MIB1001 Lab Manual 2015-16

THE UNIVERSITY OF TECHNOLOGY, JAMAICA
GENERAL MICROBIOLOGY
(MIB 1001)
LAB MANUAL
TABLE OF CONTENTS
INTRODUCTION...........................................................................................................................3
LABORATORY SAFETY..............................................................................................................4
Laboratory Exercise 1......................................................................................................................6
Laboratory Exercise 2 MICROSCOPY-EXAMINING YEAST CELLS AND BACILLUS....12
Laboratory Exercise 3 MICROSCOPY- ALGAE, FUNGI & PROTOZOA............................14
Laboratory Exercise 4 UBIQUITY OF MICROORGANISMS................................................15
Laboratory Exercise 5 ASEPTIC TECHNIQUES - TRANSFER OF BACTERIAL
CULTURES ................................................................................................................19
Laboratory Exercise 6 ISOLATION OF PURE CULTURES...................................................26
Laboratory Exercise 7 & 8 MULTIPLE AND DIFFERENTIAL STAINS..............................28
Laboratory Exercise 9 DEFINED, UNDEFINED, SELECTIVE AND DIFFERENTIAL
MEDIA ................................................................................................................33
Laboratory Exercise 10 FACTORS AFFECTING MICROBIAL GROWTH –........................35
PHYSICAL FACTORS.................................................................................................................35
Laboratory Exercise 11 MICROBIAL METABOLISM...........................................................44
Laboratory Exercise 12 QUANTIFICATION OF MICROORGANISMS...............................46
Laboratory Exercise 13 CONTROL OF MICROBIAL GROWTH – CHEMICAL METHODS..
..................................................................................................................
53
Appendix 1 Gram stain and General Microscopy Troubleshooting........................................61
Appendix 2 Stains....................................................................................................................62
Appendix 3 Staining Solutions................................................................................................63
Appendix 4 Broths...................................................................................................................63
Appendix 5 Agars....................................................................................................................63
Appendix 6 Writing a Laboratory Report................................................................................65
Glossary of Microbiology Terms...................................................................................................69
References......................................................................................................................................72
General Microbiology (MIB 2001) 2

Lab Manual
INTRODUCTION
Microbiology is the study of microorganisms. This is a large and diverse group of microscopic organisms that
exist in single cells or clusters; it also includes viruses, which even though microscopic, are not cellular.
Microbial cells are different from cells of animals and plants, which are unable to live alone in nature and can
exist only as parts of multicellular organisms. In contrast, to macroorganisms, microorganisms are generally
able to live and reproduce independently of other cells.
This manual is a compilation of exercises from various Microbiology Lab Manuals. The series of experiments
will introduce you to most of the basic techniques, apparatus and methodologies of general microbiology.
Initially, you will discover the ubiquity of microorganisms, especially bacteria in your immediate environment
as well as on yourself. One organism will be chosen from these for sub-culturing to result in a pure culture.
This pure culture will then be studied – morphologically as well as biochemically, to expose you to some of
the routine tests used to identify bacteria.
You will also be exposed to the concept of defined, undefined and complex media; factors affecting microbial
growth; microbial metabolism, methods for the quantification of microorganisms and finally methods for the
control microbial growth.
P.D. McLaughlin
Biology Division
FHAS

Lab Manual
LABORATORY SAFETY
The safe and easy functioning of Science Laboratories depends on observation of specific safety rules and
regulations, and standard operating procedures. The laboratory uses a wide range of chemicals and materials
that may be toxic, carcinogenic, and or biohazardous. It is important that you read the following sections so
you can work safely and effectively without any danger not only to your safety and well-being but to that of
others in the laboratory. If you have any questions or do not understand any of the items, ask the Instructor or
Laboratory Technician for clarification.
General Rules and Regulations
All students are required to read the laboratory procedure prior to each laboratory session .
1. Only authorised personnel and or students are allowed in the laboratory. Do not invite friends or
take minors into the lab.
2. Never eat, drink, store food, or smoke in the laboratory. No chewing gum or sweets are allowed in the
laboratory. Do not bring food or drink to the laboratory.
3. Store your bags and books in the area designated. Do not clutter your workspace with personal
belongings or they may become contaminated such as laptops, tablets or cell phones.
4. To prevent contamination of your clothes, always wear a laboratory coat once in the laboratory.
Remove this protective wear before leaving the laboratory.
5. Only shoes that cover the entire foot are allowed in the laboratory, no slippers allowed.
6. Be on time. Students must attend the pre-laboratory talk at the start of the session, within the first 20
minutes. If you miss the pre-laboratory talk you will be asked to sit out the session.
7. There is to be no running or playing in the laboratory.
8. Turn cellular phones off. There are to be no cellular phone conversations in the laboratory.
9. Before and after each class period, wipe table tops with disinfectant/alcohol provided. Dispose of
the paper towels used in the bins provided.
10. When carrying equipment, chemicals or cultures do not run or handle carelessly.
11. Report any spills of cultures or chemicals, fires or personal injuries such as burns or cuts to the
instructor immediately.
12. Always double-check the label of any chemical or microbial culture you may use.
13. Keep any flammable liquids away from open flames (Bunsen burner).
14. Do not inhale the fumes of any substance directly. When required to note the odour of a culture,
for example, take a deep breath of fresh air and exhale normally. With your hand waft the vapours
towards your nose and sniff slowly.
15. Do not pipette by mouth. Always use a pipette aid or autopipette.
16. Do not use your fingers as a stopper for a tube or bottle when shaking to mix its contents.

Lab Manual
17. Conserve water and gas. Turn off water faucets and Bunsen burners when not in use.
18. Never remove equipment, media, or microbial cultures from the laboratory.
19. Leave all the laboratory facilities and equipment in good order at the close of the session. Return
all materials used to the area designated by the instructor.
20. Dispose of Chemical and Biohazardous material as directed by the instructor.
21. Place dirty glassware in the designated area
22. Wash hands thoroughly with soap before leaving the laboratory, whether it’s for a drink or
restroom break.
23. Whenever in doubt about a personal health problem or a laboratory procedure, ask the lab
technician or instructor.

Lab Manual
Laboratory Exercise 1
PART 1 Introduction to the Microscope
Biological specimens are sometimes too small to be viewed with the naked eye and a microscope has to
be used to facilitate observation. The microscope available in most laboratories is the compound light
microscope. The light microscope uses light rays that are magnified and focused by means of lenses. The
illumination is from below and the light passes through clear sections of small objects or thinly sliced
sections of an object. To improve contrast stains and dyes that bind to cellular structures and also absorb
light are normally used to stain/dye specimens.
I. Part 1 Identification of the microscope parts
1. Eyepiece (Ocular lens): Topmost series of lenses through which an object is viewed. The magnifying
power of the ocular lens is usually 10X or 15X.
2. Body Tube: Holds the eyepiece at one end and the nosepiece at the other end. It also conducts light
rays.
3. Arm: Supports upper parts and provides carrying handle.
4. Nosepiece: Revolving device that holds the objectives.
5. Objectives: Three or four series of lenses fitted to the Revolving Nosepiece and used to view the
specimen directly.
The scanning objective is the shortest of the objectives and is usually used to locate on the slide the
specimen to be viewed. The magnification stamped on the housing of the objective is usually 4X.
The low-power objective is used to view objects in greater details than the scanning objective. It usually
magnifies objects 10 times (10X).
The high-power objective (in a 3 objective microscope is the longest objective) usually magnifies the
specimen forty times (40X) and has the smallest field of view in a three objective microscope.

Lab Manual
The oil immersion objective holds a 100X lens and is used in conjunction with immersion oil to view
objects with the greatest (meaningful) magnification possible in a light microscope.
6. Coarse-adjustment knob: Knob used to bring object into approximate focus. Used only with
scanning and low-power objective while still looking through the ocular.
7. Fine-adjustment knob: Knob used to bring object to final focus.
8. Condensor: Lens system below the stage used to focus the beam of light on the object to be viewed.
9. Diaphragm: Controls the amount of illumination used to view the object.
10. Light source: An attached lamp that directs a beam of light up through the specimen.
11. Base: The flat surface of the microscope that rests on the table.
12. Stage: Holds and supports microscope slides.
13. Stage Clips: Hold slides in place on the stage.
14. Stage knobs: Two knobs below the stage that control right and left movement and forward and
reverse movements of the stage.
II. Part 2 Focusing the microscope
1. Turn the nosepiece so that the lowest power objective is in straight alignment over the stage.
[Always begin with the lowest power objective]
2. With the coarse adjustment knob raise the stage until it stops.
3. Place a slide of the letter e on the stage and stabilize with clips. Center the e as best you can on
the stage.
4. Looking from the slide, decrease the distance between the e and the tip of the objective lens until
the objective comes to an automatic stop or as close to no closer than 3 mm above the specimen.
5. While looking through the eyepiece, rotate the diaphragm to give the appropriate amount of light.
6. Using the coarse-adjustment knob, slowly rotate the knob until the specimen comes into view.
7. If object is seen and there is need to improve the contrast. To increase or decrease the contrast,
rotate the diaphragm slightly.
8. Use the fine adjustment knob to sharpen focus.
9. Practice having both eyes open when looking through the eyepiece, it reduces eyestrain.
Note: Compound light microscopes are parafocal; that is, once the object is in focus with the lowest power, it should
almost be in focus with the higher power. This means that one can normally move from one objective to the other
without losing focus and using the fine-adjustment knob to bring back specimens into sharp focus .
III. Part 3 Microscopic measurements
Your microscope may be equipped with a scale (called a reticule) that is built into one eyepiece. The
reticule can be used to measure any planar dimension in a microscope field since the ocular can be turned
in any direction and the object of interest can be repositioned with the stage manipulators. To measure the
length of an object note the number of ocular divisions spanned by the object. Then multiply by the

Lab Manual
conversion factor for the magnification used. The conversion factor is different at each magnification.
Therefore, when using a reticule for the first time, it is necessary to calibrate the scale by focusing on a
second micrometer scale (a stage micrometer) placed directly on the stage.
Conversion factor
Identify the ocular micrometer. A typical scale consists of 50 - 100 divisions. You may have to adjust the
focus of your eyepiece in order to make the scale as sharp as possible. If you do that, also adjust the other
eyepiece to match the focus. Any ocular scale must be calibrated, using a device called a stage
micrometer. A stage micrometer is simply a microscope slide with a scale etched on the surface. A typical
micrometer scale is 2 mm long and at least part of it should be etched with divisions of 0.01 mm (10 µm).
Suppose that a stage micrometer scale has divisions that are equal to 0.1 mm, which is 100 micrometers
(µm). Suppose that the scale is lined up with the ocular scale, and at 100x it is observed that each
micrometer division covers the same distance as 10 ocular divisions. Then one ocular division (smallest
increment on the scale) = 10 µm at 100 power. The conversion to other magnifications is accomplished
by factoring in the difference in magnification. In the example, the calibration would be 25 µm at 40x, 2.5
µm at 400x, and 1 µm at 1000x.
Some stage micrometers are finely divided only at one end. These are particularly useful for determining
the diameter of a microscope field. One of the larger divisions is positioned at one edge of the field of
view, so that the fine part of the scale overlaps the opposite side. The field diameter can then be
determined to the maximum available precision.
1. Following the instructions provided, calibrate the ocular micrometer provided.

2. Record the measured/calculated diameter of the fields (scanning, low power, high power, oil
immersion)
3.

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Estimating and reporting dimensions
Be aware that even under the best of circumstances the limit of resolution of your microscope is 1 or 2
µm (or worse) at any dry magnification, and 0.5 µm or so using oil immersion. No directly measured
linear dimension or value that is calculated from a linear dimension should be reported with implied
accuracy that is better than that. That includes means, surface areas, volumes, and any other derived
values. For example, suppose you measure the length of a flagellum on a Chlamydomonas cell at 400x,
and determine that it covered 3 ½ ocular divisions. The length is directly calculated as 3.5 divisions times
2.5 µm per division, which comes out to 8.75 µm. You know, however, that at 400x the absolute best you
can do is to estimate to the nearest µm, so before reporting this measurement round it to 9 micrometers
(not 9.0, which would imply an accuracy to the nearest 0.1 µm).
The microscope can be used not only to observe very small objects, but also to measure their sizes.
Microscopic measurements are expressed in units of the metric system, which is a decimal system based
on the meter.
It is easy to calculate the amount of magnification produced by a compound microscope. This is

calculated by multiplying the magnifying power of the objective lens times the magnifying power of the
ocular. For example, a certain microscope may have an ocular of 10 X power and a low power objective
lens of 5 X power. The total magnification of this particular microscope under low power is 50 X. This
means that an object viewed in this microscope would appear fifty times larger than it would to the naked
eye.
Alternate method of microscopic measurement

If we do not possess a reticule and stage micrometer, we can get perfectly good results involving our
microscopic measurements utilizing another technique that we shall utilize in this activity.
Procedure
(a) Place a plastic millimeter ruler across the midline of the microscopic field by aligning it properly
on the stage of your microscope where the light comes through.
(b) RECORD the diameter of the widest central portion of your microscopic field in the appropriate
place on your data table. (This recording will be both in millimeters and in the smaller units more
frequently used by Biologists called microns.)
(c) Record the low power and high power diameters of the microscopic field.
(d) The diameter of the high power field may be obtained quickly once the diameter of the low power
field is known. Although you could likely determine the diameter of the high power field with
the metric ruler through our microscopes, it is not always this simple. Often the scale on the ruler
is not sufficiently visible under the poorer lighting conditions of high power, and therefore the
diameter of the high power field must be calculated by means of ratios. The magnifying power is
the multiplicative inverse of this ratio.
Record the measured/calculated diameter of the fields (scanning, low power, high power, oil
immersion).

Lab Manual
Questions
1.Compare the results obtained for the two methods of obtaining the diameters of the fields. Discuss the
relative accuracy of each method
2. Compare the following types of microscopes by detailing: the method of illumination, the
treatment of specimen to be viewed, maximum magnification, resolution, appearance of the
image and application:
(i) Stereoscopic / microscope
(ii) Compound microscope
(iii) Scanning electron microscope
(iv) Transmission electron microscope
Sample microscopic measurement calculations
Calculating the Diameter of Field of View

 Use ruler or graph paper to calculate the size of the field of view at lowest power (Scanning
objective).
o 4.5mm or 4,500µm (micrometers) at x40mag
 Size of field at higher power = Size at lower power x Magnification at lower power
Magnification at higher power
For example we can use diameter of the scanning objective lens to calculate the diameter of the
low power objective lens as shown below.
o = 4,500µm x 40
400
o =450µm at x 400 mag
Magnification of field of view

 Objective magnification (4, 10, 40, or 100) x ocular magnification (x10)
o 40 x 10 = x400mag

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Size of object
 Estimate length or width of object being view by estimating what portion of the field of
view is covered by it.
 For example if you estimate that it would take 20 cells to span the diameter of the field of
view then the size of one cell takes up 1/20th of the field of view
o Length or width = 1 x size of field of view at particular magnification

20
o Length or width = 1 x 1800µm = 90µm

20
Magnification of drawing
 Magnification of drawing = Size of drawing
Actual size of object
o Say the length of the cell was 90µm and the drawing was 21 mm
o 2.1cm = 21mm = 21,000µm
o = 21,000µm = x 233mag
90µm
Biological Diagrams

Lab Manual
Laboratory Exercise 2 MICROSCOPY-EXAMINING YEAST CELLS AND BACILLUS
In this section you will acquaint yourself with the morphological characteristics of yeast cells. Yeasts are
unicellular and are at least five times the size of bacteria. Morphologically, they may be ellipsoidal,
spherical, and in some cases cylindrical. Most yeasts reproduce asexually by budding. A bud is an
outgrowth of the parent cell that pinches off, producing a daughter cell. Yeasts are important since many
are beneficial. Examples such as Saccharomyces are used for brewing beer and baking bread and the
making of wine. Also the high vitamin content of yeast makes it a valuable food supplement. However,
some yeasts are pathogenic and are responsible for urinary tract and vaginal infections and infections of
the mouth.
Materials
Yeast culture
Bacillus culture
Methylene Blue
Water – iodine solution
Microscope slides
Microscope
Procedure
1. Prepare a wet mount of the yeast suspension by adding a loopful of yeast into a drop of
water-iodine solution on a slide
2. Prepare a second wet mount using methylene blue
3. Observe your slides under the microscope at high and low power
4. Make drawings and notes of your observations.
Part 4
Observe the prepared slides provided and making clearly labeled drawings and notes.

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Lab Manual
Laboratory Exercise 3 MICROSCOPY- ALGAE, FUNGI & PROTOZOA
In this section, you will be examining the protozoa and algae prepared slides.
The protozoa are a large and diverse group of unicellular, eukaryotic organisms. Most are free living, but some
are parasitic. Their major distinguishing characteristics are: the absence of a cell wall; their ability to move, at
least in some part of their life cycle; heterotrophic nutrition; primarily asexual means of reproduction.
Taxonomically, they are classified based on their means of locomotion. The phylum is subdivided into four
classes – Sarcodina, Mastigophora, Ciliophora and Sporozoa. There are more than 20,000 known species of
free-living protozoa.
The algae are mostly aquatic, although some are found in soil or on trees when sufficient moisture is available.
They are simple eukaryotic photoautotrophs that lack tissues, such as roots, stem and leaves of plants. Algae is
a common name that includes several phyla commonly called the brown algae, red algae, green algae, diatoms
and dinoflagellates.
In this lab we will simply become familiar with the general structural characteristics of representative protozoa
and algae.
Materials
Protozoa and algae prepared slides
Microscope
Procedure
1. Examine the slide under microscope using both low power and hi power objectives
2. Make clearly labeled drawings of your observations.
Answer the following questions on a separate sheet of paper:
1. Describe two differences that you observe between protozoa and algae?
2. Compare and contrast the bacterial cell and the yeast cell.
3. From your observations, why is protozoan not considered an animal cell? Explain.

Lab Manual
Laboratory Exercise 4 UBIQUITY OF MICROORGANISMS
Microorganisms are found throughout the environment: in the air and water; on the surface of any object such
as clothes, walls, furniture; in soil and dust; and on and in our own bodies (skin and mucous membranes).
Microbes are even found in extreme, seemingly inhospitable environments: living and thriving microbes are
common in hot springs with temperatures in excess of 95°C, in Antarctic seawater below 0°C, and in acid mine
waters whose pH is below 4. Ordinarily these ubiquitous microorganisms are of no threat to healthy humans,
and quite a few are actually beneficial. Normal flora on and in our body act as an initial defense against
invading pathogens by competing with them for nutrients and space thereby preventing them from flourishing.
In the environment, many organisms in soil play important geochemical roles in processing vital elements such
as phosphorus and nitrogen, making them available to other living things. However, there are organisms in our
environment that are harmful.
As stated above, all environmental situations, including our laboratory, are populated by a wide variety of
microorganisms, most commonly bacteria, fungi and viruses. In this lab, we will inoculate growth media from
variety of natural sources. These inoculations will also serve as the beginning of your pure culture project.
Please feel free to bring in samples from home that you wish to test for the presence of bacteria, e.g., dirt from
your yard, a telephone or remote control, a cutting board, old make-up, etc.
The survival and continued growth of microorganisms depend on an adequate supply of nutrients and a
favorable growth environment. A solution containing these nutrients is a culture medium. Culture media are
liquid, semi-solid or solid. A liquid media is called a broth. A semisolid or solid medium is obtained by
supplementing a liquid media with a solidifying agent called agar. Agar is an extract of seaweed, a complex
carbohydrate composed mainly of galactose, and has no nutritional value. Agar is an excellent solidifying
agent because it liquefies at 100 C and solidifies at 40C. A completely solid medium requires an agar
concentration of 1.5 – 1.8 %. A concentration of less than 1% results in a semisolid medium.
No one medium will permit the growth of all microorganisms, since different microorganisms have different
nutritional requirements. The type of media of media used is dependent on the microorganism being cultivated.
Media can be classified as defined, complex, selective, differential, selective-differential, or enrichment,
depending on composition and purpose.
A microorganism must also be provided with a suitable environment. Temperature and pH must be maintained
within the proper ranges; oxygen must be provided or excluded.
Different microbial species have characteristic temperature ranges over which they grow best. Organisms
associated with warm-blooded animals usually prefer temperatures close 37C, which is the approximately
body temperature of most animals. Soil microorganisms generally prefer a cooler temperature of 30 C
Organisms growing on glaciers would find room temperature (about 25C) much too warm and would grow
better in a refrigerator
Optimum pH varies among microbial species, but any particular species can grow only within its own
relatively narrow range. Most bacteria grow at pH values near neutrality.
The concentration of oxygen in the environment is very critical in determining microbial growth. Some
microorganisms cannot grow without oxygen, others cannot grow in the presence of oxygen, still others can
grow in the presence or absence of oxygen

Therefore, the organisms that will grow on your plate represent only a small portion of microorganisms present
in the environment.
First Session
Materials
Trypticase Soy Agar (TSA) plates
Sterile swabs
Sterile distilled water

Lab Manual
Procedure
Part 1
1. Obtain your plates and label them with the following information: Date, type of medium, initials of team
members (or some identifying mark),How the plate is going to be inoculated (see #2 below)
Note that petri dishes should always be labeled along the edge of the bottom of the dish (Fig. 1). Dishes
are labeled on the bottom because lids can be switched between dishes and along the edge because writing
across the entire surface of the bottom may obscure the bacteria you want to look at.

bottom

bottom

Figure 1. The proper location of Petri dish label.

2. Working in groups, inoculate your plates in the following manner. (To swab, take a sterile swab, put it in
sterile water, roll the swab across the test surface, then roll the swab across the entire surface of the plate.)
Plate 1: Remove the lid and expose the plate to the laboratory air for 30 minutes.
Plate 2: Remove the lid and expose the plate to the air outside the building for 30 minutes.
Plate 3: Remove the lid and inoculate with plant material. Soft plant material such as
grass clippings or rotting fruit can be sprinkled directly on the plate. Tougher plant material can
be chopped up and placed in a test tube containing 5 ml of sterile water. After the plant material
soaks for 30 minutes or so, swirl the water then pour about ½ of it onto the petri dish. Close the
lid and swirl the water over the plate.
Plate 4: Remove the lid and sprinkle a small amount of dirt on the plate.
Plate 5: Divide the plate into four sections using a marker or wax pencil. Inoculate each section with a
sample from your body such as hair, a swabbing from the top of your nose, from your lips, from
your forehead, armpit, back of your hand etc.
Plate 6: Inoculate the plate with a swabbing from the floor near the edge of your lab table.
Plate 7: Inoculate the plate with samples from the environment such as swabbing from a door handle,
drinking fountain, shelf, bottom of shoe, and/or anywhere else you can think of.
Plate 8: Inoculate the plate with a swabbing from the inside corners of your drawer/cabinet.
3. Invert all plates and then incubate them for 48 hours. Environmental samples will be incubated at 25°C;
samples from your body will be incubated at 37°C.
Second Session

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NOTE: Handle all plates with colonies as if they are potentially disease causing – pathogens
Examine the plates prepared in the first session, noting the following characteristics:
1. Colony morphology
Colony morphology gives important clues as to the identity of the microorganisms. Important classes of
characteristics include:
1. size
2. type of margin
3. colony elevation
4. colony texture
5. light transmission
6. colony pigmentation
2. Colony size
Colony size is dependent not just on the type of organism but also on the growth medium and the number
of colonies present on a plate (that is, colonies tend to be smaller when greater than a certain amount are
present) and it also depends on culture medium characteristics.
3. Type of margin
Colonies can vary in the shape of their margins. See illustration below.
Illustration of the variation in colony margins
4. Colony elevation
Colonies can vary in their elevations. See illustration below.
Illustration, variations in colony elevation
5. Colony texture / Surface appearance:

1. Colonies can vary in their texture. Possible textures include: shiny to dull
2. smooth to wrinkled
3. rough
4. granular
5. mucoid
A shiny, smooth, and/or mucoid appearance tends to be associated with the presence of capsular material.

Lab Manual
6. Colony light transmission
The light transmission through colonies can range from:
i. complete (transparent)
ii. through intermediate (translucent)
iii. through completely lacking (opaque)
7. Colony pigmentation
Colonies can come in a variety of colours.
Record your results in a tabular format. For each sample, include the inoculum source, number of colony
types, number of colonies and a brief description of the colonies, including such features as relative size, color,
surface texture, and shape, etc.
Questions
1. List three examples of bacteria commonly found in:

a) the air
b) the soil
c) untreated water
For each example, state whether these microorganisms are pathogenic or non pathogenic
2. If sampling was done from a non sterile site but no bacterial colonies were observed, offer an
explanation
3. List the scientific names of two parasitic protozoans, detailing the symptoms and name of the diseases
they cause.

Lab Manual
Laboratory Exercise 5 ASEPTIC TECHNIQUES - TRANSFER OF BACTERIAL
CULTURES
Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile
laboratory equipment. In the laboratory, bacteria must be cultured in order to allow identification as well as to
examine their growth and metabolism. In order to do this, bacteria are introduced to into culture media with
the use of aseptic techniques to avoid contamination.
All culture media are sterilized prior to use. One of the ways this is achieved is with the use of an autoclave.
This allows for the elimination of bacterial and fungal contaminants in the media due to high heat and high
pressure (121 °C at 105kPa/15 psi). Fungal spores may survive if only heat is used. Certain media components
are susceptible to heat denaturation and therefore must be added to the media after autoclaving. To do so, one
must filter the components using a 0.22µm pore size filter that is appropriate to the solvent used.
Microbiological Transfer
Instruments
In microbiology, it is often
necessary to sterilize other
apparatus as well, to ensure
sterile conditions are
maintained. Glassware, such
as petri dishes, test tubes and
flasks can be sterilized in an
oven by placing them at 200
°C for 1-4 hours. The
glassware is covered with a
cap, cover or aluminum foil
to maintain aseptic conditions
after removing the glassware
from the oven. Test tubes,
petri dishes and flasks containing culture media are not opened until they are needed and even then, they are
not left open for extended periods of time.
Certain techniques are necessary to handle tubes of media, plated media, and inoculating loops or swabs.
Practice in the manipulations, while maintaining aseptic conditions are known as "aseptic techniques."
Transfer and inoculation of bacterial cultures are usually performed with a sterile, heat resistant, non-corroding
nichrome wire attached to a handle. When the end of the wire is into a loop it is called an inoculating loop,

Lab Manual
when straight, it id called an inoculating needle. Cultures may also be transferred with sterile cotton swabs,
pipettes, glass rods or syringes.
In this laboratory exercise, sterile inoculating loops and pipettes will be used along with aseptic techniques to
transfer bacterial cultures.
First Session
I Broth to Broth Transfer
Sterile loops are used for the transfer of small amounts (unquantified) of culture.
In this exercise, you will be transferring sterile broth from one tube to another
Materials
Nutrient Broth
Inoculating Loop
Bunsen Burner
Procedure
REFER TO THE DIAGRAM

1. Label the first tube A, and the second tube B and a third tube C and a fourth tube D
2. Hold the inoculating loop as you would a pencil, and flame the wire portion until it is red hot.
Hold it an angle in order not to burn your hand
3. After the loop has cooled for a few seconds, pick up tube A in the other hand and remove the cap
of the tube with your little finger of the hand holding the loop.
4. Flame the mouth of the tube by passing it through the Bunsen burner flame. Use the sterile loop to
obtain a loopful of liquid from the tube. Flame the tube and replace the cap.
5. Put back the first tube and pick up the second tube B. Remove the cap and flame the top of the
tube and deposit the loopful of material from tube A into tube B, flame the mouth of the tube and
replace the cap.
6. Flame the loop before setting it down.
7. After the loop has cooled for a few seconds, pick up tube C in the other hand and remove the cap
of the tube with your little finger of the hand holding the loop.
8. Flame the mouth of the tube by passing it through the Bunsen burner flame. Use the sterile loop to
obtain a loopful of liquid from the tube. Flame the tube and replace the cap.
9. Put back the first tube and pick up the fourth tube D. Remove the cap and flame the top of the
tube and deposit the loopful of material from tube C into tube D, flame the mouth of the tube and
replace the cap.
10. Flame the loop before setting it down

Lab Manual
11. The tubes will be incubated at 37ºC
Sub
culturing Procedure

Lab Manual
II Transferring Broth with a pipette
Sterile pipettes are used when it is necessary to transfer known amounts of material (sterile water, media or
culture). Sterile pipettes may be made of glass or plastic – disposable, and are individually packaged. They
may also be made of reusable glass. Substances are drawn into the pipettes with any of a variety of bulbs or
filling devices. Mouth pipetting in VERY dangerous and should never be done.
Materials
Nutrient Broth
Sterile 1 ml pipette
Pipette bulb
Procedure
1. Open a sterile pipette at the top. Insert a bulb on the end, and then carefully remove the pipette
from the package without touching the tip.
2. Pick up tube A with your other hand and remove the cap with your little finger of the hand
holding the pipette. Flame the mouth of the tube and inset the pipette into the liquid. Draw the
liquid up to the 1 ml mark, remove the pipette, flame the tube, re-cover
3. Pick up the second tube and repeat the steps used with the first tube except that the liquid should
be expelled into the second tube
4. Dispose of the pipettes
5. The tubes will be incubated at 37ºC
III Streak plate technique

Streaking is a method of applying cultures to solid medium. A sterile loop is cooled and brought into contact
with a culture. The loop is then brought into contact with the surface of solid medium whereupon it is streaked
(i.e., dragged) along the surface of the solid medium. Colonies grow along the points of the streak
Materials
TSA or NA plates
Broth culture of Staphylococcus or E. coli
Inoculating Loop
Procedure
Refer to the illustration below
1. Label two agar plates on the bottom with your name and date
2. Sterilize the loop by heating it in flame until red hot
3. Gently shake the bacterial culture to ensure that the organisms are well suspended. Aseptically remove
a loopful of culture and spread the bacteria on the plate in one corner. Be careful not to press the loop
into the surface - use a gliding motion
4. Flame the loop again to burn off the bacteria. Allow the loop to cool
5. WITHOUT going back into the broth culture, streak the plate again, by partially going into the first
streak. Make a few closely placed streaks
6. Repeat above, making a third set of streaks by partially going into the second set of streaks - turning
the plate each time until the entire plate is covered
7. Repeat the entire process on a new plate
8. The plates will be incubated at 37ºC overnight.

Lab Manual
Illustration of streaking a plate
Keep petri dishes covered as much as possible. If top must be removed completely do not LAY IT on the lab
top. This lowers the probability of contamination and prevents “false positive” results. It is important to use a
new sterile loop for each different sample taken or removed because once a sterile loop is used it is no longer
sterile.
Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to
cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and
overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying
the bench top down with a commercial disinfectant, 70% ethanol or a 10% bleach solution and allowing this to
stand for a minute. You may then wipe down the bench with the paper towel.
Second Session
1. Observe the tubes of broth for signs of turbidity and record your results
2. Observe your plates and record your results

Lab Manual
Questions
1. What is a pure culture?
2. (a) What is one media used for the culturing of fungi? What are the ingredients of this media
which makes it specific for the growth of fungi
(b) What are the standard incubation conditions for fungi?
3. If you had a contaminating culture on your plate, detail the steps you would take to purify the
culture

Lab Manual
Laboratory Exercise 6 ISOLATION OF PURE CULTURES
In nature, microbial populations do not segregate themselves by species but exist in a mixture of many other
cell types. In the laboratory, these populations can be separated into pure cultures. These cultures contain
only one type of organism and are suitable for study of their cultural, morphological and biochemical
properties.
Microorganisms are transferred from one media to another by subculturing. This technique is of great
importance in microbiology.
In this experiment, you will use the streak plate technique designed to produce discrete colonies from one of
the plates you obtained during exercise 1. The streak plate method is a rapid qualitative isolation method. It is
essentially a dilution technique. Once you have obtained these discrete colonies, you will then transfer a few of
these colonies to new agar plates sequentially, at least three times, to ensure that you do indeed have pure
cultures. Subsequently, morphological and a few chemical tests will be performed on the isolated organism
Therefore this exercise will be ongoing throughout the entire lab series
Part 1
Materials
TSA plates with microorganisms (Experiment 1)
TSA plates – uninoculated
Inoculating loop
Bunsen Burner
Procedure
1. Using the plates obtained from Experiment 1, Choose one colony, record its characteristics
2. Using a sterile inoculating loop, pick up the colony and transfer it to a new agar plate by using the
streaking method practiced in Exercise 2.
3. The plates will be incubated at the appropriate temperature
Part 2
Procedure
Repeat the above procedure in the next lab session
Part 3
Procedure
Repeat the above procedure in the next lab session
At the end of this series of subculturing you may assume you have a pure culture if all the colonies on the plate
have the same morphological characteristics.
If necessary repeat the subculturing of your isolate once more
The characteristics of the isolated, pure culture thus obtained will be studied using the following tests: (refer to
experiments and appendices for the protocols)
1. Gram Stain
2. Carbohydrate metabolism
3. Starch hydrolysis
4. Urease
5. Catalase
6. Oxidase
7. Methyl Red Test
8. TSI

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Lab Manual
Laboratory Exercise 7 & 8 MULTIPLE AND DIFFERENTIAL STAINS
Simple staining of bacterial cells (previous lab) does not provide much information to aid in the identification
of bacterial cells. In addition, all cells, both prokaryotic and eukaryotic will stain with the dyes used in simple
staining. The Gram staining method, named after the Danish bacteriologist who originally devised it, Hans
Christian Gram, is one of the most important staining techniques in microbiology. Essentially, Dr. Gram found
that bacteria fell into two distinct categories when stained sequentially with crystal violet followed in sequence
by iodine solution, a wash with a de-staining agent and a counter-stain. One group of cells resisted the removal
of the crystal violet when washed with ethanol or acetone (decolorizing agents), whereas the second group was
readily decolorized by a brief rinse with these reagents. To visualize the decolorized cells Gram briefly
exposed them to a counter-stain or a stain of a different color from the crystal violet. Gram settled on the red
counter staining dye safranin. Thus cells which resisted decolorization remained deep purple or blue and came
to be referred to Gram-positive cells, whereas cells that easily lost the crystal violet dye were red after counter
staining. These red cells came to be called Gram-negative cells.
This type of stain is referred to as a differential stain because one group of organisms reacts to the stain one
way and another group of organisms reacts a different way. We now know that Gram-positive bacterial cells
are very different from Gram-negative cells. The gram characteristic is almost as fundamental to a bacterial
description as its morphology.
The ability of the Gram stain to differentiate microorganisms into two broad categories is a result of
differences in the cell walls of the organisms. The cell walls for gram-negative microorganisms have higher
lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Iodine
is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be
removed too easily. This step is commonly referred to as fixing the dye. The actual mechanism of de-
colourization is currently not well understood and remains controversial. However, it is generally agreed that
the subsequent treatment with the decolourizer, which is a mixed solvent of ethanol and acetone, dissolves the
lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary
stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker gram-positive
cell wall, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the stain-
iodine complex out from the cell is obstructed, and the cells remain stained.
HOW THE GRAM STAIN WORKS:

Color of Gram Color of Gram
Stain Step Gram reagent What Happens Dyes Present in Cell
Positive Cells Negative Cells
Primary Crystal violet Dye enters cell Purple Purple Crystal violet
Crystal violet-iodine (CV-I)
Mordant Gram's Iodine Purple Purple CV-I
complex formed in cells
Gram positive wall shrinks
95% alcohol or Gram positive: CV-I
Decolorizer Lipids removed from Gram Purple Colorless
acetone Gram negative: none
negative walls
Gram positive:
Purple
all dyes
Counterstain Safranin Dye enters cell (the pink of the Pink
Gram negative:
counterstain is hidden)
safranin
Performing a good gram stain is easy, but it does require some experience. For example, the original "bacterial
smear" must not be too thick or too thin, as this will influence penetration of the staining reagents.

Lab Manual
Also the age of the culture is important. Very young and very old cells often produce poor results, whereas
mid-log cells that are healthy and growing optimally tend to give dependable results. The media the cells are
grown in and the environmental conditions may also affect the outcome of a gram stain because these
ultimately reflect on the chemical nature of the cell.
While the majority of bacteria are stainable by either simple or Gram Stain technique, a few, especially those
of the genera Mycobacterium are resistant and can only be visualized by the acid fast method. This is due to
the presence of a thick waxy wall that makes the penetration of stains very difficult. However, once the stain
has penetrated it cannot be removed even with the use of acid alcohol. Because of this property, these bacteria
are referred to as acid fast. The acid-fast stain uses three different reagents – carbol fuschin, acid alcohol and
methylene blue
Structural stains can be used to identify and study the fine structure of bacteria. Currently, most of the
structural details are examined using an electron microscope, but historically, staining techniques have given
much insight into bacterial fine structure. A few structural stains are still useful today. These stains are used
to observe endospores, capsules, and flagella.
Members of the anaerobic genus Clostridium and the aerobic genus Bacillus can exist either in the vegetative
state or as the highly resistant, inactive cells called spores. When the environment is unfavorable for
vegetative activities, these cells undergo sporogenesis, resulting in the formation of endospores. As the
condition worsens the endospores are released from the degenerating vegetative cell and are then referred to as
spores. Unlike most vegetative cells, the spores’ impervious coats will not accept a primary stain easily.
Therefore, heat is required in the first step of Spore staining. The primary stain is malachite green and the
counterstain is safranin.
Life Cycle of spore-forming bacteria

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Many bacteria secrete chemicals that adhere to their surface, forming a viscous coat. This structure is called a
capsule when it is round or oval in shape, it is called a slime layer when it is irregular in shape. Because of the
capsule’s non-ionic nature, simple stains will not adhere to it. The capsule staining technique stain the bacteria
and the background leaving the capsule unstained – a “negative” stain.
Flagella, the most common means of motility, are thin proteinaceous structures that originate in the cytoplasm
and project out from the cell wall. They are very fragile and are not visible with a light microscope. They can
be stained after carefully coating them using a mordant, which increases their diameter. The presence and
location of flagella are helpful in the identification and classification of bacteria.
Materials
Crystal Violet
Gram’s Iodine
95% alcohol
Safranin
Malachite green
Bunsen burner
Overnight cultures of E coli, Bacillus subtilis, Staphylococcus
Procedure
GRAM STAIN
1. Prepare a heat fixed smear of the culture you wish to examine
2. Flood the smear with crystal violet (30 sec. to 2 min)
3. Quickly and gently wash off excess stain (2 seconds)
4. Flood the smear with Grams iodine (1 minute)
5. Decolourize with alcohol (10-20 seconds or until the excess alcohol which flow off
the slide is colorless)
7. Flood the smear with safranin (30 sec to 2 min.)
9. Blot dry with paper
10. Examine your slide under the microscope using the oil immersion lens. Record your
observations with clearly labelled drawings noting the organisms, size, colour,
morphology, and culture identification.
ENDOSPORE STAIN
1. Make a smear of a Bacillus culture, air dry and heat fix
2. Tear a small piece of paper towel and place it over the smear to prevent the
evaporation of the stain. Ensure that the paper is smaller than the slide
3. Flood the smear and paper with malachite green; steam for 5 minutes. Add more stain as
needed – KEEP THE SLIDE WET
4. Remove the paper towel and discard carefully. DO NOT PUT IN THE SINK
5. Wash the stained smears well with distilled water
6. Counterstain with safranin for about 30 seconds.
7. Wash with distilled water and blot dry
8. Examine under the microscope and record your observations

Lab Manual
Gram staining procedure
Questions
1. State why the gram stain is said to be a differential stain
2. What step(s) of the gram stain procedure could be omitted without losing the ability to
distinguish between gram positive and gram negative? Explain
3. Describe the conditions that may result in a gram-positive cell staining gram negative.

Lab Manual
Laboratory Exercise 9 DEFINED, UNDEFINED, SELECTIVE AND
DIFFERENTIAL MEDIA
Microbiologists have developed several different types of media for a variety of purposes
General-purpose media are designed to grow most organisms and do not contain growth inhibitors. Standard
Methods Agar and Blood Agar Bases are examples of general-purpose media.
Differential Media contain a component that allows an observable change when a specific chemical reaction
takes place. Simmons Citrate Agar is an example of a differential medium. In Simmons Citrate Agar there is a
pH indicator that turns from green to blue when citrate is utilized as the sole carbon source.
Selective media encourage the growth of some organisms and suppress the growth of others. Dyes,
antimicrobials, and salts are all examples of selective agents used for this purpose. Bile Salts are used to inhibit
the growth of Gram-positive organisms on MacConkey Agar.
A medium can be both selective and differential depending on the formula. Hektoen Enteric Agar is an
example of a selective and differential medium. Hektoen Enteric Agar contains Bile Salts, the selective agent
used to suppress Gram-positive organisms, and pH indicators, Acid Fuchsin, and Bromthymol Blue.
Enrichment media contain nutrients that encourage the growth of organisms. Media containing enrichments
can be selective or general purpose. Universal Pre-enrichment Broth is a general-purpose enrichment broth for
Salmonella and Listeria, and is particularly successful at resuscitating low numbers of injured cells.
Chemically defined media are made of specific types and amounts of pure chemicals. Tissue culture media
are examples of chemically defined basal formulations.
Agar is the gelling agent most commonly used in culture media. The term “agar” frequently refers to a solid
surface, plated medium. Plated media are used for isolating pure colonies or counting numbers of colonies.
Organism identification is possible after obtaining pure colonies. Agar containing media that have solidified in
tubes in a slanted position are referred to as slants. Triple Sugar Iron Agar is an example of a medium that is
prepared in slants and used for organism identification.
Broths are referred to as any liquid culture medium. Tryptic Soy Broth is an example of a widely used broth
formula.
Semisolid media are between a solid and a liquid state. Agar added in low concentrations achieve fluidity but
not at a concentration that will result in a firm gel. Motility Test Medium is an example of a semisolid
medium. Bacterial motility may be observed macroscopically as a diffuse zone of growth spreading from the
line of inoculation.
In this exercise, the growth of microorganisms will be compared on defined, rich, selective and differential
media. You will be using Glucose salts agar (GSA) a simple defined media; trypticase soy agar (TSA) a rich,
undefined media and esoin methylene blue (EMB) a selective, differential medium
First session
Materials
TSA plates
GSA plates
EMB plates
Inoculating loop
Bunsen burner
Cultures – Staphylococcus, Bacillus, Pseudomonas, E coli
Procedure
1. Divide the bottom of three agar plates into thirds. Label each plate with the names of the three
microorganisms being used
2. Using aseptic technique inoculate each third of each plate with a loopful of each microorganism
3. Invert the plates. They will be incubated at 37ºC for 48 hrs

Lab Manual
Second Session
1. Observe and compare the growth on the three plates
2. Record your results in a tabular form indicating the amount of growth, per microorganism for
each type of media, using the following:
- no growth lac- lactose fermentation on EMB
+ slight growth lac + no lactose fermentation on EMB
++ good growth
+++ excellent growth
Questions
1. Why are carbon and nitrogen macronutrients while cobalt is a micronutrient?
2. Why is the following not considered a chemically defined medium: glucose (5 grams), NH 4Cl
(1g), KH2PO4 (1g), MgSO4 (0.3g), yeast extract (5g), distilled water 1L
3. If you have a liquid culture containing three different species of bacteria, how would you
generate pure cultures of each organism?

Lab Manual
Laboratory Exercise 10 FACTORS AFFECTING MICROBIAL GROWTH –
PHYSICAL FACTORS
a. Temperature
Bacteria have a minimum, optimum, and maximum temperature for growth and can be divided into 3 groups
based on their optimum growth temperature: Psychrophiles are cold-loving bacteria. Their optimum growth
temperature is between -5C and 15C. They are usually found in the Arctic and Antarctic regions and in
streams fed by glaciers. Mesophiles are bacteria that grow best at moderate temperatures. Their optimum
growth temperature is between 25C and 45C. Most bacteria are mesophilic and include common soil bacteria
and bacteria that live in and on the body. Thermophiles are heat-loving bacteria. Their optimum growth
temperature is between 45C and 70C and are commonly found in hot springs and in compost heaps. Another
group of bacteria, Hyperthermophiles are bacteria that grow at very high temperatures. Their optimum
growth temperature is between 70C and 110C. They are usually members of the Archaea and are found
growing near hydrothermal vents at great depths in the ocean.
Materials
TSA plate
Cultures of E. coli, Pseudomonas, Streptococcus, Bacillus
Inoculating loop
Bunsen burner
Procedure
First Session
1. Streak each culture provided onto a TSA plate.
2. Repeat until four such plates are prepared for each culture
3. Label the petri dishes with the name of the microorganism and the temperature of incubation as
follows: 4ºC, 20ºC, 37ºC, 55ºC
4. The inoculated plates will be incubated at the appropriate temperatures
Second Session
1. Record the size of the isolated colonies at each of the temperatures fro each microorganism, as an
indication of growth rate. Be sure to note the temperature that produces the largest colony for each
microorganism
2. Represent your data in a tabular form
Questions
1. What metabolic and structural adaptations for extreme temperatures have psychrophiles and
thermophiles made?
2. How does filtration control microorganisms? Does filtration sterilize? Explain
b. Oxygen requirements
The presence or absence of molecular oxygen can be very important to the growth of bacteria. They show a
great deal of variation in their requirements for gaseous oxygen. Most can be placed in one of the following
groups: Obligate aerobes are organisms that grow only in the presence of oxygen. They obtain their energy
through aerobic respiration. Microaerophiles are organisms that require a low concentration of oxygen (2% to
10%) for growth, but higher concentrations are inhibitory. They obtain their energy through aerobic
respiration. Obligate anaerobes are organisms that grow only in the absence of oxygen and, in fact, are often

Lab Manual
inhibited or killed by its presence. They obtain their energy through anaerobic respiration or fermentation.
Aerotolerant anaerobes, like obligate anaerobes, cannot use oxygen to transform energy but can grow in its
presence. They obtain energy only by fermentation and are known as obligate fermenters. Facultative
anaerobes are organisms that grow with or without oxygen, but generally better with oxygen. They obtain
their energy through aerobic respiration if oxygen is present, but use fermentation or anaerobic respiration if it
is absent. Most bacteria are facultative anaerobes.
Materials
TSB cultures of E coli, Pseudomonas, Clostridium
TS +5% glucose tubes of agar
TSA slants
Inoculating needle
Bunsen burner
Boiling water bath
Procedure
First Session
1. Place the tubes of agar into a beaker filled with water to the height of the agar. Boil the tubes for
10 minutes. This will melt the agar as well as drive out all the dissolved oxygen. After the agar
hardens, air will gradually diffuse into the tube so that the top half inch of the agar will be aerobic,
but the rest of the tube will be anaerobic
2. Cool the agar in a 50ºC water bath for about 10 minutes. Be careful that the agar does not cool
much lower than 50 ºC or it will solidify. The tube should feel hot but you should be able to hold
it
3. Label the tubes and slants
4. Inoculate the melted agar with one culture with a loopful of culture into the agar and mix by
rolling between the hands. Allow the agar to harden
5. Inoculate a slant by placing a loopful of broth on the bottom of the slant and making a
wiggly/wavy line on the surface to the top of the agar
6. Repeat with the two remaining cultures
7. The slants and agar deeps will be inoculated at 25ºC for at least 48 hrs
Second Session
Observe the surface of the slants and record the growth. Observe the deeps and compare to the diagram below:

Lab Manual
Procedure for determination of oxygen requirements
Questions
1. If two cultures of E. coli were growing in media of the same composition, one exposed to air and
one anaerobically, which would have the larger biomass when growth ceased? Why?

Lab Manual
2. (a) Outline the metabolic process which allows anaerobes to obtain energy for growth
(b) Why is oxygen toxic to obligate anaerobes?

c. pH
Microorganisms can be placed in one of the following groups based on their optimum pH requirements:
Neutrophiles grow best at a pH range of 5 to 8. Acidophiles grow best at a pH below 5.5. Alkaliphiles grow
best at a pH above 8.5.
Materials
Tubes containing nutrient broth adjusted to pH 2.5, 5.0, 7.0, 9.5
Cultures of Staphylococcus aureus, E. coli, Serratia marcescens, Saccharomyces
Inoculating loop
Bunsen burner
Procedure
First Session
1. Inoculate a loopful of each organism into one of each pH test broth
2. Incubate at 35ºC for at least 48 hours
Second Session
Record the growth observed fro each pH test broth in a tabular form by using the following:
- clear
+ slightly turbid
++ moderately turbid
+++ very turbid
Questions
1. Besides being able to tolerate a range of pH, some bacteria change their environment by
producing acid or alkali substances. How would you use microbiological media to determine a
change in the pH of culture media due to the metabolic activities of bacteria?
d. Osmotic pressure
Osmosis is the movement of water across a membrane from an area of higher water concentration (lower
solute concentration) to lower water concentration (higher solute concentration). No energy is required. To
understand osmosis, one must understand what is meant by a solution. A solution consists of a solute dissolved
in a solvent. In terms of osmosis, solute refers to all the molecules or ions dissolved in the water (the solvent).
When a solute such as sugar dissolves in water, it forms weak hydrogen bonds with water molecules. While
free, unbound water molecules are small enough to pass through membrane pores, water molecules bound to
solute are not. Therefore, the higher the solute concentration, the lower the concentration of free water
molecules capable of passing through the membrane.

Lab Manual
A cell may be in one of three environments: isotonic, hypertonic, or hypotonic. (The prefixes iso-, hyper-, and
hypo- refer to the solute concentration). In an isotonic environment, both the water and solute concentration
are the same inside and outside the cell and water goes into and out of the cell at an equal rate. If the
environment is hypertonic, the water concentration is greater inside the cell while the solute concentration is
higher outside. Water goes out of the cell. In an environment that is hypotonic, the water concentration is
greater outside the cell and the solute concentration is higher inside. Water goes into the cell.
Most bacteria require an isotonic environment or a hypotonic environment for optimum growth. Organisms
that can grow at relatively high salt concentration (up to 10%) are said to be osmotolerant. Those that require
relatively high salt concentrations for growth like some of the Archea that require sodium chloride
concentrations of 20 % or higher are called halophiles.
Osmotic conditions
Materials
Petri dishes containing Nutrient Agar +5%, 10%, 15% sucrose
OR
Petri dishes containing Nutrient Agar +5%, 10%, 15% sodium chloride
Nutrient Agar
Inoculating loop
Bunsen burner
Broth cultures of E coli, Staphylococcus aureus, Saccharomyces, Bacillus
Procedure
First Session

Lab Manual
1. Use a complete set of nutrient agar plus salt (or sucrose) plates. Divide the plate into quadrants
2. Inoculate each quadrant with one microorganism, and appropriately labeled
3. Inoculate one nutrient agar plate in a similar manner as a control.
4. Incubate at 35ºC for 24- 48 hrs
Second Session
Record the relative amounts of bacterial growth. The culture having the best growth is given a ++++ others
are evaluated relative to that result.
Questions
Most bacteria require an isotonic environment or a hypotonic environment for optimum growth. Explain
how a bacterial cell would compensate from a hypotonic environment and explain why it is easier to
compensate for a hypotonic environment in contrast to a hypertonic environment.
e. Carbon dioxide
Carbondioxide is essential for autotrophic organisms, but it is also used by many heterotrophic
bacteria as a single carbon-building block, and some species of Neisseria and Streptococcus
grow better in an atmosphere enriched with about 5% carbon dioxide. This is achieved by the
use of the candle jar. A lighted candle and plates are sealed in the candle jar and incubated. The
candle flickers out when oxygen is reduced from about 20% to about 16% in the atmosphere and
the carbon dioxide increased from about 0.4% to 4%. Although this is not an anaerobic culture
method, it encourages the growth of microaerophillic organisms and of organisms requiring
carbon dioxide.
Materials:
Candle jar
4 TSA plates
Streptococcus
Procedure – First Session
Carbon dioxide
1. Inoculate two TSA plates with Streptococcus
2. For each organism, label one plate “aerobic” and one plate “candle jar”.
3. Place appropriately labeled plates in candle jar and incubator at 37ºC for 3 days.
Second Session
Carbon dioxide
1. Examine the two cultures for each species.

Lab Manual
2. Compare the size and appearance of the colonies incubated under different atmospheric
conditions.
3. Record differences.

Lab Manual
Laboratory Exercise 11 MICROBIAL METABOLISM
The term metabolism refers to the sum of the biochemical reactions required for energy generation and the use
of energy to synthesize cell material from small molecules in the environment. Hence, metabolism has an
energy-generating component, called catabolism, and an energy-consuming, biosynthetic component, called
anabolism. Catabolic reactions or sequences produce energy as ATP, which can be utilized in anabolic
reactions to build cell material.
Heterotrophy (i.e. chemoheterotrophy) is the use of an organic compound as a source of carbon and energy. It
is the complete metabolism package. The cell oxidizes organic molecules in order to produce energy
(catabolism) and then uses the energy to synthesize cellular material from these the organic molecules
(anabolism). Many Bacteria (but just a few Archaea) are heterotrophs, particularly those that live in
associations with animals.
Bacteria, like most cells, use glucose and glucose derivatives for cellular respiration. But, most environments
do not have a large supply of free glucose. To cope with this, most bacteria have metabolic pathways that
allow them to use other materials as sources of energy. Since bacteria are not capable of phagocytosis, they
must break down large food particles outside of the cell. Bacteria accomplish this by the use of exoenzymes.
Exoenzymes are proteinaceous catalysts that bacteria secrete into the environment. Then the bacteria take up
the products of the enzyme-catalyzed reaction by passive diffusion or active transport. In this exercise, we will
look for the presence of three exoenzymes, gelatinase, caseinase and amylase, in several species of bacteria.
Gelatinase is a protease. Proteases hydrolyze the peptide bonds of proteins, producing short polypeptides and
free amino acids. As its name implies, gelatinase degrades gelatin. Gelatin is an incomplete protein; it lacks
tryptophan. However, the ability to hydrolyze gelatin is a well established bacterial classification
characteristic. Normally, gelatin produces a gel in water below 25°C. When gelatin is hydrolyzed, it loses its
ability to form a gel. In this experiment, you will use nutrient gelatin in place of nutrient agar. If a given
microbe produces gelatinase, the nutrient gelatin will liquefy.
Caseinase hydrolyzes the protein, casein. Casein is the primary protein in milk, and it gives milk its opaque
white color. Inoculating bacteria into milk agar or milk broth tests the presence of caseinase. Clear zones in the
medium indicate the presence of caseinase or other proteases.
Amylase hydrolyzes starch. Starch is a polysaccharide-a long chain of glucose molecules linked by glycosidic
bonds. Amylase breaks the glycosidic bonds, producing small oligosaccharides and free glucose. Culturing
organisms on starch agar is a test for amylase production. After incubation, the starch agar is flooded with
Gram's iodine. The iodine reacts with starch to produce a dark purple or brown color. If amylase is present,
clear zones will appear in the starch agar where hydrolysis has occurred.
Materials
Starch agar plate

Skim milk tubes
Nutrient gelatin deeps
Gram's iodine.
Cultures of E. coli, Bacillus subtilis, Pseudomonas, Streptococcus
Procedure
First Session

Lab Manual
1. Divide a starch agar plate into quadrants. Label these for the four bacterial species listed
above. (Remember to label plates on the bottom).
2. Using the inoculating loop and proper sterile technique, make a single streak of each
bacterium on the agar corresponding to the lines drawn on the petri dish.
3. Incubate the plates upside-down at 37°C for 24-48 hours.
4. Using a needle, inoculate each of the above bacteria into separate nutrient gelatin deeps.
5. Using a loop, inoculate each of the above bacteria into separate skim milk broth tubes.
6. Incubate the deeps and broths at 37°C for 24-48 hours.
7. After incubation, place the deeps in the refrigerator for at least three hours before examining
for gelatinase activity. Tubes that are still liquid after three hours are positive for gelatinase.
Session 2
1. Examine the tubes for gelatinase activity. Tubes that are still liquid after three hours are
positive for gelatinase
2. Without shaking or otherwise disturbing them, examine the skim milk tubes. Clear areas at
the top of the broth indicate the presence of caseinase.
3. Flood the starch agar plate with IKI solution or Gram's iodine. Wait for one or two minutes.
Clear zones around colonies indicate starch hydrolysis, and the presence of amylase.
4. Record your results in a tabular form

Lab Manual
Laboratory Exercise 12 QUANTIFICATION OF MICROORGANISMS
The determination of the number of cells or concentration of cells in a sample plays numerous and important
roles in microbiological characterization and experimentation. The methods of cell quantification can be
divided between direct and indirect (estimation) counts Direct count is the actual counting of microorganisms
by methods in which a reasonably statistically precise measurement of cell quantification is achieved. The
basis of a direct count is the actual counting of every organism (or every living organism-often limited to a
single type of microorganism) present in a sub-sample of a population. Direct counts include: plate counts (a
viable count), most probable number method (a viable count) and direct microscopic count (a total count). A
viable count is a direct counting method in which only viable cells are counted. Viable counts can be
accomplished by such techniques as pour plating, spread plating or most probable number method. Total
count is a direct counting method in which all cells are counted, whether dead or alive. Generally taking total
counts requires the employment of microscopes.
Estimation of cell numbers uses various correlations of cell number rather than direct counting. Such
methods are often preferable either for convenience or because direct counting is difficult or even impossible
in many situations (for example, when quantifying filamentous organisms). Estimates of cell number include
determinations of turbidity, metabolic activity, or dry mass
Plate counts (pour plate, spread plate) Colonies grown in petri dishes by various methods (not including
streaking) may be used to determine the count of viable microorganisms. Plate counts assume that a single cell
forms every colony. It is clear that the cell must have been alive in order to grow and form a colony.
Problems with plate counts are that they require lengthy incubation for colonies to become visible; cell
clumping can lead to an undercount of viable cells; it is very easy to have too many or too few colonies on a
plate to accurately measure viable count; prevention of crowding often requires serial dilution. Also too few
cells requires concentrating, e.g., by centrifugation or filtration . It should be noted that depending on your
organism, and other circumstances, you will want to have no more than 300 to 500 colonies per plate (e.g., for
big colonies 300 will be many, for small colonies 500 may still work). Typically one uses a minimum number
which is 10-fold smaller than the maximum number, but greater than 30.
Filtration allows for the Concentrating cells. If too few colonies are present then the original culture must be
concentrated prior to determining its plate count. Filtration, which sieves microorganisms out of the medium,
is one method of concentrating microorganisms.
Most probable number method [MPN] is a “No Plate” viable count. The most probable number method is a
way of determining approximate viable count by diluting cultures then growing the dilution cultures in broth
tubes. The concentration of the culture is then taken to be equal to the amount of dilution necessary to have
reached this point. MPN is especially useful in situations where there is an advantage to using broth over solid
medium.
For example, many organisms are not good at forming colonies, such as highly motile organisms. Also, when
samples contain too few organisms to give reliable measures of population size by the standard plate count
method, as in food and water sanitation studies, or when organisms will not grow on agar, the most probable
number method is used.
Direct microscopic count is a determination of the number of microorganisms found within a demarcated
region of a slide known to hold a certain volume of culture. This total count method of cell quantification is
very rapid but has the problem of requiring high cell concentrations (e.g., 10 7 / ml) as well as potentially
counting dead and living cells with equal probability.
Turbidity is the cloudiness of a culture and is caused by the individual cells scattering light. The degree of
turbidity is a direct correlate of cell mass. The average cell mass of individual cells in a culture must first be
determined if the turbidity of a culture is to be used to estimate cell count. This standardization usually works
best for cultures growing in exponential phase.

Lab Manual
The Metabolic activity - output or input of a culture may be used to estimate viable count. The rationale is that
the rate at which metabolic products such as gases and/or acids are formed by culture reflects the mass of
bacteria present, as well as the rate at which a substrate such as glucose or oxygen is used up also reflects cell
mass.
Determination of dry mass has all of the problems of turbidity and metabolic activity with the added problem
of having to dry cultures before employing it. In order to calculate the dry weight of cells, they must be
separated from the medium by some physical means such as filtration or centrifugation. The cells are then
dried, and the resulting mass is weighed. For filamentous organisms this method works sufficiently well
compared to the other methods listed that dry mass determination is employed.
In this lab we will be using the Serial dilution method, followed by pour and spread plate techniques to
quantify a bacterial culture.
Since you are not sure of the sample concentration, to be on the safe side, you choose to use dilution factors of
105, 106, and 107. You do this by removing 0.1 ml of sample and diluting it into 9.9 ml of buffer to obtain a
100-fold dilution. You make sure you mix this diluted sample well with the buffer and then remove 0.1 ml and
mix that with a second 9.9 ml buffer. This gives you a second 100-fold dilution or 100 x 100 = 10 4-fold overall
dilution. You repeat this a third time to achieve a 100 x 100 x 100 = 106-fold overall dilution
You plate 0.1 ml from the 10 4-fold dilution (second dilution) to achieve a 10 5-fold overall dilution. You then
plate 1.0 ml from the 106-fold dilution, and finally 0.1 from the same dilution to give you your 10 6 and 107
overall dilution factors, respectively. The plates are incubated and colony counts taken.
Ideally, of course, you should do the entire dilution series and plating in duplicate or triplicate to be more
confident that you haven't introduced any serious errors.
The concentration of organism in the original sample, therefore, is equal to the number of colonies counted
multiplied by the dilution factor. This is your viable count, i.e., the number of living organisms you estimate to
be in the original sample.
There are ways of minimizing serial dilution errors:
I Errors in technique:
Obvious errors in technique while serial diluting can include:
1. improperly measuring diluent and volumes to be diluted
2. potential for some volume carry over on the outside of pipettes
3. dilutions may not be fully mixed prior to your removing volumes from them
4. making more than one plating per dilution
Minimizing the total number of dilutions you employ can help you to minimize many of the above errors.
II Independent duplication:
Random variation, independent of your technique, can render individual enumerations less than fully precise.
To guard against such imprecision it is always a good idea to do your measurements in duplicate.
Note that in performing duplicate measurements it is important to keep your duplications as independent as
possible. In this exercise, you will be using serial dilutions and the plate count methods to enumerate bacteria

Lab Manual
Making a serial dilution

Lab Manual
First Session
Materials
99 ml aliquots of sterile distilled water
9.9 ml aliquots of sterile distilled water
9.0 ml aliquots of sterile distilled water
1 ml pipettes
Sterile petri dishes
Broth culture of E coli
Procedure
Pour plate Technique
1. Label all the water blanks with the appropriate dilutions.
2. Ensure that the TS agar is melted and kept at approx 50º C.
3. Make serial dilutions of the bacterial suspension as follows:
i) Mix the bacterial culture by rotating between the hands and aseptically transfer 1 ml to the 99.0
ml water blank, labeled 10-2. Discard the pipette.
ii) Mix well and transfer 0.1 ml of the 10-2 dilution to the 9.9 ml water blank labeled 10-4. Discard the
pipette.
iii) Mix well and transfer 0.1 ml of the 10-4 dilution to the 9.9 ml water blank labeled 10-6. Discard the
pipette.
iv) Mix well and transfer 1.0 ml of the 10-6 dilution to the 9.0 ml water blank labeled 10-7.
Using the same pipette transfer 1.0 ml of the 10-6 dilution into an empty petri plate labelled ‘Pour
plate 106’.
Using the same pipette transfer 0.1 ml of 10-6 dilution into an agar plate labelled ‘Spread plate 10-
6
’. Discard the pipette and spread the plate using the plastic spreader under the flame.
v) Mix well and transfer 1.0 ml of the 10-7 dilution to the 9.0 ml water blank labeled 10-8.
Using the same pipette transfer 1.0 ml of the 10-7dilution into an empty petri plate labelled ‘Pour
plate 107’.
Using the same pipette transfer 0.1 ml of 10-7 dilution into an agar plate labelled ‘Spread plate 10-
7
’.
Discard the pipette and spread the plate using the plastic spreader under the flame.
vi) Mix well and transfer 1.0 ml of the 10-8 dilution to the 9.0 ml water blank labeled 10-9.
Using the same pipette transfer 1.0 ml of the 10-8dilution into an empty petri plate labelled ‘Pour
plate 108’.
Using the same pipette transfer 0.1 ml of 10-8dilution into an agar plate labelled ‘Spread plate 10-8’.
Discard the pipette and spread the plate using the plastic spreader under the flame.
vii) Mix well and transfer 1.0 ml of the 10-9 dilution into an empty petri plate labelled ‘Pour plate 10
9
’.
Using the same pipette transfer 0.1 ml of 10-8dilution into an agar plate labelled ‘Spread plate 10-9’.
Discard the pipette.

Lab Manual
4. After placing samples of the dilutions 10-9, 10-8, 10-7, 10-6 into sterile labeled empty petri dishes,
add a tube of melted agar and swirl the plate gently by moving the plate in a “figure 8” pattern on
the bench.
Pour Plate Technique
5. After the agar has hardened, invert the plate and incubate at 37ºC for approx 24 hours.
Notice all plates are to be spread as soon as the 0.1 ml of each sample is added.
Spread Plate Technique

Using a “hockey stick” spread the diluted culture over the surface of the plate as illustrated below:

Lab Manual
After the surface of the inoculated plates have dried, invert the plates and at 37ºC for approx 24 hours.

Lab Manual
Second Session
Observe the plates from session 1 and record the number of colonies on each plate and record in a tabular
format
Questions
1. If you wanted know how many dead cells were present in a bacterial culture, what would you do?
2. How would you determine how many nitrogen fixing bacteria are present in 1 g of soil?

Lab Manual
Laboratory Exercise 13 CONTROL OF MICROBIAL GROWTH – CHEMICAL
METHODS
Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. Antimicrobial agents
include chemical preservatives and antiseptics, as well as drugs used in the treatment of infectious diseases of
plants and animals. Antimicrobial agents may be of natural or synthetic origin, and they may have a static or
cidal effect on microorganisms.
Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane; should not
be taken internally. Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents.
Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe for application to
living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine,
hypochlorites, chlorine compounds, lye, copper sulfate, quaternary ammonium compounds.
Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to
mucous membranes. Often, safety depends on the concentration of the compound. For example, sodium
hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent
disinfectant, is hardly safe to drink.
Common antiseptics and disinfectants
Chemical Action Uses

Denatures proteins and
Ethanol (50-70%) Antiseptic used on skin
solubilizes lipids
Denatures proteins and
Isopropanol (50-70%) Antiseptic used on skin
solubilizes lipids
Reacts with NH2, SH and
Formaldehyde (8%) Disinfectant, kills endospores
COOH groups
Tincture of Iodine (2% I2 in 70%
Inactivates proteins Antiseptic used on skin
alcohol)
Forms hypochlorous acid
Disinfect drinking water; general
Chlorine (Cl2) gas (HClO), a strong oxidizing
disinfectant
agent
General antiseptic and used in the eyes of
Silver nitrate (AgNO3) Precipitates proteins
newborns
Inactivates proteins by Disinfectant, although occasionally used
Mercuric chloride
reacting with sulfide groups as an antiseptic on skin
Detergents (e.g. quaternary ammonium
Disrupts cell membranes Skin antiseptics and disinfectants
compounds)
Phenolic compounds (e.g. carbolic acid, Denature proteins and disrupt Antiseptics at low concentrations;
lysol, hexylresorcinol, hexachlorophene) cell membranes disinfectants at high concentrations
Disinfectant used to sterilize heat-sensitive
Ethylene oxide gas Alkylating agent
objects such as rubber and plastics
Preservatives are also used to control microbial growth. These are static agents used to inhibit the growth of
microorganisms, most often in foods. If eaten they should be nontoxic. Examples: calcium propionate, sodium
benzoate, formaldehyde, nitrate, sulfur dioxide.
Common food preservatives and their uses
Preservative Effective Uses

Lab Manual
Concentration
Propionic acid and
0.32% Antifungal agent in breads, cake, Swiss cheeses
propionates
Sorbic acid and sorbates 0.2% Antifungal agent in cheeses, jellies, syrups, cakes
Benzoic acid and benzoates 0.1% Antifungal agent in margarine, cider, relishes, soft drinks
Sodium diacetate 0.32% Antifungal agent in breads
Antimicrobial agent in cheeses, buttermilk, yogurt and
Lactic acid unknown
pickled foods
Sulfur dioxide, sulfites 200-300 ppm Antimicrobial agent in dried fruits, grapes, molasses
Sodium nitrite 200 ppm Antibacterial agent in cured meats, fish
Sodium chloride unknown Prevents microbial spoilage of meats, fish, etc.
Prevents microbial spoilage of preserves, jams, syrups,
Sugar unknown
jellies, etc.
Wood smoke unknown Prevents microbial spoilage of meats, fish, etc.
Chemotherapeutic agents: antimicrobial agents of synthetic origin useful in the treatment of microbial or
viral disease. Examples: sulfonilamides, isoniazid, ethambutol, AZT, chloramphenicol.
Antibiotics: antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms. There
is also a more broadened definition of an antibiotic which includes any chemical of natural origin (from any
type of cell), which has the effect to kill or inhibit the growth of other types cells. Most clinically-useful
antibiotics are produced by microorganisms and re used to kill or inhibit infectious bacteria.
Antibiotics are produced as secondary metabolites, mainly by microorganisms that live in the soil. Most of
these microorganisms form some type of a spore or other dormant cell, and there is thought to be some
relationship between antibiotic production and the processes of sporulation. Among the molds, the notable
antibiotic producers are Penicillium and Cephalosporium. Among the bacteria, the Actinomycetes, notably
Streptomyces sp., produce a variety of types of antibiotics. Endospore-forming Bacillus sp. produce antibiotics
such as polymyxin and bacitracin. The table below is a summary of the classes of antibiotics and their
properties including their biological sources.

Lab Manual
Classes of antibiotics and their properties
Spectrum
Chemical class Examples Biological source Mode of action
(effective against)
Beta-lactams Inhibits steps in cell wall
Penicillium notatum and
(penicillins and Penicillin G, Cephalothin Gram +ve bacteria (peptidoglycan) synthesis
Cephalosporium sp
cephalosporins) and murein assembly
Inhibits steps in cell wall
Gram+ve and Gram-ve
Semisynthetic penicillin Ampicillin, Amoxycillin (peptidoglycan) synthesis
bacteria
and murein assembly
Gram+ve and Gram-ve Inhibit translation (protein
Aminoglycosides Streptomycin Streptomyces griseus
bacteria synthesis)
Gram +ve and Gram-ve
Inhibit translation (protein
Gentamicin Micromonospora sp bacteria esp.
synthesis)
Pseudomonas
Inhibits steps in murein
Gram+ve bacteria, esp.
Glycopeptides Vancomycin Streptomyces orientales (peptidoglycan)
Staphylococcus aureus
biosynthesis and assembly
Gram-+ve bacteria,
Gram-ve bacteria not Inhibits translation
Macrolides Erythromycin Streptomyces erythreus
enterics, Neisseria, (protein synthesis)
Legionella, Mycoplasma
Damages cytoplasmic
Polypeptides Polymyxin Bacillus polymyxa Gram-negative bacteria
membranes
Inhibits steps in murein
Bacitracin Bacillus subtilis Gram-positive bacteria (peptidoglycan)
biosynthesis and assembly
Gram +ve Gram-ve Inhibits transcription
Rifamycins Rifampicin Streptomyces mediterranei bacteria, Mycobacterium (eubacterial RNA
tuberculosis polymerase)
Gram +ve and Gram -ve Inhibit translation (protein
Tetracyclines Tetracycline Streptomyces sp
bacteria, Rickettsias synthesis)
Gram +ve and Gram-ve
Semisynthetic Inhibit translation (protein
Doxycycline bacteria, Rickettsias
tetracycline synthesis)
Ehrlichia, Borellia
Gram+ve and Gram-ive Inhibits translation
Chloramphenicol Chloramphenicol Streptomyces venezuelae
bacteria (protein synthesis)
Kinds of Antimicrobial Agents and their Primary Modes of Action
Cell wall synthesis inhibitors Cell wall synthesis inhibitors generally inhibit some step in the synthesis of
bacterial peptidoglycan. Generally they exert their selective toxicity against eubacteria because human cells
lack cell walls. These cell wall inhibitors include: Beta lactam antibiotics, Natural penicillins, such as
Penicillin G or Penicillin V, Semisynthetic penicillins, Cephalolsporins, Bacitracin
Cell membrane inhibitors disorganize the structure or inhibit the function of bacterial membranes. The
integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds that disorganize the
membranes rapidly kill the cells. However, due to the similarities in phospholipids in eubacterial and
eukaryotic membranes, this action is rarely specific enough to permit these compounds to be used
systemically. The only antibacterial antibiotic of clinical importance that acts by this mechanism is
Polymyxin. Polymyxin is effective mainly against Gram-negative bacteria and is usually limited to topical
usage.
Protein synthesis inhibitors Many therapeutically useful antibiotics owe their action to inhibition of some
step in the complex process of translation. The most important antibiotics with this mode of action are the

Lab Manual
tetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g.
streptomycin). Other protein inhibitors include aminoglycosides such as streptomycin, kanamycin, tobramycin
and gentamicin. Another protein inhibitor, Chloramphenicol has a broad spectrum of activity but it exerts a
bacteriostatic effect. It is effective against intracellular parasites such as the rickettsiae.
Effects on Nucleic Acids Some chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to
DNA or RNA so that their messages cannot be read. Either case can block the growth of cells. The majority of
these drugs is unselective, however, and affects animal cells and bacterial cells alike and therefore has no
therapeutic application. Two nucleic acid synthesis inhibitors that have selective activity against procaryotes
and some medical utility are nalidixic acid and rifamycins.
Competitive Inhibitors The competitive inhibitors are mostly all synthetic chemotherapeutic agents. Most are
"growth factor analogs" which are structurally similar to a bacterial growth factor but which do not fulfill its
metabolic function in the cell. Some are bacteriostatic and some are bactericidal. They include Sulfonamides
The sulfonamides have been extremely useful in the treatment of uncomplicated UTI caused by E. coli, and in
the treatment of meningococcal meningitis Examples of sulfonamides are Gantrisin and Trimethoprim
Part A
Materials
Antiseptic mouths wash eg Listerine; 70% alcohol; 3% hydrogen peroxide
filter paper discs
petri dishes
molten NA; tubes of nutrient broth
forceps
1.0 ml pipettes
ruler
Nutrient broth cultures of Staphylococcus, E. coli
5.0ml pipettes
sterile test tubes
Procedure
i) Filter Disc technique (Bacteriostatic activity)

1. Divide petri dishes into quadrants and label each quadrant 1 to 4
2. Label one dish S aureus, and the other E coli
3. Inoculate a tube of melted NA with 0.5 ml of S aureus and mix well. Pour into the plate with the
corresponding label. Repeat with E coli
4. Allow the agar dishes to solidify
5. Sterilize forceps by dipping in 70% alcohol and passing through the flame of the Bunsen burner
6. With the sterile forceps remove one of the paper discs from the container and dip into solution 1
(mouthwash)
7. Drain the disc thoroughly on a piece of paper towel and place it in the centre of quadrant No. 1of
the petri dish labelled S aureus. Repeat for E coli
8. Repeat steps 5-7 for the remaining compounds
9. Incubate the petri dishes at 37ºC for 48 hours. DO NOT INVERT THE PLATES
Second Session
Observe each plate, and with a ruler, measure the diameter of the clear zone on inhibition around each disc.
Record your results in a tabular form. NOTE if isolated colonies are present within the zone of inhibition of
any disc

Lab Manual
Questions
1. Which disinfectants or antiseptics would be used to treat the following?

oral thermometer, laboratory bench top, drinking water, patch of skin before surgery, small medical
instruments (probes, instruments, etc). Provide a rationale for your choice as well as the
mechanism of action of the chemical
2. Destruction of microorganisms and inhibition of microbial growth are not simple matters. Describe
three factors which affect the efficiency of an antimicrobial agent

Lab Manual
Part B
Antibiotic Susceptibility of Bacteria
Materials
Sterile cotton swabs

Antibiotic disks
Forceps
Alcohol
Ruler
TSA plates
Broth cultures of Staphylococcus aureus, E. coli, Pseudomonas
Procedure
1. Aseptically swab the culture onto the appropriately labelled plate. Swab in three directions to
ensure complete plate coverage
2. Let stand for 5 minutes
3. Sterilize forceps by dipping in alcohol and burning off the alcohol in the Bunsen flame
4. Obtain a disc with an antibiotic and place it on the surface of the agar. Gently tap it with the
forceps to ensure better contact with the agar. Repeat with the remaining antibiotic discs
5. Incubate the plates inverted at 35ºC until the next lab period
Second Session
Measure the zone of inhibition in millimetres using a ruler on the underside of the plates, using the table,
determine whether the organisms are sensitive, intermediate or resistant to the various antibiotics tested
Questions
1. Many microbiologists feel it will become increasingly more difficult to find effective new
antibiotics. Why?

Lab Manual
2 (a) How does Penicillium escape the effects of the penicillin it secretes?
(b) How might a colony of Bacillus licheniformis escape the effects of its own bacitracin
3. Less than 1% of known antibiotics have any practical value in treatment of disease. Why is this
so?
4. Clostridium difficile is an obligate anaerobic opportunistic pathogen. It can cause serious, life-
threatening pseudomenbranous colitis in patients undergoing long-term antimicrobial
therapy. Why might aminoglycosides be used to treat this disease more often than
tetracyclines

Lab Manual
Lab Manual
Appendix 1 Gram stain and General Microscopy Troubleshooting
Problem Probable Cause Corrective Action
There should be enough oil to make a seal between the

Slide is almost focused, but is hazy or unclear Inadequate or no immersion oil
slide and the 100X objective
Dirty Objective Contact instructor to clean the objective
Slide is clear under 10X but cannot be focused under
Slide is upside down Check slide and flip over if necessary
oil
Organisms appear to have a halo around them or Oil was added before slide was Make a new slide. Allow slide to dry longer or blot more
cannot be focused dry carefully before adding oil
Slide is in focus, then a shadow appears Air bubble trapped by lens Rotate objective back and forth
Clean objective; gently remove oil from slide and refocus
Floating material is seen Objective scraped the smear
more carefully
Nothing is visible on the slide Smear too thin Remake with a thicker smear
Slide wiped to dry it Blot slides; never wipe
Smear was not heat fixed Repeat; heat fix the smear
Repeat; wash off one stain completely before adding the
Excessive deposits of stain crystals on slide Inadequate rinsing between steps
next
Stain was allowed to dry on slide
Repeat; watch timing of staining steps more carefully
before rinsing
Repeat: Do not allow dye to dry on the slide. Do not allow
Gram positive organisms have odd angular shapes Viewing crystal violet crystals crystal violet to remain on slide longer than suggested
times
Gram negative organisms look more like needles
Viewing safranin crystals
than rods
Specimen appears mixed with large oval cocci when Yeast growing in the staining
Repeat after the instructor has filtered the reagents
it should be one organism reagents
Gram positive cells appear gram negative Over-decolorization Decrease decolorizartion time
Non-viable organisms Use a fresher culture
Failure to include mordanting
Be careful to follow each step in the proper order
step (iodine)
Gram negative organism appears gram positive Under-decolorization Increase decolorization time
Be careful to follow each step in the proper order
Forgot decolorization step

Stain dried on smear
Mixture of gram positive and gram negative
Smear is too thick Remake smear
organisms from a pure culture
Culture is contaminated Follow proper aseptic technique

Lab Manual
Appendix 2 Stains
 Acid-Fast Stain
1. Ziehl's Carbol Fuchsin
A. Basic fuchsin (90% dye content), 0.3 g
Ethyl alcohol (95%), 10 ml
B. Phenol, 5 g
Distilled water, 95 ml
Mix solutions A and B and let stand several days before use.
2. Acid alcohol
Hydrochloric acid, concentrated, 3 ml
3. Methylene blue
Methylene blue, 0.3 g
 Cell-Wall Stain
1. Cetylpyridinium chloride solution
Use M/100 or 0.34% aqueous solution. (Molecular weight = 357.99)
2. Congo red solution
Use a saturated aqueous solution
3. Methylene blue solution
As in #acid-fast stain
 Flagella Stain (Gray's Method)

1. Flagella mordant (Gray's)
A. Saturated aqueous solution potassium alum, 5 ml
20% aqueous tannic acid, 2 ml. (This solution must be preserved in the refrigerator with a few drops
of chloroform if a large amount is to be kept on hand.)
Saturated aqueous solution of mercuric chloride, 2ml
B. Saturated alcoholic solution of fuchsin, 0.4ml
Mix solutions A and B on the day the mordant is to be used. Filter through paper before flooding the
slide.
2. Ziehl's carbol fuchsin
As in acid-fast stain
 Gram Stain
1. Crystal violet-ammonium oxalate (modified Hucker's)
A. Crystal violet (90% dye content), 2 g
B. Ammonium oxalate, 0.8 g
Mix solutions A and B. Store for 24 h. before use. Filter through paper into staining bottle.
2. Gram's iodine
Iodine, 1 g
Potassium iodide, 2 g
3. Decolorizer
Ethyl alcohol (95%)
4. Safranin (counterstain)
Safranin O (2.5% solution in 05% ethyl alcohol), 10 ml
 Spore Stain
1. Malachite green solution
Malachite green, 5 g
2. Safranin (counterstain)
As in #Gram stain

Lab Manual
_______________________________________________________________________________________
Appendix 3 Staining Solutions
 Carbol fuchsin (dilute), Carbol fuchsin (Ziehl's), 1 ml, Distilled water, 19 ml
 Loeffler's methylene blue (alkaline)

1. Methylene blue (90% dye content), 0.3 g; Ethyl alcohol (95%), 30 ml
2. Dilute KOH (0.01% by weight), 100 ml
Mix solutions A and B.
 Dorner's nigrosin solution

Nigrosin, water-soluble, 10 g, Distilled water, 100 ml
Immerse in boiling water bath for 30 min; then add, as a preservative, formalin, 0.5 ml
Filter the solution through double filter paper. Store in small tubes.
 Kinyoun carbol fuchsin

Basic fuchsin, 4 g, Ethanol (95%), 20 ml, Phenol crystals, 8 g, Distilled water, 100 ml
_______________________________________________________________________________________
Appendix 4 Broths
 Nutrient broth: General purpose growth medium
o Peptic digest of gelatin ... 5.0 g
o Beef extract ............... 3.0 g
 LB broth: Maintenance and propagation of E. coli

o Pancreatic digest of casein 10.0 g
o Yeast extract, autolyzed, low sodium ... 5.0 g
o Sodium chloride ............... 5.0 g
 Luria broth: Maintenance and propagation of E. coli

o Sodium chloride .............. 10.0 g
 Terrific broth: Buffered, enriched medium high yield plasmid DNA transformed E. coli
o yeast extract .................... 23.6 g
o Dipotassium hydrogen phosphate ... 9.4 g
o Potassium dihydrogen phosphate ... 2.2 g
 Glucose broth
o Peptone ........... 10.0 g
o Glucose ........... 5.0 g
o Sodium chloride ... 5.0 g
 Yeast extract broth

o Peptone ........... 5.0 g
o Beef extract ...... 3.0 g
o Yeast extract ..... 5.0 g
_______________________________________________________________________________________
Appendix 5 Agars
 Nutrient agar: general purpose growth medium

Lab Manual
o Peptic digest of gelatin ... 5.0 g
o Beef extract ............... 3.0 g
o Agar ....................... 15.0 g
 Potato dextrose agar: Isolation and cultivation of yeast and molds found in dairy and other food products
o Potato infusion (dehy.) ..4.0 g
o Dextrose ....................... 20.0 g
o Agar ........................... 15.0 g
 LB agar: Maintenance and propagation of E. coli

o Sodium chloride ................. 5.0 g
o Bacteriological agar.......... 12.0 g
 Desoxycholate agar: pH 7.2

o Polypeptone ............. 10.0 g
o Lactose ................. 10.0 g
o Sodium chloride ......... 5.0 g
o Dipotassium phosphate ... 2.0 g
o Ferric citrate .......... 1.0 g
o Sodium citrate .......... 1.0 g
o Sodium desoxycholate .... 1.0 g
o Neutral red ............. 0.033 g
o Agar .................... 16.0 g
 Phenylethyl alcohol (pheynlethanol) agar: pH 7.3

o Trypticase ............ 15.0 g
o Phytone ............... 5.0 g
o Sodium chloride ....... 5.0 g
o Phenylethyl alcohol ... 2.5 g
o Agar .................. 15.0 g
 Motility agar: pH 7.0

o Peptone ........... 10.0 g
o Agar .............. 3.5 g

Lab Manual
Appendix 6 Writing a Laboratory Report
Practical exercises would be of little or no value if they were not properly recorded and analyzed. In this course
students are required to write a full lab report on the experiments performed. This requires detailed understanding of
the purpose, procedure and principles applied in the laboratory exercise. Lab reports are usually written under the
following headings – Title, Aim, Material/Apparatus, Procedure, Results/Observation, Discussion and
Conclusion. A Reference section should also be placed at the end of the lab report. The following discourse
outlines what should be included under each of these headings.
Title his part of the lab report should be straightforward, concise and informative. It should in less than ten
words clearly state what is being investigated.
Aim his in effect outlines the purpose of conducting the experiment. It is also a concise statement which usually
includes the variables being investigated. It often begins with the phrases, “To investigate”, “To determine”, “To
find out” etc. For example, “To investigate the effect of heat on the function of the enzyme, amylase.”
Apparatus/Material his is a complete accurate list of the equipment and material used when conducting the
experiment.
Method or Procedure his is an account of the activities carried out during the practical exercise. It should be
written in paragraph form in chronological order. The method should be concise and yet explain all steps as they
occurred not as they were supposed to occur. It should be written in past tense and should not be written in the first
person. For example, do not write
“I put a beaker of water to boil.” Instead write, “A beaker of water was put to boil.” If you were instructed the
teacher to write in this section, “As outlined in the lab manual”, the page number has to be quoted and the lab
manual should be listed in the reference section of the lab report. If any changes were made to the procedure in the
lab manual for the particular exercise it should also be noted in this section. If written correctly another researcher
should be able to read the procedure and duplicate the experiment.
Results and Observations This section should include data derived from the practical exercise. This data
may be qualitative or quantitative and should be presented as clearly as possible. The results may take the form of
verbal descriptions, tables of data collected, graphs, drawings, pictures, etc. (See page for guidelines on how to
present drawings, tables and graphs).
Discussion his is one of the most important sections of the lab report as it demonstrates understanding of the
experiment and ties it in with theoretical knowledge. In the discussion the student is expected to explain, analyze
and interpret results and not verbally repeat the results recorded in the preceding section of the report. The
discussion nevertheless should not be long-winding but concise and straightforward. In the discussion answers to
such questions as, “What do the results clearly indicate?” “What is the significance or meaning of the results?”
should be answered.
The discussion should be written in paragraph form not in point form.
Background information or theoretical knowledge of principles behind the practical exercise should be tied in with
the results collected in the discussion. If there were flaws or anomalies resulting from experimental design this
should be recorded and suggestions on how to improve the design discussed. If the results obtained are different
from those expected, this should be noted and an account given for the differences.
In some cases you may compare your results with those of other researchers (whether published ones or your
classmates), not to change your results but to compare, i.e. point out similarities and anomalies and explain the
latter.
Conclusion or undergraduate purposes this section should be a very short statement/s, stating simply what you
know for sure as a result of observations made in the laboratory exercise.

Lab Manual
References References should be made of all text, articles etc. used in writing up the laboratory report. These
should be listed in alphabetical order in an appropriate format e.g.
Taylor, D.J., Green N.P.O., Stout, G.W., 1997. Biological Science 1 & 2, third ed.
REPRESENTATION OF RESULTS
Data collected may be qualitative – drawings, pictures, verbal descriptions, etc. or quantitative – usually represented
in tables and graphs. Below is a discourse designed to give students guidelines on how to make appropriate
biological drawings, construct tables and graphs.
I Biological Drawings
1. Drawing must be done on plain sheets only. No lined sheets may be used.
2. Drawings must be done with a sharp pointed pencil producing distinct lines, not ones too feint to be seen.
3. The drawing should be large enough to clearly represent specimen or equipment being studied or recorded.
It should also be large enough to accommodate labels and annotations. Biological drawings should not fill
less than half of a letter size plain sheet of paper. The greater the number of parts, the larger the drawing
should be.
4. Drawing lines must be sharp, clear and continuous – each line should be drawn without removing the point
from the paper. Coloured pencils should not be used and shading should be avoided.
5. All drawings must be labeled and annotated. Labels and annotations should be on the right hand side of the
drawing only. Label lines should be drawn using a ruler and should be horizontal and parallel to each other.
Label lines should not cross.
6. Labels and annotations should be appropriately spaced to avoid clustering of information and confusion.
Annotations should be short and straightforward. They must be in note form and not complete sentences.
7. Drawings must have an appropriate title which clearly states what the drawing represents. The title of the
drawing should make the representation sufficient to stand on its own (reader should be able to understand
what is being represented) if removed from the lab report.
8. The title of the drawing must be written in pencil, in BLOCK CAPITAL letters and should be underlined.
The title should be below the drawing and should include the view and magnification of the drawing.
9. The drawing should be accompanied be a scale bar drawn to the left side of the drawing on which is written
the actual size of the specimen, not the size of the drawing itself.
10. All markings on the drawing, including title, scale bar, magnification, view, labels, annotations, the
drawing itself etc. MUST be written with a pencil.
II Tables
1. The table should display results very clearly and show the trends being portrayed very efficiently. The
reader should not be confused when reading the table.
2. Each table must be given an appropriate, concise, informative title. Should the table with title be removed
from the report the reader should still be able to grasp what is being represented with little or no room for
questions.
3. The title must be written above the table and must be in block capital letters and underlined. The title must
include the variables being measured and the units in which they are measured in brackets.
4. The table itself must be full enclosed with the table lines drawn distinctly using a ruler.
5. Table headings, indicating what variables are being recorded should be included along with the units in
which the variables are measured. The units should be written as a part of the heading. The remainder of
the table should include only values with no need to rewrite units, e.g.

Lab Manual
TABLE SHOWING THE CHANGE IN TEMPERATURE ( ° C) OF A CUP OF ICE WITH TIME ( mins) AFTER
BEING LEFT OUT ON THE BENCH
Time (mins) Temperature (° C)
0 5
5 7
10 9
15 14
20 21
25 22
30 23
35 23.5
40 24
Note that the units (in bold) are only placed in the headings and not included in the section of the table containing
the values.
III Graphs
1. Graphs should represent data very clearly so that trends may be observed at a glance – even more so that in
a table.
2. As with presenting a table, the graph should be accompanied by an appropriate, concise, informative title
such that should the graph with title be removed from the report the reader should still be able to grasp
what is being represented with little or no room for questions.
3. The title should be written in block capital letters and underlined and should be placed above the graph.
The title should be written with a pencil and must include the variables being measured and the units in
which they are measured in brackets.
4. The x- and y- axes should be drawn in with a pencil and must be labeled. The label must include the units
in which the variables are being measured.
5. The scale of the graph, i.e. of both axes should be written neatly on the graph sheet, preferably in a box
towards the lower right hand corner of the graph sheet away from the graph itself.
6. The points being plotted to be joined should not be drawn too large as this makes the presentation of the
graph unsightly.
7. The pointed should be joined with a sharply pointed, high quality pencil very neatly. Presentation is
important!
8. All markings on the graph must be in pencil. This includes the line/ curve itself, title, axis labels,
numbers, scale etc.
An example showing how a graph should be presented is given on page.

Lab Manual
Lab Manual
Glossary of Microbiology Terms
acidophile- Organism that grows best under acid conditions (down to a pH of 1).
aerobic- (i) Having molecular oxygen as a part of the environment. (ii) Growing only in the presence of molecular
oxygen, as in aerobic organisms. (iii) Occurring only in the presence of molecular oxygen, as in certain chemical or
biochemical processes such as aerobic respiration.
aerotolerant anaerobes- Microbes that grow under both aerobic and anaerobic conditions, but do not shift from one
mode of metabolism to another as conditions change. They obtain energy exclusively by fermentation.
agar- Complex polysaccharide derived from certain marine algae that is a gelling agent for solid or semisolid
microbiological media. Agar consists of about 70% agarose and 30% agaropectin. Agar can be melted at
temperature above 100ºC; gelling temperature is 40-50ºC.
agarose- Nonsulfated linear polymer consisting of alternating residues of D-galactose and 3,6-anhydro-L-galactose.
Agarose is extracted from seaweed, and agarose gels are often used as the resolving medium in electrophoresis.
alga (plural, algae)- Phototrophic eukaryotic microorganism. Algae could be unicellular or multicellular. Blue-
green algae are not true algae; they belong to a group of bacteria called cyanobacteria.
alkalophile- Organism that grows best under alkaline conditions (up to a pH of 10.5).
anabolism- Metabolic processes involved in the synthesis of cell constituents from simpler molecules. An anabolic
process usually requires energy.
anaerobic- (i) Absence of molecular oxygen. (ii) Growing in the absence of molecular oxygen, such as anaerobic
bacteria. (iii) Occurring in the absence of molecular oxygen, as a biochemical process.
anaerobic respiration- Metabolic process whereby electrons are transferred from an organic, or in some cases,
inorganic compounds to an inorganic acceptor molecule other than oxygen. The most common acceptors are nitrate,
sulfate, and carbonate.
anoxic- Literally "without oxygen." An adjective describing a microbial habitat devoid of oxygen.
antibiotic- Organic substance produced by one species of organism that in low concentrations will kill or inhibit
growth of certain other organisms.
antibody- Protein that is produced by animals in response to the presence of an antigen and that can combine
specifically with that antigen.
antigen- Substance that can incite the production of a specific antibody and that can combine with that antibody.
antiseptic- Agent that kills or inhibits microbial growth but is not harmful to human tissue.
Archaea- Evolutionarily distinct group (domain) of prokaryotes consisting of the methanogens, most extreme
halophiles and hyperthermophiles, and Thermoplasma.
archaebacteria- Older term for the Archaea.
aseptic technique- Manipulating sterile instruments or culture media in such a way as to maintain sterility.

Lab Manual
bacillus- Bacterium with an elongated, rod shape.
bacteria- All prokaryotes that are not members of the domain Archaea
bacteriophage- Virus that infects bacteria, often with destruction or lysis of the host cell
binary fission- Division of one cell into two cells by the formation of a septum. It is the most common form of cell
division in bacteria
capsid- Protein coat of a virus.
capsule- Compact layer of polysaccharide exterior to the cell wall in some bacteria.
coccus- Spherical bacterial cells
colonization- Establishment of a community of microorganisms at a specific site or ecosystem.
colony- Clone of bacterial cells on a solid medium that is visible to the naked eye.
colony forming units (cfu), when microorganisms do grow on agar media they commonly form visible
distinguishable structures composed mainly of cellular material which are called colonies. Each of these colonies is
considered to have formed from a single colony-forming unit, which may be a single cell or a clump of cells. By
appropriate mathematical relationships of the dilution of the sample and the area of the agar inoculated, it is possible
to predict a population as: cfu/ml (for liquids), cfu/g (for solids) or cfu/cm2 (for surfaces).
complex medium- Medium whose precise chemical composition is unknown. Also called undefined medium.
constitutive enzyme- Enzyme always synthesized by the cell regardless of environmental conditions.
culture- Population of microorganisms cultivated in an artificial growth medium. A pure culture is grown from a
single cell; a mixed culture consists of two or more microbial species or strains growing together.
cyanobacterium- Prokaryotic, oxygenic phototrophic bacterium containing chlorophyll a and phycobilins, formerly
the "blue-green algae."
defined medium- Medium whose exact chemical composition is quantitatively known.
differential medium- Cultural medium with an indicator, such as a dye, which allows various chemical reactions to
be distinguished during growth
direct count- Method of estimating the total number of microorganisms in a given mass of soil by direct
microscopic examination.
disinfectant- Agent that kills microorganisms.
endospore- Differentiated cell formed within the cells of certain Gram-positive bacteria and extremely resistant to
heat and other harmful agents.
Eubacteria- Old term for the Bacteria.
flagellate- protozoan that moves by means of one to several flagella.

Lab Manual
flagellum (plural, flagella)- Whiplike tubular structure attached to a microbial cell responsible for motility.
Gram stain- Differential stain that divides bacteria into two groups, Gram-positive and Gram-negative, based on the
ability to retain crystal violet when decolorized with an organic solvent such as ethanol. The cell wall of Gram-
positive bacteria consists chiefly of peptidoglycan and lacks the outer membrane of Gram-negative cells.
halophile- Organism requiring or tolerating a saline environment
incubation- The act of growing an organism under conditions that will encourage rapid growth (compared to natural
conditions).
in vitro- Literally "in glass"; it describes whatever happens in a test tube or other receptacle, as opposed to in vivo.
When a study or an experiment is done outside the living organism, in test tube, it is done in vitro.
in vivo- In the body, in a living organism, as opposed to in vitro; when a study or an experiment is done in the living
organism, it is done in vivo.
mesophile- Organism whose optimum temperature for growth falls in an intermediate range of approximately 15 to
40ºC
microaerophile- Organism that requires a low concentration of oxygen for growth. Sometimes indicates an
organism that will carry out its metabolic activities under aerobic conditions but will grow much better under
anaerobic conditions.
micrometer- One-millionth of a meter, or 10-6 meter, the unit usually used for measuring microorganisms.
microorganism (microbe)- Living organism too small to be seen with the naked eye (< 0.1 mm); includes bacteria,
fungi, protozoans, microscopic algae, and viruses.
mold- A filamentous fungus
mushroom- Large, sometimes edible, fruiting body produced by some fungi.
pathogen- Organism able to inflict damage on a host it infects
pure culture-Population of microorganisms composed of a single strain. Such cultures are obtained through
selective laboratory procedures and are rarely found in a natural environment.
serial dilution- Series of stepwise dilutions (usually in sterile water) performed to reduce the populations of
microorganisms in a sample to manageable numbers.
slime layer- Diffuse layer of polysaccharide exterior to the cell wall in some bacteria..
spread plate- Method for performing a plate count of microorganisms. A known amount of a serial dilution is
spread over the surface of an agar plate. After growth the number of colony-forming units is counted.
viable count- Measurement of the concentration of live cells in a microbial population
xerophile- Organism adapted to grow at low water potential, i.e., very dry habitats.

Lab Manual
Acknowledgements
Ms. Nikki Bramwell kindly provided the contents of Appendix 9
References
Cover photo: http://picturethis.pnl.gov/picturet.nsf/f/uv?open&SMAA-3V9T37
Cappuccino, J.G, Sherman, N, 1992 Microbiology: A Laboratory Manual, third ed.
Madigan, M.T, Martiniko, J.M, Parker, J, 2003 Brock Biology of Microorganisms, tenth ed
Totora, G.J.; Funke, B.R.; Case, C. L., 2003 Microbiology An Introduction, eighth ed.
http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/Lab2_Simple_staining.html, May 19, 2004
http://www.mansfield.ohio-state.edu/~sabedon/biol4038.htm May 23, 2004

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THE UNIVERSITY OF TECHNOLOGY, JAMAICA

General Microbiology (MIB 2001) 2

General Microbiology (MIB 2001) 3

General Rules and Regulations

7. There is to be no running or playing in the laboratory.

15. Do not pipette by mouth. Always use a pipette aid or autopipette.

General Microbiology (MIB 2001) 4

20. Dispose of Chemical and Biohazardous material as directed by the instructor.

21. Place dirty glassware in the designated area

General Microbiology (MIB 2001) 5

PART 1 Introduction to the Microscope

I. Part 1 Identification of the microscope parts

3. Arm: Supports upper parts and provides carrying handle.

4. Nosepiece: Revolving device that holds the objectives.

General Microbiology (MIB 2001) 6

7. Fine-adjustment knob: Knob used to bring object to final focus.

9. Diaphragm: Controls the amount of illumination used to view the object.

12. Stage: Holds and supports microscope slides.

13. Stage Clips: Hold slides in place on the stage.

II. Part 2 Focusing the microscope

III. Part 3 Microscopic measurements

General Microbiology (MIB 2001) 7

1. Following the instructions provided, calibrate the ocular micrometer provided.

General Microbiology (MIB 2001) 8

It is easy to calculate the amount of magnification produced by a compound microscope. This is

Alternate method of microscopic measurement

General Microbiology (MIB 2001) 9

Sample microscopic measurement calculations

Calculating the Diameter of Field of View

o 4.5mm or 4,500µm (micrometers) at x40mag

Magnification of field of view

General Microbiology (MIB 2001) 10

o Length or width = 1 x size of field of view at particular magnification

o Length or width = 1 x 1800µm = 90µm

o 2.1cm = 21mm = 21,000µm

General Microbiology (MIB 2001) 11

General Microbiology (MIB 2001) 12

Answer the following questions on a separate sheet of paper:

General Microbiology (MIB 2001) 14

General Microbiology (MIB 2001) 15

General Microbiology (MIB 2001) 16

Illustration of the variation in colony margins

Colonies can vary in their elevations. See illustration below.

Illustration, variations in colony elevation

5. Colony texture / Surface appearance:

General Microbiology (MIB 2001) 17

1. List three examples of bacteria commonly found in:

General Microbiology (MIB 2001) 18

General Microbiology (MIB 2001) 19

REFER TO THE DIAGRAM

6. Flame the loop before setting it down.

10. Flame the loop before setting it down

General Microbiology (MIB 2001) 20

General Microbiology (MIB 2001) 21

III Streak plate technique

General Microbiology (MIB 2001) 22

General Microbiology (MIB 2001) 23

1. What is a pure culture?

General Microbiology (MIB 2001) 24

If necessary repeat the subculturing of your isolate once more

General Microbiology (MIB 2001) 25

HOW THE GRAM STAIN WORKS:

General Microbiology (MIB 2001) 27