytic
o Incubation at 10% CO2 promotes the growth of Nocardia species
o Nocardia species may not always survive the decontamination procedures for
mycobacteria
o Tap water agar is used to observe the morphology of actinomyctes, and to differentiate
branching Nocardia species from non-branching Rhodococcus species

Rhodococcus, Gordonia, and Tsukamurella

 These are aerobic, catalase-positive, branching, filamentous, Gram-positive bacteria that can
fragment into rods and cocci
 They can be isolated from the soil, fresh and marine water, and organic matter
 They are primarily acquired throught inhalation
 They can grow on most of the non-selective media for bacterial, mycobacterial, and fungal
siolation
 Related infections and diseases: Skin infections, pneumonia, perotinitis, catheter-associated
sepsis

Rhodococcus equi
 It is non-motile and partially acid-fast, and is composed of mycolic acid with longer carbon
chains
 It can persist and replicate within macrophages
 It can be acquired through the respiratory route and exposure to infected animals
 It can infect immunocompromised individuals (HIV patients) and cause slowly progressive,
granulomatous pneumonia
 Microscopy: Coccobacilli with a ―zigzag‖ pattern and a filamentous form
 Culture: BAP-Colonies exhibit a pale pink or yeloow color
 Differential test: Susceptible to lysozyme

Gordonia
 The species of this genus vary from Gram-positive to Gram-variable rods
 They are partially acid fast, non-motile and catalase-positive
 Culture: Colonies are smooth and slimy with irregular edges; but may appear as dry or rough;
and exhibit the presence of mycelia
 Differential test: Susceptible to lysozyme

Tsukamurella
 The species of this genus are Gram-positive and shaped like long rods that fragments into three
parts
 The members are slightly acid-fast when the kinyoun method is used
 Culture: Colonies are circular with rhizoid edges; has no aerial hyphae; and exhibit white or
orange pigmentation

Non-acid-fast Aerobic Gram-positive Actinomycetes

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Streptomyces
 Its species are Gram-positive and rod-shaped with branching filaments
 They are found in the soil
 Human pathogen: S. somaliensis (agent of actinomycotic mycetoma)
 Differential test: Susceptible to lysozyme
 Culture media for isolation: BHI agar and SDA
 Culture: Colonies are dry to chalky and heaped; some colonies exhibit a grayish white color and
a ―musty basement odor‖

Actinomadura
 The microscopic and colony morphology of the species of this genus is very similar to that of the
Nocardia species
 It causes fungal wound infection that is known as eumycetoma
 The most common form of eumyctetoma is known as mycetoma pedis in which the infection is
localized on the patient‘s foot
 Species: A. madurae and A. pelletieri
 Culture: Routine agar-Colonies exhibit a ―molar tooth‖ appearance

Tropheryma whipplei
 It is a gram-positive actinomycete and a facultative, intracellular pathogen
 It is the etiologic agent of Whipple‘s disease that affects the gastrointestinal tract joints, and
muscles and is characterized by abdominal pain and diarrhea
 It is isolated from human feces, saliva, and gastric secretions
 Diagnostic test: (+) periodic acid Schiff staining (PAS) macrophages

Topic: Aerobic spore-formers

Time allotment: 4 hours

Learning objectives
At the end of the learning session, the student should be able to
1. Differentiate Bacillus anthracis from Bacillus cereus

Aerobic-Spore Formers
Spore-Forming, Nonbranching Catalase-Positive Bacilli

Bacillus
 The species of this genus are apore-forming, aerobic or facultatively anaerobic, rod-shaped
bacteria, and can be isolated from the soil
 The species only form endospores aerobically
 The species are motile with peritrichous flagella except for B. anthracis and B. mycoides
 The species can survive in extreme environmental conditions due to their endospores
 Microscopy: Large, boxcar-shaped, Gram-positive rods with clear, unstained, central spores or
―empty spaces‖
 Biochemical test: (+) Catalase; ferments glucose; hydrolyzes starch
 The most clinicall significant species include: B. anthracis, B.cereus, B. thuringiensis, and B.
mycoides

Bacillus anthracis (Anthrax Bacillus)

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  It is the causative agent of anthrax
 It is not part of the indigenous human microbiota
 It can grow aerobically or anaerobically
 It is not highly contagious
 It is non-motile and is considered as a halophilic organism that can withstand up to 7% NaCl
 It can be used as a biological weapon of mass destruction, and therefore a vital concern in the
campaign against global terrorism
 It grows in a low-pH (<6.0) environment, produces lecithinase, and ferments glucose
 Virulence factor: D-glutamic acid capsule and protein exotoxins
 Microscopy: Gram positive, large, encapsulated, and square-ended rod; has a ―bamboo fishing
rod‖ appearance on the unstained central spore
 Culture:BAP-Colonies have a ―Medusa head‖ appearance with swirling projections; are non-
hemolytic‘ and gain a ―beaten egg white‖ appearance when an inoculating loop is used
 Growth factor: Thiamine B1
 It gains a ―string of pearls‖ appearance when penicillin, to which it is susceptible, is used
 Related disease: Anthrax
o Anthrax
 It is a disease that primarily affects animals, such as goats and sheeps, by
feeding on plants that are contaminated with spores
 Modes of acquisition: Inhalation of spores during exposure to infected animals
and contaminated animal products, or through skin cuts
 Person-to-person transmission has not been documented
 The protein exotoxins causes the signs and symptoms of anthrax
 Three forms of anthrax:
 Cutaneous anthrax
 It is acquired through skin cuts and abrasions
 A small papule appears at the site of the spore inoculation two to five
days after exposure
 It is characterized by the appearance of a ―black eschar‖ which is a black,
necrotic, and painless central area that does not produce pus
 Pulmonary anthrax/Woolsorter‘s disease
 It is acquired when spores are inhaled into the pulmonary parenchyma
 It resembles an upper respiratoy tract infection
 Signs and symtoms: Mild fever, fatigue, malaise, and dyspnea
 Gastrointestinal anthrax
 Spores are inoculated into a lesion on the intestinal mucosa following
their ingestion
 Signs and symptom: Abdominal pain, nausea, anorexia, vomiting and
bloody diarrhea
Laboratory Diagnosis
 Specimens: Malignant pustule, sputum and blood
 B. anthracis is typically isolated from normally sterile sites such as blood, lung tissue, and CSF
 Processing of samples for B. anthracis should be done in a biological safety cabinet with
biosafety level 3
 1. Staining
o The use of old cultures in Gram staining may result in gram-variable cells
o Spore stains: Malachite green and McFadyean stain
o Capsule stain: India ink
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 o Fluorescent microscopy can provide a rapid presumptive diagnosis
 2. Culuture media
o Culture media: BAP, CAP, egg yolk agar (EYA), phenylethyl alcohol (PEA), polymyxin-
lysozyme-EDTA-thallous acetate (PLET), bicarbonate agar, and nutrient broth
o Enrichment and selective technique: Application of heat or alcohol shock technique
before plating on media
o PEA is recommended for identification of B. anthracis in fecal specimens
o PEA and PLET are used in isolating Bacillus species from contaminated specimens
o EYA is used to determine if the B. anthracis has produced lecithinase, in which case the
medium will have an opaque zone
o A gelatin medium is utilized to observe the ―inverted-pine-tree‖ appearance of B.
anthracis
o Cultures can also be incubated with an increased CO2 to stimulate capsule formation
 3. Diagnostic test
 A. Ascoli test (precipitin test)
o It detects thermostable anthrax antigens
o It uses rabbit antiserum to observe precipitin formation
o Sample solution: A 2-g sample in a 5 mL saline that is placed in a 1/100 final
concentration of acetic acid
o (+) Result: Formation of precipitin band after less than 15 minutes
 B. Catalase test
o Differentiates Bacillus species (catalase-positive) from Clostridium species (-)
 C. Oxidase test
o Gives variable results
 Direct fluorescent antibody test
o Positive cell wall polysaccharide and capsule antigen

Bacillus cereus (―Fried rice‖ bacillus)

 It causes food poisoning due to the ingestion of contaminated cooked rice dishes (typical
source) or other food products
 It is the most commonly encountered Bacillus species in opportunistic infections that causes eye
and ear infections
 It exhibits motility and resistance to penicillin
 Culture: BAP-colonies are large and feathery; have a spreading growth; have a ―frosted-glass‖
appearance; and are Beta hemolytic
 Biochemical test: Ferments salicin; (+) lecithinase
 Virulence factors: Enterotoxins (heat-stable and heat-labile), cerelysin, phospholipase C, and
pyogenic toxin
 Best specimen for testing: Suspected food (> 105 cells/gram)
 There are 2 types of food poisoning or gastroenteritis that are caused by B cereus:
o A. Diarrheal type
 It is associated with the ingestion of contaminated meat, poultry, and vegetables
 Incubation period: 8 to 16 hours
 Symptoms: Abdominal pain and watery diarrhea without fever
 (+) Production of heat-labile enterotoxin
o Emetic type
 It is associated with the ingestion of improperly stored cooked rice.
 It is caused by B. cereus type 1
 Incubation period: One to 6 hours
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  Symptoms: Abdominal cramps and profuse vomiting
 (+) Production of heat-stable enterotoxin

Bacillus subtilis ( Hay Bacllus)

 It is the most commonly encountered laboratory contaminant
 It is a halophilic organism that can tolerate up to 7% NaCl
 It is the source of the bacitracin antibiotic
 It can cause an eye infection among prohibited drug users
 Culture: BAP-colonies are larhe. Flat and dull with a ground glass appearance; may be B-
hemolytic; and may exhibit pigment atoms
 Biochemical test: Ferments mannitol, xylose, and arabinose

Bacillus pumilus
 It is used as a biological indicator in sterilization methods
 Culture; BAP-Colonies are large and moist; have a blister-like appearance and can be Beta
hemolytic

Bacillus thuringiensis
 It is an insect pathogen
 It produces parasporal crystals that can be utilized as a pesticide

Topic: Anaerobic-Spore-Formers
Time allotment: 4 hours

Learning objectives
At the end of the learning session, the student should be able to
1. Describe anaerobic bacteria, including their sensitivity to oxygen, why they are sensitive to oxygen,
and where they might be found in the environment and human body.

Clostridium
 The species of this genus are obligate anaerobes, catalase-negative, Gram-positive, spore
forming bacilli
 They are frequently encountered in exogenous anaerobic infections or intoxications
 The toxins produced by the species are acquired through ingestion or open wounds that have
been contaminated with soil
 Virulence contributors: Collagenase, hyaluronidase, lecithinase (cell destruction), and
phospholipase
 Species: C. perfringens, C. noyvi, C. septicum, C. histolyticum, C. bifermentans, C. sordellii, C.
innocuum, C. botulinum, and C. tetani
 Histotoxic Clostridium species that cause myonecrosis: C. perfringens, C. noyvi, C. septicum, C.
histolyticum, and C. bifermentans
Distinguishing characteristics
 They form endospores anaerobically.
 They are motile with peritrichous flagella except for C. perfringens, C. ramosum, and C.
innocuum
 They have swollen sporangia except for C. perfringens
 They are non-encapsulated except for C. perfringens
 They are carbohydrate fermenters except for C. tatami and C.histolyticum

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Clostridum perfringens(Gas gangrene bacillus)
 It is the most commonly isolated member of Clostridum in blood culture
 Virulence factor: alpha toxin and enterotoxin
 Microscopy: ―Boxcar-shaped‖ bacilli with oral, subterminal spores
 Culture:
o a. BAP-Colonies are dome-shaped and grayish white with double zones of hemolysis
(alpha and beta zones)
o b. litmus milk- Colonies exhibit a stormy fermentation of milk
 Biochemical test: Very fermentative
o a. (+) Lecithinase- detected using egg yolk agar
o b. (+) Nagler test- lecithovitellin reaction on EYA
o c. (+) Reverse CAMP test-formation of an ―arrowhead-shaped‖ zone of hemolysis
towards the test organism
Related Infection and Disease
 1. Gas gangrene
o It is a life-threathening destruction of muscle and other tissues
o It is accompanied by bullae, pain, swelling, serous discharge, discoloration and tissue
necrosis
 Clostridial necrotizng enteritis or enteritis necroticans
o It is caused by the ingestion of Beta-enterotoxin in contaminated food
o Improperly stored food allows the germination of the spores and growth of vegetative
bacteria
o Symptoms: Bloody diarrhea and abdominal pain

Clostridium tetani (Tack head bacillus)

 It is a soil and environmental inhabitant
 The endospores are found in the soil, dust, and feces of many farm animals
 Virulence factor: Tetanospasmin (neurotoxin)
 Microscopy: cells are with terminal spores and swollen sporangis that have a ―drumstick‖ or
―tennis racket‖appearance
 Culture: BAP-Colonies exhibit a slow, anaerobic, heavy, smooth, and swarming growth and
have a matte surface with a narrow zone of Beta hemolysis
 Biochemical test: (+) gelatinase, and indole; (-) lecithinase and lipase
 Related infection: Tetanus
Tetanospasmin
 It is an endopeptidase that selectively cleaves the synaptic vesicle membrane protein,
synaptobrevin
 It causes tension or cramping and twisting in skeletal muscles that surround the wound, and
tightness of the jaw muscle
Tetanus
 It is characterized by trismus or lock jaw and risus sardonicus or distorted grin
 It occurs when the organism enters an open wound and spreads a potent toxin that mediates
generalized muscle spasm
 Symptom: Muscular rigidity, difficulty in swallowing, rigidity of the abdomen, chest, back, and
limbs
 Incubation period: 3 to 21 days
 Tetanus neonatorum is contracted through the use of contaminated instruments on newborns

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Clostridium botulinum (―Canned food‖ bacillus)
 It is found in soil and aquatic sediments
 It is a potential bioterrorism agent
 It is characterized by the presence of subterminal spores
 Virulence factor: Botulism toxin-ia a neurotoxin that is considered as one of the most potent
natural toxins known to man
 Culture: BAP-beta-hemolytic colonies
 Related infection: Botulism
Botulism toxin
 It selectively cleaves the synaptic vesicle membrane protein, synaptobrevin, thus preventing
exocytosis and the release of the neurotransmitter, acetlcholine
 It requires a small amount to produce paralysis and death
 Botulism antigens: A to G
 The antigens that cause human diseases are A, B and E
Botulism
 It is characterized by double or blurred vision, impaired speech, difficulty in swallowing,
weakness, and paralysis
 There are 2 types of botulism:
o Foodborne botulism
 It results from the ingestion of preformed toxin in preserved or meat-based food
 It is commonly caused by botulism toxin A
o Infant botulism
 It is an actual infection that is caused by ingesting the organism from honey or
through breastfeeding for infants
Clostridium difficile
 It is the most common cause of antibiotic-associated diarrhea and pseudomembranous colitis
 It is acquired in the hospitals by individuals who are receiving antibiotics
 It is an ―infection control dilemma‖ among hospitalized patients
 It ferments fructose-producing formic acid that changes the color of medium from pink to yellow
 Virulence factor: Toxin A (enterotoxin) and toxin B (cytotoxin)
 Microscopy: Chains up to 6 cells that are aligned from end to end with oval, subterminal
endospores
 Culture:
o Cycloserine-cefoxitin-fructose agar (CCFA)-colonies exhibit a yellow color and a
―ground-glass‖ appearance
o BAP-Colonies exhibit a ―horse-stable‖ odor; are non-hemolytic‘ and produce a
fluorescent chartreuse under a long UV light wave

Laboratory Diagnosis
 1. Collection, transpot, and storage of specimens for anaerobic culture
o In case it cannot be processed immediately, all specimens for anaerobic culture should
be kept at a room temperature because refrigeration exposes the specimen to oxygen
o The specimen must be transported promptly to the laboratory under anaerobic
conditions or with minimal exposure to oxygen
 2. Recommended specimens for anaerobic culture
o To ensure the isolation of anaerobic clostridia, the specimens must be collected from the
actual site of the infection; swabbing of the mucosal surface is insufficient

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 o Needle aspiration is the best specimen for anaerobic culture
o Swabs should only be used when performing aspiration is not possible or if a biopsy
specimen is not available; the swabs should be transported under anaerobic conditions
in this case
o The swab should be placed into a 0.5 mL sterile thioglycollate broth
o Food and fecal specimens that are suspected of C. perfringens food poisoning should be
transported at 4C
 3. Unacceptable specimes for anaerobic culture
o a. swabs
o b. sputum
o c. bronchial washings
o d. feces and effluents from ileostomy and colostomy
o gastric and small bowel contents
 Gram stain
o Spores are not observed in Gram-stained smears of clinical specimens that contain
clostridia or in smears of colonies from an agar plate, unless the culture has been
incubated for many days
 Culture
o Culture media: Anaerobic blood agar, thioglycollate, EYA, CCFA, PYG, brucella blood
agar, PEA, and CAN
o Transport media: Pre-reduced anaerobically sterilized (PRAS) medium and Amies
medium
o A transport tube with PRAS medium is used for aspirates and tissue specimens
o Cycloserine and cefoxitin in CCFA inhibit Gram-negative coliforms
o Neutral red is the pH indicator in CCFA
o EYA is used to detect lecithinase and lipase activity
o PYG detects volatile fatty acid
o Brucella blood agar is used to observe swarming growths and the formation of double
zones of hemolysis, and supports nearly all obligate and facultative anaerobes
 Differential test
o Catalase test
 It is used to differentiate the aerotolerant strains of Clostridum (catalase negative
from Bacillus (catalase-positive)
 Reagent: 15% H2O2
 (+) Result: Formation of bubbles
o Direct Nagler test
 It is performed using an EYA plate and C. perfringens type A antitoxin.
 (+) Result: Inhibition of the lecithinase reaction that is produced by C. perfringens
o Mouse neutralization test
 It is a definitive identification test for C. botulinum
 It detects the presence of neurotoxins in serum or feces
o Reverse CAMP test
 It is used to confirm the presence of C. perfringens
 (+) Result: Formation of an ―arrowhead-shaped‖ zone of hemolysis at the
intersection of the 2 streaks toward the Clostridum isolates
o Cell culture cytotoxicity test
 It is the gold standard test for the detection of the C. difficile toxin type B
 It requires two to three days to achieve a positive result
o Lipase and lecithinase test

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  Medium: EYA
 (+) Lecithinase reaction: Formation of an opaque zone in the medium that
surrounds the colonies
 (+) Lipase reaction: Colonies exhibiting a ―mother-of-pearl‖ or ―gasoline-on-water‖
appearance
 Lecithinase-positive species: C. perfringens, C. bifermentans, and C. novyi
 Lipase-positive species: C. bitulinum and C. novyi

Bacteroides fragilis
 It is the most commonly isolated anaerobes from blood cultures
 It is a Beta-lactamase producer; and is non-motile and saccharolytic
 It is a significant cause of intra-abdominal abscesses
 Culture: Bacteroides bile esculin (BBE) agar-Colonies exhibit a gray color and growth witj 20%
bile, and cause the blackening of the originally yellow-colored agar
 Biochemical test: (+) Esculin hydrolysis

Actinomyces israelii
 It is the most common cause of actinomycosis
 It is part of the indigenous microbiota of the oral cavity

Propionobacterium acnes
 It is part of the indigenous microbiota of the skin
 It is frequently isolated from blood cultures, but its presence also indicates contamination of the
patient‘s skin due to the improper cleansing of the site prior to a phlebotomy

Lactobacillus
 The species of this genus are pleomorphic, Gram-positive rods
 It is non-motile, and may also be considered as an aerotolerant anaerobe
 Species: L. acidophilus, L fermentum.L.vaginalis, and L.salivarius

Lactobacillus acidophilus
 It is part of the indigenous microbiota of the mouth, GIT, and vaginal canal
 It protects the female genital tract from urogenital infections
 Related infection: Bacterial vaginosis
 Differential medium: Tomato juice agar (pH 3 to 4)
 Biochemical test: (-) Catalase , H2S and esculin hydrolysis

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Week 12: Gram-negative bacilli
Topic: Enterobacteriaceae
Time allotment: 12 hours

Learning objectives
At the end of the learning session, the student should be able to
1. Discuss the general characteristics of the family Enterobacteriaceae
2. Specify the distinct features of some species on selective and nonselective media
3. explain the antigenic structures and their purpose in bacterial identification
4. define the principle of the different biochemical test

Gram-negative Bacilli

Enterobacteriaceae

General Characteristics
 The common term that is used to refer to the members of this family is ―enterobacteris‖
 They are facultatively anaerobic, non-spore-forming, Gram-negative bacilli
 All members are non-encapsulated except for Klebsiella and Enterobacter
 All members ferment glucose and reduce nitrate to nitrite
 Most of them are present in the intestinal tract as commensal microbiota except for
Plesiomonas, Salmonella, Shigella, and Yersinia
 Some organisms like Serratia and Yersinia may grow at 1C to 5C
 Microscopy: Straight Gram-negative rods or coccobacilli with rounded ends
 Culture: BAP-Colonies appear as large, smooth, and gray except for Klebsiella and
Enterobacter with mucoid colonies, and are non-hemolytic except for some strains of E.coli
 Biochemical test: (+) catalase; (-) oxidase except for Plesiomonas shigellosis

Groups of Enterobacteria
 1. Opportunistic pathogens
o They are part of the intestinal microbiota of both humans and animals
o They generally do not initiate disease in healthy, uncompromised human host
o They may produce serious extraintestinal infection outside their normal body sites
o They produce significant virulent factors
o Some examples are: E.coli, Citrobacter, Enterobacter, Klebsiella, Proteus, and Serratia
 2. Overt/True Pathogens
o They are not present as commensal microbiota of the human GIT
o They are acquired through ingestion of contaminated food or water
o Their presence in specimens is considered as very significant
o Some examples are: Salmonella, Shigella, and Yersinia pestis

Antigen-determinants for Serological Identification of Enterobacteria

 Somatic O antigen-heat-stable; located in the cell wall; used for E.coli and Shigella serotyping
 Flagellar H antigen- heat-labile; found in the flagellum; used for Salmonella serotyping
 Capsular K antigen-heat-labile polysaccharide; found as K1 antigen of E.coli and Vi antigen of
S. entrica subsp. enterica serotype typhi

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Escherichia

Escherichia coli

 It is the most significant species in the genus Escherichia

 It may inhabit the female genital tract, although it is a microbiota of the large intestine
 It is a primary indicator of fecal contamination in water purification
 It is the leading cause of nosocomial urinary tract infection
 It has both the sex pili and adhesive fimbrae
 Culture:
o MAC-Colonies appear flat and dry, and exhibit a pink color ; some strains may non-
lactose fermenters
o BAP-Most strains are non-hemolytic; some strains are beta-hemolytic
o EMB-Colonies exhibit a greenish metallic sheen
 Virulence factor: Endotoxin, common pili, K1 antigen, and intimin
 Antigenic determinants: O, H, and K antigens
 IMViC- ++/--
 TSIA reaction: A/A (acidic slant/acidic butt), (+) gas, (-) hydrogen sulfide or H2S

Escherichia hermanii
 It is formerly called E.coli atypical or enteric group II
 It has been isolated from CSF, wounds, blood
 Culture: Colonies have yellow pigmentation

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Klebsiella
 The species of this genus are usually found in the GIT of humans and animals
 Culture: MAC-Colonies exhibit pink color

Klebsiella pneumonia subsp. Pneumonia (Friedlander‘s bacillus)

 It is the most commonly isolated species of Klebsiella
 It is the causative agent of community-acquired pneumonia; afflicted patients cough up ―currant
jelly-like‖ sputum
 It is the frequent cause of lower respiratory tract infections among hospitalized patients and in
immunocompromised hosts such as newborns, elderly patients, and patients on respirators
 Virulence factor: Polysaccharide capsule
 Culture: MAC-colonies exhibit a pink color and are mucoid (LF)
 Differential test: String test
 Neufeld-Quellung test: Positive
 Growth on media with potassium cyanide (KCN): Positive
 IMViC reactions: --/++

Enterobacter
 The members of this genus resemble those of Klebsiella when gron on a McConkey agar
 Culture: MAC-Colonies exhibit a pink color and are sometimes mucoid
Biochemical tests
 Ornithine decarboxylase test: Positive
 Lysine decarboxylase test: Positive (Except for E. cloacae and E. gergoviae)
 Growth on media with KCN: Positive
 Sorbitol fermentation: Positive
 Urease test: Positive
 Malonate test: Positive
 IMViC reaction: --/++
 TSIA reaction: A/A. (+) gas, (-) H2S

Enterobacter gergoviae
 It is found in respiratory samples and is rarely isolated from blood cultures

Enterobacter cancerogenus (formerly E. taylorae)

 It has been isolated with osteomyelitis following traumatic wounds

Cronobacter
Cronobacter sakazakii
 It is formerely known as Enterobacter sakazakii
 It is found as a contaminant of powdered infant formula
 It is isolated from individuals with brain abscess, and respiratory and wound infcetions
 Culture:
o MAC-Colonies exhibit a pink color
o BHIA-Colonies exhibit a yellow pigmentation
o IMViC reaction:--/++
o TSIA reaction: A/A, (+) gas, (-) H2S

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Pantoea
Pantoea agglomerans
 It is formerly known as Enterobacter agglomerans
 It causes nosocomial outbreaks of septicemia due to contamination of IV fluids
 It shows a triple decarboxylase negative reaction
 IMViC reaction: - v/+ v
 TSIA reaction: K/A, (-) gas, (-) H2S

Serratia
 The species in this group are opportunistic pathogens that are usually associated with
nosocomial outbreaks
 The species are resistant to a wide range of antibiotics
 Culture: MAC- Colonies are clear and colorless. Some strains may show a slow or late lactose
fermentation
 Biochemical test: (+) DNAse, gelatinase, lipase, and ONPG
 IMViC reaction: --/++
 TSIA reaction: K/A, (+) gas, (-) H2S
 S. marcescens, S. rubidaea, S. liquifaciens, S. plymuthica produce pink to red colonies after
incubation at 25C
 S. odorifera has a musty and pungent odor or a ―rotten potato-like‖ odor
 S. liquefaciens ferments arabinose and exhibits growth in a culture medium with KCN

Serratia marcescens
 It is the most clinically significant species of the genus
 It causes bacteremic outbreaks in nurseries, cardiac surgery units, and burn units
 A few strains of this species are late lactose fermenters
 Biochemical test: (+) urease, gelatinase and ONPG; (-) arabinose fermentation

Proteus
 The species of this genus are isolated from urine, wound, and ear infections
 The species can infect the proximal kidney tubules and can cause acute glomerulonephritis,
particularly in patients with UTI in catheterixation
 The species are rapid urease producers; the urease that they produce splits urea in urine,
raises urine pH, and encourages renal stone formation
 Human pathogens: P. mirabilis and P. vulgaris
 Common isolate: P. mirabilis
 Culture: MAC-Colonies are clear and colorless; exhibit a ―swarming phenomenon‖; and have a
―burnt-chocolate‖ or ―burnt-gunpowder‖ odor
 Phenylalanine deaminase test: Positive
 IMViC reaction:
o a. P. mirabilis: -+/vv
o b. P. vulgaris: ++/-v
 Lysine iron agar reaction: R/A
 TSIA reaction:
o a. P. mirabilis: K/A, (+) gas, (+) H2S
o b. P. vulgaris: K/A, (+/-) gas, (+) H2S

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Providencia
 The species of this genus are the cause of nosocomial outbreaks involving burn units
 Culture: MAC-Colonies are clear and colorless
 PAD test: Positive
 IMViC reactions: ++/-+
 LIA reaction: R/A
 TSIA reaction: k/A, (-) gas, (-) H2S

Providencia rettgeri
 It is a pathogen of the urinary tract
 It also causes diarrheal disease among travelers
 It is mostly resistant to antimicrobial agents

Providencia stuartii
 It is found in cosocomial outbreaks in burn units
 It is also mostly resistant to antimicrobial agents

Providencia alcalifaciens
 It is most commonly found in the feces of children with diarrhea

Morganella
 The species of this genus have the same biochemical reaction as those of P. vulgaris, except
that the latter is citrate-negative.
 Species: M. morganii
 Culture: MAC- colonies are clear and colorless
 PAD test: Positive
 IMViC reaction: ++/--
 LIA reaction: R/A
 TSIA reaction: K/A, (+) gas, (-) H2S

Citrobacter
 The species of this genus produce colonies in MAC agar that are similar to those of E.coli and
have a biochemical resemblance to those of Salmonella
 All species grow in Sinon‘s citrate (SC) agar
 Culture: MAC-Colonies become clear and colorless after 24 hours; colonies exhibit a ligh pink
color after 48 hours
 Species: C. freundii and C. koseri
 Urease test: Positive
 IMViC reaction:
o C. freundii: - + - +
o C. koseri: + + - +
 TSI reaction
o C. freundii: A/A or K/A, (+) GAS, (+) H 2S
o C. koseri: K/A, (+) gas, (-) H2S

Citrobacter freundii
 It can be isolated in diarrheal stool cutures
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  It produces group 1 cephalosporinase

Citrobacter koseri (formerly C. diversus)

 It causes outbreaks of neonatal meningitis and brain abscess in nursery units

Salmonella
 The species of this genus are the most pathogenic enterobacteria that cause enteric fever and
acute gastroenteritis to humans
 They are not part of the large intestine microbiota
 They inhabit the GIT of humans and animals
 They may also be transmitted by human carriers
 Mode of acquisition: Ingestion of contaminated animal food products or improperly cooked
poultry, milk, eggs, and dairy; and direct human contact
 Virulence factors: Fimbrae and enterotoxin
 Culture:
o MAC-Colonies are clear and colorless
o SSA- Colonies are colorless with black centers
 Antigenic structure
o Somatic O and flagellar H- for serologic grouping
o Vi antigen-antiphagocytic
 The main etiologic agent of enteric fever is Salmonella serotype typhi
 The etiologic agents of paratyphoid fever are Salmonella serotype Paratyphi A, B, and C, and
Salmonella serotype Choleraesuis

Salmonella bongori
 It is named after the town of Bongor in Chad, Africa where it was isolated from a host lizard in
1966
 It can also be isolated from other cold-blooded animals aside from lizards

Biochemical Characteristics of Salmonella species

 All species are motile except for Salmonella serotype Pullorum and Salmonella serotype
Gallinarum
 All special produce gas except for Salmonella serotype Gallinarum and Salmonella serotype
Typhi
 All species produce H2S except for Salmonella serotype Paratyphi A
 Urease: Negative
 Growth on media with KCN: Negative
 IMViC reaction: - + -+
 - + - - Salmonella serotype Typhi
 TSIA reaction: K/A (+) gas, (+) H2S
 K/A, (-) gas, (+) H2S Salmonella serotype Typhi

General Categories of Salmonella Infection

1. Gastoenteritis
 It is one of the most common forms of poisoning
 It is commonly caused by S. enterica subsp. Enterica that comes from animals

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  The Salmonella serotype Typhimurium outbreak in the US in 2009 came from contaminated
peanut butter crackers
 Sources of infection: Poultry products, milk, and handling of pets
 Mode of dissemination: Contaminated kitchen utensils
 Infective dose: 106 bacteria
 Symptoms: Nausea, vomiting, fever and chills, watery diarrhea, and abdominal pain
2. Enteric fever (Typhoid fever)
 It is a febrile disease that develops from eating contaminated food prepared by infected
individuals or carriers
 Direct transmission through fomites is also possible
 Causative agent: Salmonella enterica subsp. enterica serotype Typhi
 Sources of infection: Human carriers, contaminated food, and water
 Causes of outbreaks: Improper sewage disposal, poor sanitation, and lack of clean water
source
 Symptoms:Malaise, anorexia, myalgia, and severe frontal headache
 Complication: Necrosis in the gallbladder and Peyer‘s patches
 The hallmark of its infection is the appearance of ―rose spots‖ during the second week of fever
 The site of long-term carriage is the gallbladder
3. Bacteremia
 It occurs with and withour extraintestinal infection that is caused by non-typhoidal Salmonella
species
 It is characterized by prolonged fever and intermittent bacteremia.
 Its causative agents are Salmonella serotype Typhimurium, Salmonella serotype Paratyphi, and
Salmonella serotype Choleraesuis

Specimens for Salmonella Identification

1. Blood-first week of infection
2. Stool-second to third week of infection
3. Urine- third week of infection

Shigella
 The species of this genus are closely related to those of Escherichia
 The species are not members of the indigenous GI microbiota
 These species are non-motile, intracellular pathogens that multiply within the cells of the
intestinal epithelium
 Most of the species can cause bacillary dysentery
 Its reservoir is the humans
 Modes of transmission: Flies, fingers, food, and feces; and from water from infected persons
 Species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei
 The most virulent is S. dysenteriae
 S. flexneri is one of the causes of gay bowel syndrome
 Serogroups: A, B, C, D
 Antigenic structure: Somatic O
 Culture
o MAC-Colonies are clear, fragile, and colorless
o SSA-Colonies are colorless without black centers
 IMViC reaction: v + - -
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  TSIA reaction: K/A, (-) gas, (-) H2S

Biochemical Characteristics of Shigella Species

 All species do not produce gas from glucose except for S. flexneri
 All species are mannitol fermenters except for S. dysenteriae
 All species do not decarboxylate lysine
 All species do not decarboxylase ornithine except for S. sonnei
 S. sonnei is a late lactose fermenter and has a positive reaction with the ONPG test

Shigella dysenteriae
 It is the most virulent of the species and causes bacillary dysentery
 Virulence facor: Shiga toxin
 Urease test: Negative
 LDC test: Negative
 IMViC: v + - -
 TSIA reaction: K/A, (-) gas, (-) H2S

Shigella sonnei
 The infection from this organism is self-limiting, and usually characterized by fever and watery
diarrhea
 It has one serotype as opposed to the other species, which have several serotypes

Bacillary dysentery
 It is an infection that is most commonly caused by S. dysenteriae type I
 It is characterized by acute inflammatory colitis and bloody diarrhea
 It is highly communicable because of the low infective dose that is required to produce the
disease
 In young children, rectal prolapse occurs due to the excessive straining
 Source of infection: human carriers
 Modes of transmission: Person-to-person contact, fecal-oral route, and contaminated water from
infected persons
 Symptoms: Fever, chills, abdominal cramps, painful bowel movement, and tenesmus
 Complications: Ileus, seizure, and hemolytic uremic syndrome

Yersinia
Yersinia pestis
 It is also known as the ―plague bacillus‖
 It is a class A bioterrorism agent
 It is not part of the indigenous microbiota of human GIT and is non-motile enterobacterium
 It is the only enterobacterium that is transmitted to humans through the bite of an infected flea
 It is the causative agent of the bubonic plague
 It can be isolated on a routine culture media, and grows best at 25C to 30C
 Its vector is the Xenopsylla cheopis (Oriental Rat Flea)
 Its reservoir is the rats
 Virulence factors: Endotoxin, coagulase, and fibrinolysin
 Microscopy: Short, plump rod with a bipolar body or a ―closed safety pin‖ appearance
 Culture:
o MAC- Colonies are colorless and clear
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 o BAP- Colonies are pinpoint at 24 hours
o Broth- Colonies have a ―stalactite-shaped‖ pattern
 IMViC reaction: - + - -
 TSIA reaction: K/A, (-) gas, (-) H2S

Plague
 It is a disease of the rodents that is caused by Yersinia pestis and is transmitted to humans
through flea bites
 Humans may also acquire it by ingestion of contaminated animal tissues and inhalation of
contaminated airborne droplets
 Once inside the human body, the bacteria multiply in the blood and lymph
 There are 2 forms of plague
o Bubonic plague
 It results from the bite of infected flea
 It is associated with high fever and painful inflammatory swelling of the axilla and
groin
o Pulmonary plague
 It is acquired by close contact with infected individuals
 It occurs secondarily to bubonic plague

Yersinia enterocolitica
 It is the most commonly isolated species of Yersinia
 It is the causative agent of enterocolitis or waterborne gastroenteritis
 It is motile in SIM at 22C but not at 35C
 It has been isolated from contaminated packed RBC units, and is thus considered as a blood
transfusion hazard
 It has the ability to survive in cold temperatures
 Mode of acquisition: Ingestiom of undercooked food and dairy products, and handling of pets
 Its reservoirs are swines, dogs, cats, rabbits, and cows
 Related infections: Appendicitis-like syndrome, arthritis, and erythema nodosum
 Selective medium: Cefsulodin-irgasan-novobiocin (CIN) agar
 Microscopy: Coccobacilli with bipolar bodies
 Culture:
o MAC-Colonies are clear and colorless
o CIN- Colonies exhibit a ―bull‘s eye‖ appearance or dark red or burgundy centers with
transparent borders at 48 hours of incubation
 IMViC reaction: v + - -
 TSIA reaction: K/A, (-) gas, (-) H2S

Yersinia pseudotuberculosis
 It is a pathogen of the rodents, particularly guinea pigs
 It is motile in SIM at 18C to 25C but not at 35C
 Modes of acquisition: Direct contact with infected animals or their feces, and ingestion of
contaminated food and water
 Its reservoirs are farm and domestic animals (usually birds)
 Culture: MAC-Colonies are clear and colorless
 Biochemical test: (+) Urease and rhmnose fermentation
 TSIA reaction: K/A, (-) gas, (-) H2S
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Plesiomonas
Plesiomonas shigelloides
 It is the only species in the genus
 It is not part of the indigenous human microbiota
 It causes secretory diarrhea
 It ofeten cross-agglutinates with Shigella, hence the species name shigelloides
 It is the only oxidase-positive member of the Enterobacteriaceae
 Its motility is attributed to its polar flagella
 It has been associated with HIV-positive individuals with inflammatory bowel disease
 Mode of acquisition: Ingestion of undercooked seafood and contaminated water
 Microscopy: Straight bacilli which can occur singly, in pairs, in short chains, or filamentous
 Vibriostatic test: O/129: Sensitive
 Cultural characteristics
o MAC-Colonies are clear and colorless
o BAO-Colonies are shiny, opaque, smooth, and non-hemolytic
o Inositol-brilliant green-bile salt agar: Colonies exhibit white or green to pink color for
other enterics
o HEA- Colonies exhibit growth
o TCBS- Colonies do not exhibit growth
o Media with NaCl- Colonies do not exhibit growth
 Some strains will not grow on MAC
 The use of inositol-brilliant green bile agar enhances the recovery of plesiomonads from
specimen
 Biochemical and Serological Characteristics
o Oxidase test: Positive
o Decarboxylase test: Positive trio decarboxylase test
o Inositol fermentation: Positive
o IMViC: + + - -
o TSIA reaction: K/A, (-) gas, (-) H2S
o Antigenic structures: O and H antigens

Edwardsiella
 The species of this genus have been isolated from cold-blooded and warm-blooded animals
 Species: E. tarda
 Culture: MAC-Colonies are clear and colorless
 Urease test: Negative
 LDC test: Positive
 IMViC reaction: + + - -
 TSIA reaction: K/A, (+) gas, (+) H2S

Laboratory Diagnosis of Enterobacteriaceae

 Specimen: Stool, rectal swab, blood, urine
 The members of Enterobacteriaceae are commonly isolated from stool specimens, because
identification should only be performed with true intestinal pathogens
 Enterics that are isolated from sterile sites, such as Salmonella from bone marrow aspirates, are
highly significant
 Gram Stain

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 o The members of Enterobacteriaceae are seen as straight Gram-negative rods or
coccobacilli with rounded ends
o Wayson stain can be used to obseve the bipolar bodies of Y. pestis
o Direct smears of stool specimens are not routinely performed so it does help in the
identification of enterics
 Culture
o Culture media: BAP, CAP, MAC, HEA, XLD agar, CIN agar, Salmonella-Shigella agar
(SSA0, eosin methylene blue (EMB, bismuth sulfite agar (BSA), Selenite F, and GN
broth
o Transport media: Amies, Cary-Blair, and Stuart transport media
o Colony morphology: BAP and CAP-Colonies are large, gray, and smooth
o Fecal pathogens are generally non-lactose fermenters
o Salmonella species produce colonies with black centers in media with H 2S indicators
such as HEA, BSA, and XLD agar
o The optimal temperature for growth of entrobacteria is between 35C to 37C, except for
Serratia and Yersenia (1C to 5C) and E.coli which can also grow at 45C to 50C
o Plated and tube media should be read within 18 to 24 hours of incubation to avoid false
results
o Rapid lactose fermenters: Enterobacter, Eschericihia coli, Klebsiella
o Late lactose fermenters: Citrobacter freundii, Shigella sonnei, and Serratia
o Non-lactose fermenters: Citrobacter koseri, Edwardsiella, Morganella, Proteus,
Providencia, Slmonella, Shigella, and Yersinia
o 1. MacConkey-Sorbitol agar (MAC-SOR/SMAC)
 It is used to differentiate E. coli O157:H7 (sorbitol negative) from other strains of
E.coli
 pH indicator: Neutral red
 Result: E. coli O157:H7 exhibits clear or colorless colonies while the other strains
remain with pink colonies
o 2. Hektoen enteric agar (HEA)
 It contains bile salt, bromthymol-blue dye, alicin, lactose, and sucrose
 The bile and dye inhibit the growth of other Gram-negative bacilli in the GIT and
promote the isolation of Salmonella and Shigella
 pH indicator: Bromthymol blue
 H2S indicator: Ferric ammonium citrate
 Growthon HEA
 LF-Colonies exhibit a yellow color
 NLF-Colonies exhibit a blue-green color
 Non-enteric pathogens-Colonies exhibit an orange to pinkish orange color
o 3. Xylose-lysine deoxycholate (XLD) agar
 It is used for the isolation of Salmonella and Shigella species from heavily
contaminated specimens such as stool
 It contains lactose, sucrose, xylose, lysine, and sodium deoxycholate
 pH indicator: Phenol red
 H2S indicator: Sodium thiosulfate and ferric amoonium sulfate
 Growth on XLD agar
 Shigella-Colonies exhibit a pink to red color
 Salmonella-Colonies exhibit a pink to red color with black centers
 Other enterobacteria- Colonies exhibit a yellow to red color
o 4. Eosin methylene blue

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  It contains eosin Y, methylene blue, lactose, and sucrose
 pH indicator: Eosin and methylene blue
 Results:
 LF-Colonies of E.coli exhibit a greenish metallic sheen while those of
other LFs exhibit purple color
 NLF-Colonies are colorless
 Other coliforms-Colonies exhibit pink color
o Salmonella-Shigella agar (SSA)
 It differentiates Salmonella and Shigella species from other enteric bacteria
 Its bile salts and brilliant green dye contents inhibit Gram-positive bacteria and
some lactose fermenters that are found in stool specimens
 Carbohydrate source: Lactose
 pH indicator: Neutral red
 H2S indicator: Sodium thiosulfate and ferric ammonium citrate
 Result:
 Salmonella-Colonies are pink or colorless with black centers
 Shigella-Colonies are pink or colorless without black centers
 Serotyping (Slide agglutination test)
o It is a serological test that identifies strains (serovars or serotypes) of microorganisms
that differ in their antigenic composition
o Commonly tested microorganisms: Salmonella, Shigella, and E.coli O157:H7
o Preferred medium for testing: BAP with 5% sheep‘s blood
o 1. Salmonela and Shigella serotyping
 Salmonella serotyping is based on the heat-stable somatic O antigen and heat-
labile flagellar H antigen while Shigella serotyping is based on the somatic O
antigen
 Salmonella serotype Typhi also produces heat-labile capsular polysaccharide V-
antigen and carries the D serogroup
 Most of the Salmonella isolates from humans belong to the serogroup A to G
o 2. E. coli serotyping
 Sorbitol-negative E. coli can be serotyped to identify whether the somatic O
antigen O157 and the flagellar H antigen are present
 E. coli O55, O111, and O127 serotypes are most frequently associated with
infantile diarrhea
 Result: (+) agllutination

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Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Activity No. 14
Enterobacteriacceae
Introduction:
The enteric bacteria are gram-negative rods under the family Enterobacteriaceae. They are found in the
intestinal tract of man and other animals, in the soil, and on plants. Some are considered part of the
normal intestinal florawhile others are considered regularly pathogenic of man.
The enteric pathogens are present in appreciable number only during the stage of diarrheal diseases.
Therefore specimens should be obtained within this period whenever possible.
Objectives

At the end of the of the laboratory session, the student should be able to
1. Given the key reactions for identification, place an unknown organism in its proper tribe, genus, and
species.
2. Develop an algorithm using biochemical tests to presumptively identify clinically significant
Enterobacteriaceae

Materials
Selenite F broth Slides
MacConkey Plate Inoculating loop/needle
EMB plate Alcohol lamp
IMViC Reagents Slides
LIA Tube Microscope
Urease Tube
Urease Tube

Procedure

TSIA
1. Inoculate the TSIA medium with isolated organism, using a straight needle, stabbing to bottom of the
butt and streaking the slant
2. Incubate at 37C for 18-24 hours
3. Interpret results as follows
Alkaline (K): formation of reddish violet color on the slant or in butt
Acid (A): formation of yellow color on slant or the butt
Cracks in the medium: indicates gas production
Blackening of the medium: indicates formation of hydrogen sulfide

SIM- This test is used for the determination of sulfide production, indole formation, and motility
1. Pick up colonies from TSIA medium
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2. Stab the semi-solid medium carefully so that only one stab is made
3. Incubate at 35C for 24 hours
4. Interpret results as follows:
a. blackening of the medium along the line of inoculation: sulfide production
b. diffusion of growth from the line of inoculation: motility

To test for indole formation:

1. Add 0.5 mL of Erlich‘d reagent
2. Positive reaction is the development of brilliant red ring indicationg production of indole from
amino acid

IMViC Test
A. Indole production
Indole is produced as a putrefactive product from tryptophan and other amino acids containing the
indole ring

B. Methyl Red test

1. With the inoculating loop previously used in the SIM test inoculate the MR-VP broth.
2. Incubate at 35C for 18-24 hours
3. Add a drop of methyl red indicator
3. Interpret result: a distinct red color is positive and a yellow color is negative

C. Voges-Proskauer test
1. With the loop previously used in the MR procedure, inoculate another MR-VP broth
2. Incubate at 35C for 18 to 24 hours
3. Add a drop of methyl red indicator
Interpret result: a distinct red color is positive and a yellow color is negative

D. Citrate utilization
1. With the loop previously used in the VP test, inoculate the surface of the citrate agar medium
2. Incubate at 37C for 18-24 hours
3. Examine the tube for growth and color change. Positive reaction is the formation of a blue color

LIA test
1. Inoculate the LIA medium, using a straight needle, stabbing the butt and streking the slant
2. Incubate at 35C for 18 to 24 hours
Results:
Lysine deaminase positive-red slant
Lysine deaminase negative- purple slant
Lysine decarboxylase positive-purple butt
Lysine decarboxylase negative- yellow butt

Urease test
1. Inoculate the organism on the urea medium
2. Incubate at 37C for 18-24 hours
Result: formation of red/pink color

Illustration
1. Illustrate the positive and negative result of the above tests

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Questions for Research
1. What is the principle of each of the tests mentioned above?
2. Describe the API 20E and API 20NE
3. What are the differences between enterics and non-enterics
4. What are the advantages and disadvantages of using sem-automated from conventional methods of
bacterial diagnosis
5. What is the principle of the following tests:
a. starch hydrolysis
b. casein hydrolysis
c. fat hydrolysis
d. gelatin hydrolysis

Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

EXPERIMENT NO. 15
BLOOD CULTURE

The presence of microorganisms in the circulating blood, a condition known as bacteria, is

potentially dangerous situationwhich may cause immediate consequences such as shock,
disseminated intravascular Coagulation and death organisms can also reach all major body organs and
tissues by way of circulatory system using disseminated infection. Bacterimia may be transient,
intermediary or continuous condition depending on the source of organisms, the magnitude of the
infection and on the ability of the elements of the reticuloendothelial system, primarily in the liver and
spleen, to clear the blood stream of the circulating microorganisms.

Continuous bacteremia develops when the infection is intravascular as in endocarditis or when

the rate of bacterial multiplication exceeds the ability of the RES to removed the organisms. This
continuous circulation of microorganisms may result to septicemia, mark by chills, fever, and
prostration. The presence of microorganisms in the blood cand be detected by blood culture.

For blood culture, tryptic sot broth for aerobic organisms and thioglycollate medium for
anaerobic organisms are used. Usually 5 ml of blood is added to forty five ml of the bottled medium.

We can also commercially prepared medium that contains sodium polyanethosulfonate (SPS) to
conteract phagocytes, antivodies, and other antagonists. SPS alsoacts as an excellent anticoagulant
and shows no toxicity to bacteria in appropriate concentration. In some newer media, SPS is being
replaced by sodium amylosulfate(SAS). Sucrose may also be added to the culture medium to provide
osmotic stability. This is required by bacteria with damage cell wall or thse which are transitional forms
whose survival rate will be very poor without osmotic protection. There are also media that contain
carbon dioxide in the atmosphere.

For the collection of specimen, sterile syringes and needles are needed. It is very important to
emphasize at this stage the value of proper skin preparation and septic collection of blood. The skin

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should be washedwith soap and water, dried, swab with seventy percent alcohol followed by concentric
application of 3.5% tincture of iodine solution.

IMPORTANCE OF BLOOD CULTURE

1. To detect the presence of organism in the blood.
2. To establish the phase of infection.
3. To determine the effectiveness of antibiotic treatment.
4. To assess the prognosis of the patient.

Procedures:
1. Obtain the sample of blood with aseptic technic.
2. Deliver 5 ml. of blood to a tube of TSB and thioglycollate btorh.
3. Incubate the cultures aerobically at 35C for 24 hours.
4. Useful information can be obtained by observing cultures for typical appearances.
a. Gram negative rods: frequently produce turbidity with greenish tint in the medium above
the red cell.
b. Pneumococci and meninggococci produce turbidity with greenish tint in the medium.
c. Streptococci in thioglycollate medium produce clear upper layer broth with ‗cotton ball‘
colonies below but on the top of sediment red cell.
d. Beta-hemolytic streptococci and othe hemolyticorganisms produceturbidity with
hemolysis of red blood cells.
e. Staphylococcus produces turbidity with large jelly-like coagulum throughout the broth.
f. Bacillus species or saprophytic fungi: pellicle formation at the surface with hemolysis of
red blood cells.
g. Anaerobes: bacteroides and clostridium grow readily in thioglycollate medium.
5. If these characteristic are observed, subculture on clood agar, chocolate agar and mac conkey.
6. The BAP and CAP are incubated at 35C with increased carbon dioxide tension. The mcConkey
agar is incubated aerobically.

INTERPRETATION
A. Growth on BAP and CAP, no Growth on Mac Conkey (refer to pyogenic cocci for
identification
B. Growth on BAP, CAP amd Mac Conkey: (refer to Enterobacteriaceae for ID).

LIMITATIONS OF BLOOD CULTURE:

Some blood Cultures may be negative due to the presence of antimicrobioal substances in the
blood specimen

The absence of anticoagulant in the epecimen may cause the organisms present in the blood to
become trapped in the clot and go undetected, or the time required to recover them maybe greatly
increased.

The suse of a single broth for a specimen is not recommended

Premature discarding of apparently negative cultures or infrequent or infrequent observation

may result in failure to detect the presence of pathogenic microorganisms

Cloudiness may develop in negative culture as a result of frequent shaking. Thioglycollate

media may develop turbidity on prolonged incubation due to fibrin or agar particles
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 The roll tube method involves the use of a flamed gassing canalicula for delivering
oxygen-free gas into the test tube containing the medium to maintain reduced conditions.

The most elaborate and most expensive method is the anaerobic glove box in which
there is a large plastic chamber where anaerobic condition is maintaines and wwhere all manipulations
are carried out

Questions for Research

1. Give 5 specific examples of disease affecting blood. Give their bacterial etiologic agents
2. Enumerate some of the commercially available culture media for blood culture and give a brief
description of it
3. Why is the collection of blood taken from different sites?

Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Experiment No. 16
Cerobrospinal Fluid Culture
Cerebrospinal fluid, which completely surrounds the brain and spinal cord, is normally a sterile
fluid. Microorganism, however, can gain access to areas of the Central Nervous System and cause
meningitis, an inflammatory infection involving the meninges of the brain and spinal cord. Meningitis
occurs either primary disease or secondarily to infection in some other areas of the body. Since it is an
acute, life threatening disease, the microbiological examination of CSF is an emergency procedure

The CSF must be collected under sterile conditions and transported to the laboratory without
delay. Since the volume of CSF obtained is usually small, the specimen should be centrifuged for 10
minutes for 10 minutes at 2500g. The supernate should be removed with sterile capillary pipette and
the sediment shpuld be used for culture, gram stain and India ink procedure. The following organisms
in the order of their frequency; are isolated from CSF.
Haemophilus influenza Pseudomonas and Proteus species
Neisseria meningitidis Bacteroides
Streptococcus pneumonia Listeria monocytogenes
Mycobacterium tuberculosis Leptospira
Cryptococcus neoformans

ISOLATION AND IDENTIFICATION PROCEDURE

1. Obtain a spinal fluid specimen
2. Centrifuge the specimen at 2500g for 10 minutes
3. Carefully remove the supernate with a sterile capillary pipette
4. Use the sediment for gram stain

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 5. For culture, inoculate blood agar plate, chocolate agar plate, Mac Conkey and thioglycollate
broth
6. Incubate the plates in the candle jar, while the thioglycollate medium and the Mac Conkey plate
is done in ordinary conditions at 35C
7. Identify the organisms with the help of biochemical tests

Illustration/s
1. Draw the microscopic appearance of Leptospira interrogans

Questions for Research

1. If the specimen could not be processed immediately, where is the CSF stored
2. What is meningitis and what are the most common bacteria causing this infection

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Topic: Spirochetes
Time allotment: 2 hours

Lesson 1: Spirochetes

Learning objectives
At the end of the learning session, the student should be able to
1. Differentiate

Introduction:
The recognition of spirochetes as human host-associated organisms is believed to date from
nearly 400 years ago, when Van Leeuwenhoek described spiral, nimble ―animalcules‖ in human oral
plaque. Determination of taxonomic relationships among spirochetes has been complicated by their
fastidious nature and the refractoriness of many to cultivation. Numerous phenotypic traits have been
examined in attempts to establish taxonomic hierarchies. Paster et al. demonstrated using 16S rRNA
sequences that spirochetes can be grouped into a single phylum containing five clusters: Treponema,
Spirochaeta, Borrelia, Serpula (now Brachyspira), and Leptospira.
The relatedness among the members of individual clusters varied considerably. Interspecies
similarities among borreliae were >97%, suggesting recent evolutionary divergence. In contrast, the
∼10% sequence differences among treponemes pointed toward divergence over a greater evolutionary
time frame, a conclusion consistent with the diversity of vertebrate and invertebrate hosts known to
harbor treponemes as symbionts. (Arlene C, Sena, Pillay, Cox, Radolf, 2015)

SPIROCHETES

General Characteristics
- They belong to the order Spirochaetales
- Motile, long, slender, helically curved, gram-negative bacilli
- They have an unusual morphologic feature of axial fibrils and outer sheath
- Fibrils/axial filaments – flagella-
like organelles that wrap around
the bacteria‘s cell wall
o Enclosed within the
outer sheath, and
facilitate motility
(corkscrew-like winding)
of the organism
- insertion disk – platelike
structures where fibrils are
attached, located near the ends
of the cell
- differentiation of general within
the outer Spirochaetales is
based on the number of axial
fibrils, the number of insertion
disks and biochemical and
metabolic features
(https://www.quora.com/What-are-spirochaetes)

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TREPONEMA
- derived from the Greek owed meaning ―turning thread‖
- it has 6 – 10 axial filaments and 1 insertion disk
- characterized with corkscrew motility
- infect only humans and have not been cultivated for more than one passage in vitro
- they are killed rapidly at 42C and are used as a basis for syphilis therapy
- they remain visible in whole blood or plasma for at least 24 hours, which is of potential
importance in blood transfusion

2 types of Antibodies
o Treponemal antibodies
o Non-treponemal (reagin) antibodies

- Direct observation techniques

o Most species stain poorly with Gram‘s stain or Giemsa‘s methods
o Best observed with the use of dark-field or phase-contrast microscopy

- Important members:
o Treponema pallidum
o T. pertenue
o T. endemicum
o T. carateum

Treponema pallidum
- Etiologic agent of syphilis
- Fine spiral organism with 3 periplasmic flagella
- Appears white against a dark background
- Microaerophilic and survives longer in the presence of 3-5% oxygen
- This organism enters the host by either penetrating intact mucous membrane or entering
through breaks in the skin
o Not highly contagious
- Has a remarkable tropism (attraction) to arterioles – infection leads to endarteritis
- They die rapidly on drying and susceptible to disinfectants
- Generation time: 30 hours

- Clinical infection
o Syphilis
 French disease/ Italian disease/ the great pox
 Also known as the ―great imitator‖ because it can copy and assume many clinical
manifestations
 A disease of blood vessels and of the perivascular areas
 It can cross the placenta
 It is transmitted by direct/sexual contact or congenital
 It is characterized by chancre, fever, sore throat, headache and rash (palms and
soles), gummas in skin, neurosyphilis
 Chancre – a single erythematous, painless lesion that is non-tender and
firm with a clean surface and raised border
 If untreated, T. pallidum can disseminate to other parts of the body such as the
bones
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 Stages of Syphilis
 Primary Syphilis
o Characterized by the appearance of a chancre (hunterial
chancre) usually at the site of inoculation, most commonly the
genitalia
o Within 3 – 6 weeks, the chancre heals
o Dissemination of the organism occurs during this stage
o This stage is highly contagious
 Secondary syphilis
o Starts when the organism reached a sufficient number, after 2 –
24 weeks
o Fever, weight loss, malaise, loss of appetite and skin rashes
o skin rash spreads from the palms and soles towards the trunk
o condylomas develop at the anogenital region, axilla and mouth
o rash lasts 2 – 6 weeks
o a period of latency follows lasting for several years
 Latent stage
o The stage wherein the disease becomes subclinical but not
necessarily dormant
o Diagnosis can be made only be serologic tests
 Tertiary stage
o It is the tissue destructive stage-highly disfiguring
o Appears 10 – 25 years after the initial infection
o Complications arise at this stage – central nervous system
disease, cardiovascular abnormalities, eye disease, granuloma-
like lesions (gummas)
o affects all parts of the body
 cardiovascular and neuromuscular are most common
cause of death
o gummas
 large granulomas resulting from hypersensitivity reactions

 Laboratory Diagnosis
 Microscopic examination
o Skin lesions by dark field examination or fluorescent antibody
staining and microscopic examination
o Dark-field examination under 400x, high-dry magnification for the
presence of motile spirochetes
o Long, slightly larger than RBC, consists of 8-14 tightly-coiled, even
spirals
o Stains used: Levaditti‘s Impregnation Stain and Fontana
Tribondeau
 Serodiagnosis
o Non-treponemal flocculation tests
 Venereal Disease Research Laboratory (VDRL) test
 Rapid plasma reagin (RPR) test
 Automated Reagin test (ART)
 Wasserman, Kolmer test
o Treponemal tests

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  FTA-ABS – performed by overlaying whole treponemes
that are fixed to a slide with serum from patients suspected
of having syphilis because of previous (+) VDRL or RPR
test
 MHA-TP – utilize RBC from a turkey or other animal that
are coated with treponemal antigens
 (+) formation of flat mat across the bottom of the
microdilution well
 Treponema pallidum Hemagglutination (TPHA)
 Treponema pallidum Immobilization (TPI)
Treponema pallidum subsp. Pertenue
- Etiologic agent of Yaws/Frambesia Tropicana/ parangi/ paru/bua/bouba
- Traumatized skin comes in contact with an infected lesion

Yaws – a nonvenereal infection, characterized by chronic ulcerative sores anywhere on the

body with eventual tissue and bone destruction leading to crippling if untreated

Lesions
o Primary lesion – mother of yaws/frambesia
o Secondary lesion – daughter of yaws
o Tertiary lesion - gangosa

Treponema carateum
- Causative agent of pinta/carate/mal del pinto/azul
- common in tropical America

- Pinta
o Infection of the skin
o lesions heal slowly unlike syphilis and yaws
o Primary lesion is a slowly enlarging painless papule on the hands, scalp and feet with
regional lymph node enlargement followed in 1-12 months by a generalized red to slate-
blue macular rash

Treponema pallidum subsp. Endemicum

- Causative agent of endemic non-venereal syphilis/bejel
- Bejel – transmitted by direct contact with active lesions and contaminated fingers

ASSIGNMENT:
1. What is Congenital Syphilis? How is this disease contracted and what are the symptoms,
include photos if possible.
2. What is the Jarisch-Herxheimer reaction? Describe completely.
3. Describe the principle of the following serological tests for T. pallidum.
a. TPHA
b. TPI
c. VDRL
d. RPR

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BORRELIA
- Blood spirochetes
- Composed of 3 – 10 loose coild and are actively motile
- Has 30-40 axial filaments and 2 insertion disks.
- Stains well with Giemsa stain; slow growing spirochetes.
- Species that have grown in vitro are microaerophilic.
- Species: B. recurrentis, B. Dutoni, B. Hermsii, B. turicatae, B. parkeri, B. afzelii, B. burgdorferi,
B. garinii

Borrrelia Recurrentis
- Agent of louseborne or Epidemic relapsing fever/ European relapsing fever
- Transmitted by Pediculus humanus (louse)
- Humans are the only reservoir.

Borrelia hersmii/ B. turicatae/ B. dutoni / B. Parkeri

- Agents of tickborne relapsing fever/ Endemic or American Relapsing fever
- Transmitted via tick bites – Ornithodoros (soft ticks)

Borrelia burgdorferi (sensu stricto), B. garinii, B. afzelii

- Agents of Lyme disease
- Transmitted by the bite of Ixodes ticks – Ixodes pacificus, Ixodes dammini (deer ticks), Ixodes
persulcatus
- Deer and rodents (Peromyscus leucopus – white-footed mouse) – ticks‘ natural hosts
- All stages of ticks -, larva, nymph and adult, can harbor the spirochete and transmit disease.
- It is maintained in the environment by horizontal transmission from infected nymphal ticks.

Clinical Infection:
5. Relapsing Fever
- An acute infectious disease marked by recurrent febrile episodes; it has 2-10 relapses.
- Fever, headache, myalgia 2-15 days after infection.

6. Lyme disease
- An acute, recurrent inflammatory infection involving the large joints like the knees, with local
inflammation and swelling.
- It was first observed and described in 1975 among people of Old Lyme. Connecticut, USA.

3 Stages:
a. First Stage – Erythema Migrans
- characteristics red, ring-shaped lesion w/ a central clearing.
- appears at the site of the tick bite but may also develop at other sites.
- signs and symptoms: headache, fever, muscle, joint pain and malaise
b. Second stage
- it may start weeks to months after infection.
- symptoms: arthritis, neurologic disorders (meningitis) and carditis
c. Third Stage
- chronic arthritis and may continue for years.
- infected individuals may also develop demyelination of neurons with symptoms of
Alzheimer‘s disease and multiple sclerosis.

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Laboratory Diagnosis
1. Microscopic Examination
- Darkfield microscopy can be used for detection in blood cultures after 2-3 weeks of incubation at
34-37C.
a. Relapsing fever
 Peripheral blood – specimen of choice
 Organisms can be seen in wet preparations of peripheral blood – mixed w/ equal
parts of sterile, nonbactereiostatic saline.
 Can also be stained w/ Wright‘s or Giemsa stains.
b. Lyme Disease
 Blood, biopsy specimens and CSF
 For tissue sections, Warthin-Starry stain is used.
 For blood and CSF, acridine orange or Giemsa stain is used.
 PCR is also important in diagnosis of B. burgdorferi DNA in urine.
2. Culture
- Agents of relapsing fever can be cultured in nutritionally rich media under microaerophilic
conditions.
- For lyme disease, Keller‘s medium or chick embryo may be used.

3. Serodiagnosis
a. Relapsing fever
 Increased titers to Proteus OX K antigens (up to 1:80)
b. Lyme Disease
 Serology is the standard method for diagnostic testing
 IgM and IgG antibodies are detected
 ELISA, IFA, Western blot

LEPTOSPIRA
- Tightly coiled, thin, flexible, obligate aerobic spirochete; spimming motility.
- Often seen as a chain of cocci (dark-field microscope)
- Have ―question mark-like‖ shape.
- Organisms have books rather than tapering off.
- Live in the lumen of the renal tubules (shed info the urine).
- Can survive in neutral or slightly alkaline waters for months
- Can be grown in artificial media.
- Hamsters and guinea pigs are the animal of choice for cultivation
- Species: L. interrogans (pathogenic), L. biflexa (non-pathogenic)
- Generation time: 6-16 hrs
- Animal reservoirs: rats, dogs

Clinical Infection

Leptospirosis/Infectious Jaundice
- A zoonotic disease in human caused by L. interrogans.
- Hosts acquire infections directly by contact with the urine of carriers or indirectly by contract with
bodies of water contaminated with the urine of the carriers.
- Leptospires enter the human host through breaks I the skin, mucous membranes or conjunctiva.
- Rapidly invade the bloodstream, spread throughout CNS and kidney can be acquired in home
and recreational settings (swimming).
- Symptoms: fever, headache, myalgia, anorexia and vomiting
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 - A pretibial rash has been associated with serovar autumnalis (ft. Bragg fever)

2 Syndromes:
1. Anicteric Leptospirosis
 Septicemic stage, high fever and severe headache (3-7 days) followed by the
immune stage.
 Hallmark of immune stage is aseptic meningitis.
2. Icteric Leptospirosis/ Well Syndrome
 Severe form of illness characterized by liver, kidney and or vascular dysfunction.
 Death can occur in up to 10% of cases.

Laboratory Diagnosis:
1. Specimens
o Blood and CSF
o Urine – obtained after 2 weeks of illness, up to 30 days after the onset of
symptoms.

2. Microscopic Examination
 Detention of motile leptospires in the specimens by dark-field microscopy.
3. Culture – Fletcher‘s or Stuart‘s media (enriched w/ rabbit serum); Bovine serum albumin
Noguchi‘s medium, Ellinghausen, Mc Cullough, Johnson & Harris (EMJH) medium.
 EMJH medium 0 is incubated in the dark for 4-6 weeks at 25-36C.
 Few drops of heparinized or oxalated blood are inoculated into culture media.
 Urine should be inoculated immediately because acidity may harm the
spirochetes.
 Organisms grow below the surface – should be examined weekly for the
presence of growth.
 200ml of 5-fluorouracil
 Saprophytes can be differentiated from pathogens by their ability to grow to 10C
and lower.
4. Serodiagnosis
 Requires a fourfold or greater rise in titer of agglutinating antibodies
 Microscopic Agglutination (MA) – standard procedure using living cells.

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Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Experiment No. 17
Cerobrospinal Fluid Culture

Bacteruria is the presence of bacteria in the urine. Even though urine is considered a sterile
body fluid, the presence of bacteria in the urine may or may not denote infection within the urinary tract.
Acute or chronic infections of the urinary tract may involve the kidneys, uretrs, bladder and urethra.
Urinary tract infections (UTI) have classically been separated into lower and upper UTIs, most of which
develop from microbes ascending from the lower tract into the upper tract. This is not always the
situation, however, since kidney infection can be caused by microorganisms carried to the kidney via
the bloodstream.

Members of the bacterial family, Enterobacteriaceae are the primary cause of UTI. Other less
frequently occurring etiological agents of UTI include gram-positive cocci, anaerobic bacteria,
Chlamydia, Mycoplasma, Mycobacteria, fungi, protozoans, and viruses. Without quantitation, It is
virtually impossible to evaluate which microorganisms recovered in the urine is associated with
infection.

In urine culture, it is best to get early morning specimen whenever possible. Bacterial counts are
highest at this time due to overnight incubation in the urinary bladder. In symptomatic patients only one
specimen is collected before therapy is initiated. In asymptomatic patients, two or three specimens are
collected to show the presence of bacteruria.

Urine may be collected by the following methods:

1. Clean voided mid-stream technique
2. Suprapubic aspiration
3. Diagnostic catheterization
4. From indwelling catheter

The usual practice at the present time is to use the clean voided mid-stream technique.

Immediately after collection of the urine, it should be sent to the laboratory where it should be
examined within 15 minutes. If the urine is allowed to remain at room temperature without
bacterial examination, in a matter of 2 hours it will give a false positive culture. In the evenet that
the urine culture cannot be examined immediately, it can be refrigerated with satisfactory results
for at least 24 hours.

Culture and Isolation

The use of MacConkey agar, blood agar and eosin methylene blue agar for the culture of urine
specimens is widely recommended.

Bacterial Colony Count

A. The use of calibrated wire loop
1. Flame the calibrated wire loop (platinum wires calibrated to deliver both 0.001 ml and 0.01 ml)
2. After mixing the urine, remove the top of the container
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3. Holding the cooled loop vertically, immerse the loop below the surface of the urine specimen
and subsequently remove loop vertically
4. Inoculate each culture medium by placing the loop at the top of the center of the medium and
making a straight line down the center of the medium
5. Without flaming the loop or reentering the urine specimen, streak the plated medium for
isolation by making a series of close perpendicular streaks, starting at the top moving
downward, through the original line and across the entire plate
6. After incubation, the number of colony forming units (CFU) is multiplied by 100 if a 0.01 ml
calibrated loop is used and 1,000 if the 0.001 mL calibrated loop is used.

B.Dilution technique
Use the pour plate method of inoculation
Urine Saline Dilution
0.01 ml 9.99 ml 1:1000
0.1 ml 9.9 ml 1:100

Incubate for 18-24 hrs

Count the CFU‘s and if 1:100 is used multiply by 1000
If 1:1000 is used multiply by 10000

Interpretation of results:
Kass defined 100,000 or greater CFU per mL form symptomatic patients is representing significant
bacteruria.

After bacterialvcolony count, try to identify the organism using the biochemical procedures based on
the gram stain result

Questions for Research

1. What are the 2 routes of UTI infection? Describe each
2. What are the indirect indices of UTI? Describe each

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Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Experiment No. 18
Water Analysis

Water is a very important commodity in man‘s life. Man cannot live very long without water. Because of
this need for water, various sources are tapped for human consumption. Some of these sources
provide wholesome clean water while others have undesirable qualities for they may be polluted,
contaminated, lack clarity, or undesirable taste.

In this exercise, the bacteriological approach will be done. There will be an estimation of the
concentration of bacteria present in water as well as the detection of coliform bacteria. The coliform
bacteria are the organisms sought for because of the ease of detection. All sources of water which are
contaminated with coliform bacteria are presumed to have been contaminated by feces, which may
harbor other more pathogenic bacteria. Sterilization procedures such as chlorination and filtration are
employed. Doos analysis can also be done in a similar way.

Procedure:
A. Colony Count
1. Label 3 petri dish with the dilution of the sample to be used
2. To the first tube of sterile water (9 ml) add 1 ml of the sample and mix
3. With a sterile pipette, transfer 1 mL from this tube to the second tube with 9 ml of sterile water and
mix
4. Repeat the procedure by taking a sterile pipette and transferring and mixing 1 ml from the second to
the third tube. This gives a 1:100 and 1:1000 dilutions of the original
5. With a new pipette, transfer 1 ml from each tube to a petri dish, beginning with the highest dilution
and proceeding to the lowest.
6. Melt 3 tubes of agar in water bath, let them cool to 42 to 45 C and pour the melted agar aseptically
into each petri dish
7. Mix with water by rotating the plate over a smooth surface, let agar solidify, invert the plate and
incubate at 35C for 24 hours
8. Count the number of colonies multiplied by a factor
B. Presumptive test
1. To the first lactose fermentation tube, add 1 mL of the undiluted sample; to the second, 1 mL of the
1:10 dilution, to the third 1 mL of the 1:100 and to the fourth 1 mL of 1:1000 dilution
2. Mix water contents of the tube by gently rolling the tube between the palms. Do not let the contents
of the tube come in contact with the plug
3. Place in the incubator at 35C and examine at the end of 24 to 40 hours. For each period, note the
percentage of gas formed in each tubeas indicated by the portion of the collecting tube that is filled with
gas
4. Record the results
C. Confirmatory test
1. From the lactose tubes showing gas containing the least amunt of sample, streak 1 EMB plate and
incubate at 35C for 24-48 hours
2. Read plates. If typical colonies have developed during the time of incubation, the partially confirmed
results may be considered.
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D. Completed test
1. From the EMB plate of the previous laboratory period, select typical coliform colonies and inoculate
one agar slant
2. Incubate for 24 hours
3. Perform IMViC test

Note: The following activity shall not be performed. All answers will be based on theoretical results.
Procedures are written for reference.
Name: ______________________________________________
Group No. ___________________________________________

Experiment 19
ENVIRONMENTAL SAMPLING

Required Materials:

1. Nutrient agar plates, blood agar plates

2. Sterile cotton swab
3. Sterile water

Procedure:

A. Surface Sampling
1. Each group will be assigned an area to take surface samples from walls, floors, or other
surface areas in the hospital.
2. To obtain sample, moisten a sterile swab with sterile water and swab the surface of the
assigned area. Use the swab to inoculate the agar plate.
3. Incubate plate at 37oC overnight.
4. Count the number of colonies. Suggested interpretation is as follows:
More than 10 colonies  UNSATISFACTORY
Less than 10 colonies  ADEQUATE DECONTAMINATION
The Infection Control Committee of the hospital can modify these values.
5. Gram stain each type of colony.

B. Air Sampling
1. Expose a blood agar plate in a particular area assigned of the hospital for one hour.
2. Replace cover of the plate.
3. Incubate for 24 hours at 37oC.
4. Count the number of colonies.
Suggested interpretation:
More than 10 colonies  UNSATISFACTORY
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 Less than 10 colonies  ADEQUATE DECONTAMINATION
5. Do a Gram stain of the different types of colonies.

Results and Observation:

Area sampled: __________________________________________________________

Method of sampling: [ ] Air [ ] Surface, specify: ______________________________
Colony Count: ________________ Interpretation: ______________________________
Recommendation: _______________________________________________________

Questions for Research:

1. Define nosocomial infection.

2. What are the different sources of nosocomial infection?
3. What are the types of nosocomial infection?
4. What are the different potential agents of nosocomial infection?

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Topics
1. Obligate Intracellular Bacteria (Ricketssia, Chlamydia)
2. Atypical Bacteria (Mycoplasma, Ureaplasma)
3. Other miscellaneous bacteria

Learning Objectives:
At the end of the laboratory period the student should be able to
1. name accurately the microorganism included in atypical bacteria
2. state correctly why they belong to atypical bacteria
3. enumerate completely the insect vectors of these microorganisms
4. understand the disease process produced by these microorganisms

Introduction
The bacteria discussed in this module differ from bacteria described in other parts of this
module by several characteristics, including lack of efficient characterization with the Gram stain
method and, except for Mycoplasma and Ureaplasma species and to a limited degree for Coxiella
burnetii and Tropheryma whipplei, the requirement for intracellular growth. Thus, the most frequently
used tests in clinical microbiology laboratories, the Gram stain and culture on artificial media, are
unable to detect these organisms if present in clinical samples.
Diagnosis of infections caused by these bacteria has traditionally been accomplished by
Romanowsky staining (Giemsa and Wright stains) of clinical samples, by detection of antibody
responses to infection using a variety of serologic tests, or by histopathologic analysis of biopsy
samples. Molecular diagnostic tools and better culture methods have significantly improved the ability
to detect these agents and to diagnose the diseases that they cause. For some of these infections,
molecular tools are standard practice. (Wiedbrauk, 2015)

RICKETTSIACEAE
Time Allotment: 1 hour
Characteristics
- Small (0.5 – 2 um) non-motile, pleomorphic bacilli, have a g(-) cell wall
- It is the simplest
bacterial form and
considered transitional
organism between
bacteria and viruses
- This group of organisms
infect wild animals, with
humans acting as
accidental hosts
- Most are transmitted
between animals by an
insect vector
- Humans become
infected following the
bite of an infected
arthropod vector
- The best means of
prevention for rickettsial
and ehrlichial infection
is to avoid contact with
Page 217 of 227
 the vectors
(researchgate.net/figure/Life-cycle-of-Ixodid-ticks-and-natural-transmission-of-rickettsiae)
- Genera that contain species pathogenic to humans: Rickettsia, Orientia, Ehrlichia
o all species require living cells for growth except for Bartonella quintana

Rickettsia
- these are fastidious bacteria, obligate intracellular parasite
- small organism, pleomorphic, gram-negative bacilli and multiplies by binary fission in the
cytoplasm of host cells (the release of mature rickettsiae results in the lysis of the host cell)
- survives only briefly outside of a host and multiply only intracellularly in the cytosol of the host
cell
- they do not undergo any type of intracellular development cycle
o following infection, organism escape the vacuole, becoming free in the cytoplasm then
multiply, causing cell injury and death

(https://microbiologybook.org/mayer/ricketsia.html)
o when humans contact the infected tick, the organism is deposited on the skin and then
rubbed or spread by way of the bloodstream and infect the endothelial cells of blood
vessels resulting to vasculitis.
- Phospholipase A2 contributes to its intracellular activity.
- Rickettsia ricketsii
o can be passed from generation to generation of ticks through their eggs (transovarian
passage)

Groups of Bacteria
- Spotted fever group
- Typhus group
- Scrub typhus group

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Orientia tsutsugamushi
- Formerly known as Rickettsia tsutsugamushi
- It was placed into a separate genus due to the absence of LPS and peptidoglycan, and the
presence of 54 – 58 kDa major surface proteins
- They grow in the cytoplasm of host cell and released via ―pinching‖ off‖ a host cell membrane-
bound rickettsia
- It is transovarially maintained in mites (Leptotrombidium)
- Humans and rats are accidental, nonessential dead-end hosts

Ehrlichia
- Small (0.5 um, gram negative coccobacilli and undergo intracellular development cycle following
infection of circulating WBC
- They undergo 3 developmental stages similar to Chlamydiae: EB, initial bodies and morulae

(researchgate.net/figure/Schematic-representation-of-the-growth-cycle-of-ehrlichiae-in-an-infected-cell-Source)
- Wright-Giemsa staining of intravacuolar microcolony resembles ―mulberry‖ - morula
- E. Chaffeensis
o Infects monocytes and causes HME (Human Monocytic Ehrlichiosis)
o It is transmitted to humans by the lone star tick (Amblyomma americanum)
o Can also be transmitted by D. andersoni and Ixodes pacificus
- E. phagocytophila
o Causes HGE (Human Granulocytotropic Ehrlichiosis)
o Transmitted by I. pacificus, I. scapularis, and I. ricinus ticks, and white footed mouse
(Peromyscus leucopus)

Coxiella burnetti

Page 219 of 227

 - This is the causative agent of Q fever – a systemic infection that affects the lungs
- Strictly intrancellular, gram-negative bacteria, smaller that Rickettsia species; more resistant to
chemical and physical agents, dessication and sunlight (able to withstand harsh environmental
conditions)
- Can survive extracellularly because of its endospore-like body – can be grown only in lung cells
- In infected animals, organisms are shed in urine, feces, milk and birth products
- Humans are infected by the inhalation of contaminated aerosols from dried animal feces
- In contrast to rickettsial infection, a rash does not develop following infection
- It does not multiply in bacteriologic culture media
- Shell vial assay with human lung fibroblasts is used to isolate the organism from the buffy coat
and biopsy specimens has not resulted in any laboratory acquired infections
- IFA, CF, EIA – serologic techniques for detection
- Animal reservoir – cattle, sheep and goats

Bartonella
- Facultative intracellular gram-negative bacilli
- Do not synthesize acid from carbohydrates
- They live within red blood cells in their natural mammalian hosts
- They can be cultivated in blood enriched with 5% CO2 (CAP) or charcoal yeast extract agar
(CYEA)
- Species: B. quintana, B. henselae, B. elizabethae, B. bacilliformis
- Trench fever is transmitted from person to louse (Pediculus humanus corporis) to another
person.
- B. henselae – negative catalase, oxidase and urease
o B. quintana – etiologic agent of trench fever
o B. elizabethae – Etiologic agent of Infective endocarditis
o B. bacilliformis – etiologic agent of Oraya fever (chronic verruga peruana) and febrile
acute hemolytic anemia

Laboratory Diagnosis
- Specimen of choice: biopsy of skin tissue, peripheral blood and CSF

Direct Detection Techniques

- Immunohistology, PCR
- Polyclonal antibodies
- Giemsa or Diff-Quik stains – for the detection of morulae during the febrile stage of Ehrlichiosis
Culture
- Can be cultured in embryonated eggs and in tissue culture
Serodiagnosis
- Weil-Felix reaction – agglutination of certain strains of Proteus vulgaris by serum from patients
with rickettsial diseases; presumptive test
- Microimmunofluorescent Dot test – excellent sensitivity for detecting antibodies to rickettsia;
early diagnosis of RMSF within 7 – 10 days after onset of symptoms
- Q fever, Ehrlichiosis and Rickettsial pox – do not induce Weil-Felix antibody in infected patients
- Antibodies to rickettsia (except R. rickettsia) cannot be reliably detected until at least 2 weeks
after the patient has become ill
- Other tests: IFA, indirect immunoperoxidase assay, latex agglutination, EIA, line blot, CF and
Western Immunoblotting, Immunocytologic staining

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Activity 1: Fill up the spaces with the data needed.

Organism Diseases/s Vector/s (common name and

Scientific name)
Spotted Fever Group
Rickettsia akari
Rickettsia conorii a.
b.
c.
Rickettsia rickettsii a.
b.
Typhus group
Rickettsia prowazekii
Rickettsia typhi
Scrub typhus Group
O. tsutsugamushi
Ehrlichia
Ehrlichia chaffeensis
Ehrlichia equi-like and E. a.
phagocytophilia-like b.
organisms
Ehrlichia sennetsu
Coxiella burnetti
Bartonella quintana

Lesson 2: Chlamydiaceae
Time Allotment: 1 hour

Introduction
Members of the Chlamydiaceae are nonmotile, obligate intracellular prokaryotic bacteria
characterized by a unique biphasic developmental cycle bearing two chlamydial forms that differ
essentially in terms of morphology and function. According to the Approved List of Bacterial Names,
published in 1980, the Chlamydiaceae contained one genus with just two species, Chlamydia
trachomatis and Chlamydia psittaci, which were separated by their capability to accumulate glycogen in
inclusions and their susceptibility to sulfadiazine.
In the 1990s, the application of DNA-based classification methods contributed to the recognition
of the emerging human pathogen Chlamydia pneumoniae (1) and of Chlamydia pecorum (2), a
pathogen of ruminants, as new species of the Chlamydiaceae. Phylogenetic analyses of the 16S and
23S rRNA genes were the rationale for the proposal of an emended description of the order
Chlamydiales and a revised taxonomy of the family Chlamydiaceae in 1999. According to this proposal,
members of the order Chlamydiales are obligately intracellular bacteria that have the unique chlamydia-
like developmental cycle and more than 80% sequence identity with chlamydial 16S rRNA genes
and/or 23S rRNA genes. The emended order now includes four families: Chlamydiaceae,
Parachlamydiaceae, Simkaniaceae, and Waddliaceae (Gaydos and Essig, 2015)

Page 221 of 227

 CHLAMYDIACEAE

General Characteristics
- Non-motile, small (0.2 – 1.5um), resembles gram-negative cell wall, obligate intracellular
bacteria that requires living cells for growth
o They stain poorly with gram stain, but they could be better visualized with the Gutstein
stain
- They contain both the DNA and RNA, ribosomes and they produce their own proteins, lipids and
nucleic acids
o They were once thought to be large viruses, but they contain both DNA and RNA
- They multiply by binary fission, replicates I the cytoplasm of infected cells
- Requires the biochemical resources of the eukaryotic host cell‘s environment to fuel their
metabolism for growth and replication – ―energy parasite‖
- They have tropism for columnar epithelial cells
- Posses a heat stable, family specific antigen that is an essential component of the cell
membrane (LPS)
- Species: Chlamydia tracomatis, Chlamydophila psittaci and Chlamydia pneumoniae

Morphologic forms
1. Reticulate body (RB) – replicative form
 Intracellular and metabolically active
2. Elementary body (EB) – infective form
 Extracellular and structurally resembles a gram-negative bacillus
 Have a rigid cell wall
 2 components of the outer membrane
 MOMP (major outer membrane protein) and lipopolysaccharide (LPS) antigen

Chlamydia trachomatis
- Major cause of pelvic inflammatory disease(PID) and ocular trachoma
- It is divided into 17 different serovars based on major outer membrane protein antigenic
differences
o Serotypes A, B, Ba, C – endemic trachoma
o Serotypes L1, L2, L3 – lymphogranuloma venereum (LGV)
o Serotypes D- K – PID, urethritis, cervicitis, epididymitis, inclusion conjunctivitis
- Humans are the only known host of all strains
- Associated with infertility and ectopic pregnancy

Clinical infection
o Primarily spread from human to human by sexual transmission; mother to infant during
birth
o Intraperitoneal spread may cause peritonitis or perihepatitis (Fitz-Hugh-Curtis syndrome)
1. Trachoma
o Chronic inflammation of the conjunctiva leading to blindness
o It can cause distortion of the eyelids – the eyelashes become misdirected and turn in
o It is transmitted by contact with individuals that carries the organism from an infected
eye, or by flies
2. Lymphogranuloma Venereum (LGV)
o A sexually transmitted disease and has multisystem involvement
Page 222 of 227
 o A small painless ulcer or papule appears initially and nodules (buboes) develop after
several weeks
o Fret‘s test – intradermal skin test
3. Inclusion conjunctivitis
o It is characterized by copious discharge from the eye, an inflamed and swollen
conjunctiva, and the presence of large inclusion bodies in the host cell cytoplasm
o Usually affects infants

Laboratory Diagnosis
 Specimen of choice: urethra, cervix, conjunctiva, nasopharynx, rectum, and
material aspirated from fallopian tubes and epididymis
1. Culture – cell culture (reference method)
o McCoy cells, HeLa 229, buffalo Green Monkey Kidney Cells, Cycloheximide-treated
McCoy cells
o Centrifugation of the specimen onto the cell monolayer – growing on a coverslip in the
bottom of a vial. ―shell vial‖ facilitates adherence of elementary bodies
o After 48 – 72 hours of incubation, monolayers are stained with iodine or
immunofluorescent stain to examine the presence of inclusion
2. Cytologic examination
o Cell scrapings from the conjunctiva of newborns or person with ocular trachoma
o Giemsa stain
3. Antigen Detection and Nucleic Acid Hybridization
o DFA staining method using the fluorescein-isothiocyanate-conjugated monoclonal
antibodies – to identify the organism in infected cells
o ELISA
o Chemiluminescent DNA probe
o More reliable in patients with symptomatic and shedding large numbers of organisms
4. Serodiagnosis
o Negative serology can reliably exclude chlamydial infection
 Complement fixation
 Genus specific antigen can be detected by this methd
 Used to diagnose LGV but not trachoma, inclusion conjunctivitis and
neonatal infection
 Single point titer > 1:64
 Microimmunofluorescence assay (Micro-IF)
 Used for type-specific antibodies of C. trachomatis
 Can be used for diagnosis of LGV, trachoma, inclusion conjunctivitis and
neonatal infection
 IgM titer of 1:32

Chlamydophila (formerly Chlamydia) psittaci

- Etiologic agent of psittacosis/ornithosis
- Endemic pathogen of all bird species – parrots, parakeets, chicken, ducks
- Outbreaks have occurred among turkey-processing workers and pigeon aficionados
- Humans acquire the disease by inhalation of infected aerosols form dried bird excreta or
handling of infected birds
- only laboratories with biosafety level 3 facilities can perform cultivation of samples

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 Laboratory diagnosis
1. complement fixation – widely used serologic test to detect psittacosis
2. Direct Microimmunofluorescence – sensitive method
3. Amplification of rDNA using PCR followed by RFLP – identity and distinguish all 9
chlamydial species including C. psittaci

Chlamydophila (formerly chlamydia) pneumoniae (TWAR strain)

- human pathogen
- transmission is by aerosol droplets via the respiratory route
- pear -shaped appearance of EB; has round and dense inclusions
- resistant to sulfonamides
- negative for glycogen staining of inclusion bodies
- uses HL of Hep-2 cell lines for culture

Activity 2: Fill up the table with the correct data

Property C. trachomatis (Group A) C. psittaci (Group B)

1. Host range
2. Elementary Body Morphology
3. Inclusion morphology and
bodies
4. Stains used
5. Glycogen-containing
inclusions
6. Susceptibility to sulfonamides
7. Disease/s produced

ATYPICAL BACTERIA

MYCOPLASMA

General Characteristics
- Belong to the class Mollicutes which means ―soft‖
- Mollicutes appear to have evolved from clostridia-loke cells of the gram-positive branch of the
eubacterial tree
- Smallest known free-living forms capable of growing on artificial media
- They have small cell size (0.3 – 0.8 um) and small genome size
o May be visualized using a dark-field or phase contrast microscope
- Pleomorphic organisms that do not possess cell wall slow grower, fastidious, facultatively
anaerobic, replicate by binary fission
o These organisms are bound by a tri-layered membrane that is responsible for
 Absolute insensitivity to penicillins
 Failure to react with organic dyes used in many stains
- They require sterols for membrane function and growth
- Part of the microbial flora of humans and found mainly in the oropharynx, URT, and
genitourinary tract.
- They are common parasites of the genital tract and their transmission is related to sexual
activity.
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 - They lack the enzymatic pathway for purine and pyrimidine synthesis – require complex media
- Species: M. pneumoniae, M. hominis, M. fermentans, M. pirum, M. penetrans, U. urealyticum
Mycoplasma pneumoniae
- Eaton agent
- Etiologic agent of primary atypical pneumonia (PAP)/ Walking pneumonia
- Pleuropneumonia-like organism (PPLO)
- Doe not occur as a normal commensal
- Primarily a respiratory tract pathogen and extremely fastidious
- Can occur singly or as outbreaks in closed populations such as families and military recruit
camps, also causes tracheobronchitis
- This organism infects children and adults and is spread by respiratory droplets produced by
coughing.
- A surface parasite – colonizes the mucosa of the respiratory tract
- It has gliding motility which allows penetration of organism through respiratory secretions
- P1 protein – responsible for attachment and interacts with neuraminic acid-containing
glycoproteins
- they produce hydrogen peroxide and ammonia as waster products, and damage respiratory and
urogenital epithelial system

Ureaplasma urealyticum
- clinical disease
o venereal transmission causing non-gonococcal urethritis
o has been linked to amnionitis, fetal wastage and congenital pneumonia
- lab diagnosis
o the T-strain of the mycoplasma (tiny)
o It has the ability to metabolize urea – produces urease which is considered to be unique
for the mycoplasmas
o When grown in the presence of manganous sulfate with 1% urea, they will develop
minute golden brown colonies

Ureaplasma urealyticum and Mycoplasma hominis

- Genital mycoplasma
- They cause invasive disease in immunosuppressed patients (agammaglobulinemia), prostatitis,
bacterial vaginosis, non-gonococcal urethritis (NGU)

Mycoplasma incognitus
- The AIDS associated mycoplasma where the organism my play a role, possibly a second
invader

Laboratory Diagnosis
o Specimen: blood, joint fluid, amniotic fluid, urine, prostatic secretions, semen, pleural
secretions, tissues, swabs of the throat, nasopharynx, urethra, cervix or vagina.
o Highly susceptible to drying and should be transported within 1 hour of collection on ice
or at 4C

1. No direct method for identification

2. Culture – beef or protein with serum, fresh yeast extract, biphasic SP-4, PPLO broth or agar
with yeast extract and horse serum, Modified New York City Medium
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  A7 to A8 agar medium – ―dark brownish clumps‖ – U. urealyticum
 Grow slowly than most other bacteria
 M. pneumoniae – ―fried egg colonies‖
 M. hominis – urease (-); colonies can be stained with the Dienes or Acridine orange
 Glucose (dextrose) is incorporated into media selective for M. pneumoniae
 Urea and/or arginine can be incorporated int media to detect U. urealyticum and M.
hominis
 Definitive identification of M. pneumoniae is by overlaying suspicious colonies with 0.5%
guinea pig RBC in phosphate-buffered saline
3. Serodiagnosis
 Nonspecific production of cold agglutinins
 ELISA – most widely used method
4. Rapid identification – manganous chloride urea test (u. urealyticum)
 Positive result: dark brown precipitate of manganese oxide around the colonies
 The reaction is observed under a dissecting microscope
 U. urealyticum utilizes manganous chloride in the presence of urea

MISCELLANEOUS BACTERIA

Streptococcus moniliformis
- Etiologic agent for rat-bite fever and Haverhill fever in humans
- Gram-negative bacillus that requires media containing blood, serum or ascites fluid
- Known to develop L forms (bacteria without cell wall)
- Shows extreme pleomorphism with long, looped, filamentous forms, chains and swollen cells –
highly pleomorphic bacteria
- Tendency to form long chains of bacilli with prominent yeast-like swelling described as
―necklace-like, string of pearls or string of beads‖
- Non-encapsulated, non-hemolytic, non-motile, facultatively anaerobic
- Normally found in the oropharynx of wild and laboratory rats
- Dienes stain is required to demonstrate the L-form colonies
- Acridine orange stain also reveals the bacteria when gram stain fails due to lack of cell wall
constituents

Transmitted by 2 routes
o Rat bite or by direct contact with rats
o By ingestion of contaminated food like unpasteurized milk or milk products and water

N.B.
- After collecting blood and joint fluids, it is mixed with equal volumes of 2.5% citrate to prevent
clotting, then inoculate to BHI-cysteine broth supplemented with Panmede (a papain digest of
ox liver)
- The organism may be cultured from blood or aspirates from infected joints, lymph nodes or
lesions
- In broth cultures, organism grows as ―puff balls of bread crumbs‖ near the bottom of the tube or
on the surface of the sedimented RBC in blood culture media
- Colonies embedded in the agar may exhibit a ―fried egg‖ appearance with a dark center a a
flattened, lacy edge.
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 - Biochemical test – catalase, oxidase, nitrate, urea, and lysine decarboxylase negative and does
not produce indole
Gardnerella vaginalis
- Etiologic agent of bacterial vaginosis, considered a sexually transmitted disease
- It is part of the normal flora of anorectal adult
- Small, pleomorphic gram-variable or gram-negative coccobacilli or short rods; non-motile
- Colonies are small and B-hemolytic on media containing rabbit or human blood
- Oxidase and catalase negative

Laboratory Diagnosis
o Specimen: vaginal secretions
o Culture media: CAP, Columbia Colistin-Nalidixic agar, human blood bilayer Tween
(HBT) agar

o Diagnostic tests
 Direct saline wet mount of vaginal secretions
 (+) clue cells – large squamous epithelial cells with numerous attached
small rods
 Whiff test
 (+) fishy amine odor – vaginal secretions plus 1 drop of 10% KOH (pH
>4.5)

Tropheryma Whippelii
- Etiologic agent of Whipple‘s disease
- Whipple‘s disease – found in middle-aged men characterized by the presence of PAS staining
macrophages
- Gram (+) actinomyces, not closely related to any other genus known to cause infection
- This microorganism cannot be cultured, it can be seen in macrophages but cannot be cultured

Klebsiella granulomatis
- Formerly known as Calymmatobacterium granulomatis
- Causes granuloma inguinale or donovanosis
o Donovanosis – STD, characterized by nodules that enlarge and evolve to form beefy,
erythematous, painless lesions that bleed easily
o Gram-negative bacilli, encapsulated, pleomorphic, non-motile
o Stains as a blue rod with prominent polar granules, giving rise to a safety pin
appearance, surrounded by a large pink capsule
o It can be cultured in yolk sac or on fresh egg yolk medium
o Capsular polysaccharides shares several cross reactive antigens with Klebsiella species

ASSIGNMENT:
1. State the principle behind the following serologic test used for the diagnosis of some
bacterial species
a. ELISA
b. RIA
c. IFA
d. CF
e. Direct microimmunoassay

ASSESSEMENTS: TBA
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