Lab Manual

RICHLAND COLLEGE
Jackie Reynolds
Mark Farinha
BIOLOGY 2420
LAB MANUAL
MICROBIOLOGY

TABLE OF CONTENTS
Study guide for lab quizzes/practicals

page number
1. Mock Epidemic 1
MICROSCOPY
2. Use of the Microscope 3
3. Preparation of Specimens 11
4. Ectoparasites 15
5. Helminths 17
6. Ubiquity of Bacteria 21
7. Fungi 25
8. Protozoa 29
STAINING TECHNIQUES
9. Simple Stain and Smear Preparation 33
10. Gram Stain 37
11. Endospore Stain 41
12. Acid-Fast Stain 43
13. Capsule Stain 45
MICROBIAL GROWTH and CONTROL OF GROWTH
14. Transfer of Bacteria Using Aseptic Technique 47
15. Pure Culture Technique 53
16. Bacterial Colony Morphology 59
17. Pipetting and Dilutions 63
18. Counting Bacteria 69
19. Environmental Conditions and Bacterial Growth 79
20. Effects of Temperature on Bacterial Growth 83
21. Evaluation of Antimicrobial Chemicals 87
22. Kirby-Bauer Test for Antibiotic Susceptibility 91
23. Surgical Hand Scrub 95
24. Bacteriophages 99
BIOCHEMICAL TESTS and UNKNOWN IDENTIFICATION
25. Identification of Unknown Bacteria 103
26. Dichotomous Key for identification of Bacteria 107
27. Motility Tests 109
28. Oxygen Requirements and Culturing Anaerobic Bacteria 113
29. Carbohydrate Utilization 117
30. Casein hydrolysis 119
31. Catalase Test 121
32. DNAse Production 123
33. Decarboxylation and Deamination 125
34. Gelatin Hydrolysis 129
35. IMViC Tests 131
36. Lipid Hydrolysis 135
37. Litmus Milk Utilization 137
38. Nitrate Reduction 139
39. Oxidase test 141
40. Starch Hydrolysis 143
41. Urea Hydrolysis 145
42. API 20E Multitest Identification for Gram – Enteric Rods 147
43. Genus Staphylococcus: Isolation and Identification 151
44. Genus Streptococcus: Isolation and Identification 155
45. Serological Testing : Latex Agglutination (Staph, Strep, and Monospot) 161
Table of Biochemical Tests---FAQs and Study Guide 165

GLOSSARY OF TERMS 167
BIOLOGY 2420 Spring 2011
STUDY GUIDE FOR LAB PRACTICALS
EXERCISE
MICROSCOPY (brightfield, darkfield, phase-contrast)
uses
parts and functions
total magnification determination
features: parfocal lens alignment resolution (and ways to increase it)
refraction and oil immersion magnification
PREPARATION OF SPECIMENS
shapes and arrangements of bacteria
how to make a wet mount vs. a stained smear
advantages/disadvantages of each
ECTOPARASITES
examples within the Arachnid and Insect classes
differences
HELMINTHS
representative groups of worms as distinguished on microscope
general features of worms in each phylum: platyhelminthes vs. nematodes
the 2 classes of platyhelminthes: cestodes vs. trematodes
FUNGI
structural differences between yeasts and molds - morphology
asexual vs. sexual spores for yeasts and molds (not specific spore names)
the major classes of fungi - differentiation and examples seen in lab
representative groups of fungi as distinguished on microscope
PROTOZOA
representative protozoa seen in lab, as distinguished on microscope
general features of protozoa as a whole and each class
UBIQUITY OF BACTERIA
shapes of bacteria
TRANSFER OF BACTERIA
advantages/disadvantages of broth vs. agar slant vs. agar plate
transfer methods
PURE CULTURE TECHNIQUES

procedure for types of isolation techniques–pours and streak plates
pros and cons of each technique
streak/pour technique and why done that way
SIMPLE STAINING & SMEAR PREPARATION

simple vs. differential stain
types of dyes - acidic vs. basic - why they bind to the cell
GRAM STAIN
reagents & procedure
interpretation of reaction, shape, and arrangement
why incorrect results
SPORE STAIN
reagents & interpretation
example of spore formers
ACID-FAST STAIN
reagents & interpretation
example of acid-fast bacteria
MOTILITY (flagella stain, SIM, TTC motility, hanging drops)

true vs. false motility (Brownian movement)
example of flagellation types
interpretation of motility in TTC media
CAPSULE STAINS interpretation
PIPETTING & DILUTIONS

solving dilution problems
use (and reading) of the pipette
COUNTING BACTERIA - STANDARD PLATE COUNT & TURBIDEMETRY

determination of bacterial counts in a specimen
use of spectrophotometer (basics of what it is reading)
use of a graph to quesstimate bacterial counts
ENVIRONMENTAL INFLUENCES: pH, osmotic pressure

effects of pH, and osmotic pressure on life
effect of hypertonic media on growth
halophiles/osmophiles
UV LIGHT: mechanism of kill
limitations of UV kill
EFFECTS OF TEMPERATURE ON GROWTH

classification based on optimal temperatures
EVALUATION OF ANTIMICROBIALS
Zones of inhibition
How to determine efficiency of chemicals
How to do a hand scrub
KIRBY-BAUER TEST FOR ANTIBIOTIC SENSITIVITIES

zones of inhibition
determination of S, R, and I (with a chart)
ANAEROBES
ways to culture according to oxygen needs
candle jar vs. GasPak jar - how they work, what atmosphere
thioglycollate broth
classification of microbes according to oxygen needs
BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA

For the following tests–the medium used, purpose, interpretation (+ vs. -), reagent used
gelatin hydrolysis sugar use (phenol red sugars)
IMViC tests (MR, VP, indole, citrate) litmus milk
urea hydrolysis hydrogen sulfide
arginine/lysine/ornithine decarboxylases phenylalanine deamination,
nitrate reduction catalase test
oxidase test starch hydrolysis
lipid hydrolysis casein hydrolysis
API20E
pros and cons
uses
STAPHYLOCOCCUS ID
tests for differentiating the genus Staph and its species
For any tests/media–purpose, interpretation (+ vs. -), reagent used
hemolytic reactions on blood agar
STREPTOCOCCUS ID
tests for differentiating the genus Strep and its species
For any media/tests run–purpose, interpretation (+ vs. -), reagent used
hemolytic reactions on blood agar
BACTERIOPHAGES
the major points in technique and reasoning
lytic vs. lysogenic infection
determination of number of viruses/ml
plaques
SEROLOGICAL LATEX SLIDE AGGLUTINATION: STAPH, STREP, and MONOSPOT

basics of the antigen-antibody test
purpose of the latex beads
interpretation
Page 1
MOCK EPIDEMIC
One person in the lab will have a talcum powder contaminated with GLO GERM POWDER.
The purpose of using this fluorescent powder is to "see" the transmission of microorganisms.
You can easily see if you have been "infected" by viewing your hand under ultraviolet light.
GLO GERM is NOT a bacterium and is not harmful.
OBJECTIVE: Get to know your classmates and appreciate the ease of disease transmission.
MATERIALS: glo-germ powder

UV light
Plate with talc powder (1 per person)
PROCEDURE:
1. Collect materials for exercise---1 plate of talcum powder per student.

2. Pick up dish with powder in your left hand and dump into your right hand. Work powder
around in the palm of your right hand WITHOUT using your left hand. Dump extra powder
back into dish.
3. Go through entire round of handshakes, making sure that your hand REALLY contacts the
other person's hand. USE THE RIGHT HAND ONLY. The instructor will direct handshake
rotations. BE SURE TO FILL IN THE HANDSHAKE ROTATIONS BELOW: you will need
the information to solve the epidemic.
HANDSHAKE ROTATIONS:
#1 #2 #3 #4 #5 #6 #7
4. AFTER ALL HANDSHAKES, do not touch anything or anyone else. Wait for the instructor
to view your hand under ultraviolet light.
5. Record the numbers of those people infected with the fluorescent powder.
6. Wash your hands after this exercise, and see if the glo germ came off of your hands using
the UV lamp.
7. Solve for the person who started the epidemic. Write out the transmission pattern for the
epidemic.
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Page 3
USE OF THE MICROSCOPE
The microscope is absolutely essential to the microbiology lab: most microorganisms cannot
be seen without the aid of a microscope, save some fungi. And, of course, there are some
microbes which cannot be seen even with a microscope, unless it is an electron microscope,
such as the viruses.
You will be using an assigned light microscope for a variety of lab exercises through the
semester, everything from viewing pond water to identification of your unknown bacterium.
Therefore, it is extremely important that you understand how to use the microscope
effectively and how to use different types of microscopy----brightfield, phase-contrast, and
darkfield. These Nikon microscopes have a revolving condenser, with specific settings for
the 3 kinds of microscopy: the settings are labeled with white letters that can be seen in the
front. Bright field is “0”, darkfield is “DF”, and phase-contrast is “PH.” There are 3 settings
of phase-contrast, one for each of the lenses—PH1 for 10X, PH3 for 40X, PH4 for 100X. If
you forget, look at the markings in red on the objective lenses.
HANDLING THE MICROSCOPE:
• Carry it with 2 hands---one on the arm and the other under the base.
• Clean ALL objective lenses and the ocular with lens paper BEFORE you even place a
slide on the stage, and it is a good idea to wipe the condenser lens also. The last
person using the microscope may have left it dirty: it is imperative to begin with clean
lenses.
• Use lens paper (ONLY) to remove any oil from the 100X lens.
• Place the microscope back into the correct spot in the cabinet, with the arm towards
you.
• Once oil has been added to the slide, do not move back to the 40X lens to focus: oil
should never get on this lens. If this happens, it will be very difficult to get all of the oil
off, and you will have to clean the lens thoroughly.
Determination of total magnification = ocular lens (10X) X objective lens (10X, 40X, 100X)
OBJECTIVES:
Identify the parts of the microscope and their functions.

Become familiar with the 3 variations of light microscopy.
Learn how to use the microscope effectively, and particularly the oil immersion lens.
Determine total magnification of the specimen, using various objective lenses.
Be able to switch objective lenses, while focused on the specimen, without moving the stage.
Handle the microscope safely and clean it.
Explain principles and terms used in microscopy and focusing.
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MATERIALS NEEDED:
microscope
lens paper
bibulous paper
oil dropper
prepared wet mounts or stained smears
THE PROCEDURES:
You will use a variety of specimens with your microscope----a wet mount, a bought
prepared smear, and a stained smear that you have made. Refer to the lab exercise
PREPARATION OF SPECIMENS.
BEFORE PUTTING A SLIDE ON THE MICROSCOPE STAGE:
1. Find all of the structures on the microscope (diagrams below) being sure that you
know their functions. Rotate the condenser so that you see all of the settings (white
letters are engraved into the front of the condenser dial). Also, move the iris
diaphragm left and right so you can see the effect on the amount of light.
2. The NIKON microscopes have 3 types of
condenser lenses for 3 types of light
microscopy:
o brightfield (O on condenser)
o darkfield (DF on condenser)
o phase-contrast (condenser settings: PH 1
for 10X, PH3 for 40X, PH4 for 100X)
3. Start with brightfield microscopy ALWAYS. The
brightfield condenser has a 0 etched in white.
4. Raise the condenser stage ALL THE WAY
UP. There is a special knob for the condenser
stage under the mechanical stage. The
condenser gathers all available light from the
lamp and directs it up to the stage. We always have the condenser stage closest to
the mechanical stage when viewing microorganisms. When viewing largest objects,
like worms or insects, you can move the condenser down to improve light density
hitting the specimen, but not for microorganisms.
5. Turn the brightness control knob ALL the way up, and then back off 1/4 of a turn.
This is where the control knob will stay: do not touch it again. Your light amount
coming up through the condenser is controlled by the iris diaphragm.
6. Rotate the revolving nosepiece until the low power 10Xobjective lens snaps into
place.
7. Bring the stage all the way up, using the coarse adjustment knob. Keep an eye on
the distance between the slide and the lens to MAKE SURE that you do not crash the
lens into the stage.
8. Clean all lenses (oculars, objective lenses, and lens on condenser) with LENS
PAPER.
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9. Set the ocular lenses to the correct distance for your face (the oculars can be moved
apart or closer together for your own needs). These ocular eyepiece lenses are both
10X magnification.
10. You will want to reduce the light coming through the condenser, so close your iris
diaphragm so that you get better contrast of the specimen.
TO VIEW A SPECIMEN:
1. Place the wet mount or prepared smear on the stage, and secure it inside of the stage
holder.
2. Try to guesstimate where the specimen is located on the slide, and place it in the
center of the hole allowing light through the stage.
3. While looking through the ocular eyepiece, lower the stage SLOWLY using the coarse
adjustment knob. Be sure that you are looking through the binocular head of the
microscope with BOTH eyes.
4. As soon as you see the specimen, STOP using the coarse adjustment, and switch
over to the fine adjustment knob. After focusing at the beginning with the coarse
adjustment knob, it is NOT TOUCHED AGAIN. All focusing will now be done with the
fine adjustment knob.
5. CHANGING OBJECTIVES:
The Nikon lenses are PARFOCAL---the objectives are

aligned so that rotation to another lens can be done
without major focusing. Rotate the 40X lens in place,
making sure that it snaps into place. Your specimen
should still be seen in the field of vision, but 4 times
larger now. Use your fine adjustment knob to clarify the
objects.
IF YOUR FIELD OF VISION IS FUZZY, AND NO
AMOUNT OF FOCUSING BRINGS THE OBJECT INTO
VIEW, YOU PROBABLY HAVE OIL RESIDUE ON THE
40x OBJECTIVE. IT HAS TO BE CLEANED WELL
WITH LENS PAPER.
TO MOVE INTO OIL IMMERSION, 100X MAGNIFICATION:
1. Do NOT MOVE the focus knobs or the stage knobs. Swing the 40X objective (high
dry) out of the way. Place a single drop of immersion oil on the slide right over where
the light is coming through the stage, and rotate the 100X objective (oil immersion)
into place. The lens will actually GO INTO THE OIL DROP.
2. Now look through the oculars, increasing your light with your iris diaphragm lever.
Your object should still be in the field of vision, probably out of focus. Use the fine
adjustment knob to focus clearly.
3. Once you have gone into oil immersion, do NOT GO BACK TO THE 40X
OBJECTIVE. The objective will get oil on it, and you will have to really clean it to get
the oil off. The 10X can be returned to, since the lens should not touch the slide
anyway.
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4. Once through with the microscope, use the lens paper to wipe the oil from the
100X objective lens.
USING DARKFIELD AND PHASE-CONTRAST:
Once you are looking at your object using brightfield microscopy, you can easily switch to
another type of microscopy: Just rotate the condenser knob. Darkfield and phase-contrast
microscopies have particular condenser lenses required for proper visualization.
HOWEVER:
Darkfield is used for wet mounts, using 10X and 40X (100X will not show well).
Be sure that your iris diaphragm is OPEN all the way.
Phase-contrast is used for wet mounts also, although SOMETIMES it is helpful
for delineating subtle shapes and colors that cannot be readily seen using
brightfield. Be sure that you are using the correct condenser setting for that
particular objective lens. Depending on whether you want to use the 10X, 40X,
or 100X objective lens, you will have to change the phase-contrast consider
lens to the appropriate setting.
PLACING MICROSCOPES BACK INTO THE CABINETS:
• YOU are responsible for your assigned microscope! There is only 1 person in each
lab who is assigned that particular microscope, so if someone else complains about it
being left with oil or a slide on the stage, you or another person who is assigned that
particular scope will be reprimanded.
• Make sure that the 10X low power lens is in place, pointing towards the stage---not the
100X oil immersion lens. The lens could hit against the stage and get scratched.
• Turn the coarse adjustment knob so that the stage is far from the lens.
• Do NOT drag it across the table, making annoying grating noises.
• Wind the electrical cord around the cord holder properly.
• Remove any slide left on the stage.
• PLACE YOUR MICROSCOPE BACK IN ITS NUMBERED POSITION IN THE
CORRECT CABINET.
TROUBLESHOOTING:
Focus fuzzy?
Probably oil on the lens. Clean with lens paper thoroughly. If that does not help, use lens
cleaner with lens paper. If that doesn’t work, ask instructor for some help. Xylene may be
used, but SPARINGLY since it can destroy the glue seating the lens.
Light off center?

Do you have the lens correctly in place? As you rotate the nosepiece, each lens should
“click” into place, and you will know that it is in the correct position.
Found the specimen on low power, but lose it when moving into a high power?
• Not focusing on the specimen, but rather dirt on slide.

• Slide has been turned upside down, with specimen facing down towards stage.
Page 7
• Specimen off-center on slide: when moving to a higher lens magnification, the
specimen is outside the field of vision.
Page 8
PART OF MICROSCOPE FUNCTION
1. coarse adjustment knob general focus, particularly for 10X lens

2. fine adjustment knob fine focus, particularly for 100X lens
3. arm infrastructure of the microscope
4. power switch/brightness control on/off switch for light, changes intensity
5. base infrastructure of the microscope
6. condenser knob condenser movement
7. iris diaphragm lever control of cone of light coming through
condenser
8. objective lens magnification of 10X, 40X, or 100X
9. revolving nosepiece attachment of objective lenses
10. ocular eyepiece lens magnification of 10X
11. stage holder hold slide in place
12. stage holder knobs movement of stage, 2 directions
13. stage placement of slide
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LABORATORY REPORT SHEET

QUESTIONS:
1. Which objective lens is also called the oil-immersion lens?
2. How do the functions of the substage condenser and the iris diaphragm within the
condenser differ?
3. What term is used to describe the feature of the microscope that makes it possible to
move among the objective lenses with just MINOR focusing?
4. What condenser setting value do you want when you are using the 100X objective
lens?
5. What would be the total magnification of a specimen using the 40X objective lens?
6. Why move the 10X objective lens into place when putting the microscope back into the
cabinet?
7. What is parfocal?
8. Why reduce your light when using the 10X objective lens?
Page 10
Page 11
PREPARATION OF SPECIMENS
Here is your first exposure to the preparation of a bacterial smear and subsequent staining of
it. However, you are making a simple stain, using only one dye. Everything on the slide will
be the same color, but you can distinguish among shapes, sizes, and arrangements of
bacteria. Most of the dyes used in the lab are basic dyes, having a + charge on the
molecule, and are attracted to the slight negative charge on the surface of microbial cells.
Methylene blue, malachite green, crystal violet, safrinin, and carbol fuschin are all basic dyes,
being used in the the gram stain, spore stain, and acid-fast stain. Acid dyes are negatively
charged and are repelled by the surface of the cell , thereby staining the background area
rather than the cell itself. Examples are nigrosine and congo red.
You will be looking at living (and often motile) microorganisms of various kinds in pond water-
--bacteria, algae, and protozoa. Although not microorganisms, but still often found in pond
water, worms and insects (or their larvae) may also be found. Unfortunately, bacteria are
difficult to see in a wet mount unstained: they are transparent and tiny. You will be staining
the bacterial suspension from your mouth, seeing a variety of bacterial species, shapes,
sizes, and arrangements of cells.
SHAPES & ARRANGEMENTS OF BACTERIA
cocci (coccus = singular)
chain pair cluster tetrad
bacilli (bacillus = singular)

(also called rod)
chain
spirals
Often, there is a hint in the name of the microorganism that gives you specific information
about shape or arrangement.
For example: Streptococcus (strepto = chain) is found as a chain of cocci.

Staphylococcus (staphylo = cluster) is seen as cocci in various cluster sizes.
Streptobacillus is seen as a chain of rods.
Streptococcus pneumoniae is a coccus found in pairs, also called diplococci.
Aerococcus is seen as cocci in a foursome, or tetrad.
Page 12
OBJECTIVES:
Prepare wet mounts using live samples.

Prepare smears of microbial suspensions and stain them.
Use a bought smear that has already been stained.
Identify the purpose of staining and the uses of a simple stain.
MATERIALS NEEDED:
pond water
microscope slides
cover slips
oil dropper
prepared slides of a bacterium (Bacillus, Streptococcus, Staphylococcus, E. coli, etc.)
toothpicks
dye kits
THE PROCEDURES:
Prepare a wet mount of pond water.
1. Go DOWN into the algae and muck to get a really good sample of protozoa and algae
with your dropper. Protozoa will be feeding at the bottom. Much of the algae will be
there also.
2. Place the drop on a microscope slide. Place a single cover slip (be sure that they are
not stuck together) on top.
3. Begin looking at the specimen using brightfield microscopy (condenser setting on 0).
Focus on the sample using 10X, then go to 40X (NOT 100X).
4. Practice with the microscope, changing condensers settings, using different lenses.
Start with brightfield, then switch over to darkfield and then phase-contrast.
5. The objective here is to become familiar with your microscope: It is NOT important to
identify protozoa or algae in the pond water.
6. Any slide that you use for a wet mount is returned to your slide box, after washing with
tap water.
7. Remove the cover slip and discard it in the red SHARPIES container on the bench.
Prepare a smear and simple stain of the material between your teeth.
1. Take a sterile toothpick, remove some solid material between your back wisdom teeth,
and mix it into a drop of water so that you have a suspension spread over the middle third
of the microscope slide. Spread the suspension over the glass so that it forms a thin layer.
NO COVER SLIP USED!
Page 13
2. Let the slide air-dry—totally. This step facilitates the fixing of the smear to the slide.
3. Heat-fix the dry smear by running the slide quickly through the flame a few times. If
your fingers get hot, you have heat-fixed TOO MUCH. The heat-fixing will coagulate
some of the protein material and cause the suspension to adhere to the glass slide.
Therefore, it will not wash off during the various steps of staining.
4. Staining the smear can be done 2 ways, depending on whether the dye is in a dropper
bottle or in a jar:
o Place the slide on the wire over the stain tray, and place the slide on the wire
mesh. Flood the smear with crystal violet: let sit for 1 minute.
o Place the slide, end up, into the jar of crystal violet for 1 minute. Be SURE that
the crystal violet dye is full in the jar (there are stock solution bottles in the
cabinets to fill them).
5. Wash the slide WELL with distilled water. Blot the smear slide with your bibulous
paper pad.
6. Focus on the sample using the 10X lens (Be SURE that you are on brightfield
microscopy): you should see masses of purple material, most of it too small to see.
7. Ready to go to 100X now? Be sure to read the directions in the MICROSCOPY
exercise so that you know how to move to the 100X objective lens using oil.
8. Identify the various shapes and arrangements of bacteria in your mouth. Most of them
will be bacillus-shaped or coccus-shaped, but it would not be uncommon to see some
spirilla. Notice the arrangements of the bacteria---pairs, clusters, chains?
9. Any slide that you use for smears is returned to your slide box, to be cleaned and used
again. The procedure for cleaning smears is simple. Make a paste with Bon Ami
cleanser powder (located in shaker bottles at each sink) and water. Use your finger to
spread the paste on the slide, cleansing each side of the slide.
Look at a bought prepared bacterial smear.

Since these are bought stained smears, cover slips are on them. You still use oil on
them with the 100X oil-immersion lens.
Be sure to REMOVE any oil before replacing them on the trays.
BE SURE TO WIPE OIL OFF OF THE 100x LENS WITH LENS PAPER---not paper
towel, not bilulous paper, not kim-wipes!
WASH YOUR HANDS AFTER WORKING WITH MICROORGANISMS AND STAINS!

Page 14
Draw your results seen in the microscope (using 100X lens)
from the mouth from the bought prepared smear
QUESTIONS:
1. What is a simple stain?
2. Do you think that all basic dyes would have the same binding affinity for different
bacteria?
3. Draw a bacillus-shaped bacterium.
4. What would happen if you forgot to heat-fix the smear?
5. Why stain bacteria rather than looking at wet mounts of them?

Page 15
ECTOPARASITES
Various arthropods, mainly from the Insect class, are vectors for infectious diseases. When
living on or interacting with the surface of the body, they are called ectoparasites.
Examples are:
o Hard body ticks (Ixodes) – Lyme disease caused by bacterium Borrelia burgdorferi
(Dermacentor) – tularemia caused by bacterium Francisella tularensis,
Rocky Mountain spotted fever caused by bacterium Rickettsia rickettsii
o Soft body tick (Ornithodoros) – Relapsing fever caused by bacterium Borrelia
recurrentis
o body louse (Pediculus) – typhus caused by various Rickettsia species, (Pithirus) pubic
louse– STD – the “crabs”
o itch mite (Sarcoptes) – sarcoptic mange –STD - “scabies” (not a microbial infection)
o mosquito (Anopheles) – malaria caused by various species of the protozoan
Plasmodium.
(Aedes) – dengue (viral) fever, viral encephalitis
o flea (Pulex) – plague caused by Yersinia pestis
These ectoparasite vectors are in the Kingdom

Animalia, generally in 2 classes---Insecta and
Arachnida. Ticks and mites are in the Class
Arachnida (relative of spiders), while the others are
in the Class Insecta. Differences between the
Insects and Arachnids include presence of wings,
main body parts, numbers of leg pairs, and
presence of antenna.
Dept. of Medical Entomology, Univ. of Sydney

http://medent.usyd.edu.au/photos/insect%20photos.htm
OBJECTIVES:
Learn about various vectors of infectious diseases.
MATERIALS NEEDED:
prepared slides of ectoparasites

flea
louse
mosquito head
tick (hard bodied and soft bodied)
fly head
THE PROCEDURES:
View various prepared slides using the low power of stereoscopic microscopes..
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QUESTIONS:
1. Record the views of the various parasites (on low power magnification)
flea louse bedbug
tick mosquito
2. How many leg pairs does the Class Arachnid have compared to the class Insecta?
3. How does the body plan of Insects differ from Arachnids based on major divisions?
4. Why is malaria not common in the United States?

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HELMINTHS
There are many worms in the kingdom Animalia, but we are looking at pathogenic
representatives in 2 phyla (a major group within a kingdom is a phylum)---Platyhelminthes
and Nematoda. Platyhelminthes are flatworms, divided into the cestode (tapeworms) class
and the trematode (flukes) class. The life cycle of a worm can be very complex, with
multiple hosts for different stages of the worm.
Helminths are multicellular, and one might wonder why they are covered in microbiology.
First, the worms are transmissible diseases, via insects, water, food, soil---similar to bacterial
and viral infectious diseases. Second, diagnosis of helminthic diseases relies on the
microscopic identification of the eggs or larvae. The adult worms are macroscopic. Mainly,
you will see genital organs inside of the adult worms under the microscope, probably with lots
of eggs. The Nematodes are roundworms. They come in separate sexes, and are a bit more
evolved than the platyhelminthes.
FEATURES OF HELMINTHIC GROUPS:
trematodes (flukes) – flat, leaf-shaped, unsegmented, separate sexes
cestodes (tapeworms) – flat, segmented, hermaphroditic
nematodes (roundworms) – unsegmented, separate sexes
Low power 10X or even scanning power (using the stereoscopic dissecting microscopes)
should be sufficient for these large organisms. The egg slides are set up as demos only:
You do not need to be able to identify the worms by their eggs. On the other hand, if
you are planning a career in clinical lab sciences (medical technology), you will definitely
have to learn the eggs later.
Excellent Resource on Parasites at Centers for Disease Control!
Laboratory Identification of Parasites of Public Health Concern

http://www.dpd.cdc.gov/dpdx/
OBJECTIVES:
Differentiate between flatworms and roundworms.

Differentiate between flukes and tapeworms.
Identify some common worms.
Recognize common features of each worm.
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MATERIALS NEEDED:
prepared slides of the adult: (use stereoscopic microscopes for scanning power and 10X
low power on your regular microscopes)
• Taenia
• Schistosoma
• Fasciola
• Enterobius
• Trichinella
• Ancylostoma or Necator
• Dirofilaria (heartworm in dogs) larvae in blood or Wuchereria (filariasis in humans)
preserved specimens of various worms

demo microscopes with eggs of adults (just for show, NOT for identification)
THE PROCEDURES:
Major features of each organism are listed to the right.
THE WORM: class and genus key features to notice
PLATYHELMINTHES
TREMATODES
Schistosoma adult
adult male and female copulating, suckers
DEMO: egg
Fasciola hepatica adult (sheep liver fluke)
eggs within body, suckers
DEMO: egg
CESTODES
Scolex head with suckers and/or hooks,
Taenia solis or T. saginata adults
proglottid segments (with reproductive
DEMO: egg
organs)
NEMATODES
Enterobius vermiformis adult (pinworm)

eggs in body
DEMO: egg
Necator or Ancylostoma adult (hookworm)
cutting teeth, tail of male vs. female
DEMO: egg
Trichinella spiralis larvae in muscle encystment
Dirofilaria immitis filarial larva in blood smear
Page 19
QUESTIONS:
1. Draw the organisms.
Taenia Schistosoma Necator/Ancylosoma
Fasciola Enterobius Trichinella
2. Is Schistosoma a trematode, cestode or a nematode?
3. Which group of helminths has a scolex?
4. The meaning of hermaphroditic?
5. Why are Schistosomes called blood flukes?
6. Which group of parasites has flat and segmented body?
7. Give one way in which roundworm Dirofilaria or Wuchereria differ from the other worms.
Page 20
Page 21
THE UBIQUITY OF BACTERIA

Bacteria far outnumber all other life forms on the planet. In fact, in your large intestine alone
you harbor more bacterial cells than the total number of human cells in your body. It is
estimated that only 3% of bacteria are pathogenic for man and animals. Bacteria are found
in a wide variety of environments---in or on animals and plants, in water, in soil, in air, or on
rock. Generally, they are contributors to the environment, decaying nutrients and recycling
the minerals (for use by plants and other organisms). Bacteria are both metabolically
diverse as well as structurally diverse.
Your body is actually its own ecosystem, containing vast numbers of bacterial species. Most
of them are part of a commensualistic symbiotic relationship (they benefit, you are neither
harmed nor do you benefit) or a mutualistic (both partners gain) symbiotic relationship.
These indigenous bacteria, living in and on your body, are part of your normal flora.
Two forms of bacteriological media will be used to culture the microorganisms---agar medium
and broth medium. The difference is the presence or absence of the complex polysaccharide
called agar agar, a solidifying agent purified from red algae. Commonly, it is added as a 1.5-
2% agar for the solidified media, giving a solid surface for the bacteria to grow on. Agar agar
is not a nutrient: it cannot be utilized by the organisms. In a broth culture, the presence of
bacteria will produce a turbidity (sterile broth medium will be clear). On an agar plate clusters
of daughter bacterial cells, piled on top of each other in a discrete area of the plate, will
produce colonies on the agar surface. Each colony started with one mother cell deposited on
the agar surface, then dividing continuously. You will undoubtedly see mixed cultures from
most of your specimens---different colony sizes, shapes, colors, etc.
OBJECTIVES:
Compare the growth of bacteria from different environments.

Identify the ways that bacteria grow on and in bacteriological media.
Become familiar with the use of various forms of media.
MATERIALS NEEDED:
3 TSA (trypticase soy agar) agar plates per table (2 for environment, 1 for body)
1 TSB (trypticase soy broth) per table (tables doing lips and sneeze do not use TSB)
THE PROCEDURES:
Each table will sample 4 habitats---2 from the body and 2 from the environment.
ISOLATION OF BACTERIA FROM THE ENVIRONMENT

1. Label the bottom of your agar plates with your name, lab section, date, and location of
specimen.
2. Your table will sample the environment in 2 ways:
a. air sample
b. surface of an object (not on the body)
3. For surfaces of objects:
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a. Taking a sterile cotton swab from the package, dip it into a tube of sterile saline
(0.9% NaCl) and squeeze the swab against the glass wall of the tube to reduce
excess fluid.
b. Roll the swab around on the assigned environmental area.
c. Rub the swab on an agar plate, using the zig-zag procedure shown below.
4. For air specimens, expose your TSA plate to your assigned area by uncovering the
agar cover.
5. Incubate the agar plate at room temperature, 25 degrees Celsius.
ENVIRONMENTAL SPECIMENS BODY SPECIMENS

Air in lab for 30 minutes Palm of hand swabbed
Air in toilet stall (at base of toilet) for 30 minutes Sole of foot swabbed
Air outside of building for 30 minutes Inside elbow swabbed
Toilet bowl inside swabbed Cheek swabbed
Toilet bowl outside swabbed Between fingers swabbed
Various coins placed on medium Armpit swabbed
Paper currency pressed onto agar medium Inside ear swabbed
Hair shaken over plate Inside nose swabbed
Doorknob swabbed Inside mouth, back teeth swabbed
Cell phone speaker swabbed Lips lightly pressed against agar medium
Cell phone earpiece swabbed Sneeze onto plate
ISOLATION OF BACTERIA FROM THE HUMAN BODY

1. Label the bottom of your agar plate with your name, lab section, date, and location of
specimen. Do the same with a tube of TSB, being sure to write on masking tape to
label the tube.
2. Taking a sterile cotton swab from the package, dip it into a tube of sterile saline (0.9%
NaCl) and squeeze the swab against the glass wall of the tube to reduce excess fluid.
3. Roll the swab around on the assigned area of your body.
4. Rub the swab on an agar plate, using the zig-zag procedure shown below.
5. Repeat steps 2 and 3, using a new swab and the same area of the body on the
opposite side.
6. Place the swab into a tube of sterile TSB and cap the tube.
7. Incubate the agar plate and broth tube at room temperature, 25 degrees Celsius.
lips and sneeze plates done on agar plates only,

no TSB.
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DATA COLLECTION:
BACTERIAL COUNTS FOR AGAR MEDIA
0 = no growth
+1 growth = 10 bacterial colonies or less
+2 growth = 10-100 colonies
+3 growth = >100 colonies
1. Check for the number of bacterial colonies.

2. Try to differentiate different species of bacteria by colony shapes, size, and pigment.
GROWTH IN BROTH MEDIA

0 = clear
+1 = light turbidity
+2 = medium turbidity
+3 = very turbid, cannot see through broth
1. Record the amounts of growth in broth cultures and agar plate cultures.
2. Check for the number of bacterial colonies.
3. Try to differentiate different species of bacteria by colony shapes, size, and pigment.
4. The entire class’s data will be listed on the board, along with the source of the specimen.
ENVIRONMENTAL SPECIMENS Amount of growth Different species #

Air in lab for 30 minutes
Air in toilet stall (at base of toilet) for 30 minutes
Air outside of building for 30 minutes
Toilet bowl inside swabbed
Toilet bowl outside swabbed
Various coins placed on medium
Paper currency pressed onto agar medium
Hair shaken over plate
Doorknob
Cell phone speaker swabbed
BODY SPECIMENS
Palm of hand
Sole of foot
Inside elbow
Cheek
Between fingers
Armpit
Inside ear
Inside nose
Inside mouth, back teeth
Lips lightly pressed against medium
Sneeze onto plate
Page 24
QUESTIONS:
1. Can you determine the number of different bacterial species in a broth culture?
Explain.
2. Which of the environmental habitats had the highest counts?
3. Which of the body areas had the highest counts?
4. Did any habitats result in no growth?
5. Of the specimens taken from the body, which one gave the great variation in bacterial
species?
Page 25
FUNGI
Fungi are eukaryotic, heterotrophic, nonphotosynthetic organisms in a separate kingdom of
the same name. The majority consists of microscopic filaments called hyphae, and the
network of filaments is the mycelium. They live either as parasites or as saprophytes,
absorbing organic material from their environment. Their cell walls contain chitin a polymer
of the sugar glucosamine. Fruiting structures arise from the mycelium, having names such
as sporangium, ascus, and basidium, to name just a few. These fruiting structures can
contain sexual spores or asexual spores. The hyphal filaments are haploid (1N).
The classes of fungi are based mainly on the type of sexual spore that is produced, i.e.
zygospore, basidiospore, ascospore. The sexual spores are produced by meiosis, and are
often contained within a structure. Even yeasts produce sexual spores, although they more
commonly reproduce by asexual budding. On the other hand, asexual spores are the more
common spores, their function being dispersal so that the fungus can disseminate itself
throughout the environment. There are various reproductive modes for production of asexual
spores---fragmentation, budding, fission, and so on.
There are quite a few classes of the kingdom Fungi---Chydridiomycota, Ascomycota,

Basidiomycota, Zygomycota, and Deuteromycota. The Deuteromycota group contains the
unclassified fungi that mycologists don't really know where to put,due to a lack of currently
defined sexual spores. In addition, fungi make up part of the composite organisms called
lichens. The lichens are actually mutualistic, symbiotic relationships between fungi and
photosynthetic algae or photosynthetic cyanobacteria.
In this lab, you will identify representatives from 3 categories of fungi:

• Basidiomycetes (representative: mushrooms)
• Ascomycetes(representative: Penicillium, Saccharomyces, various dermatophytes)
• Zygomycetes(representatives: Rhizopus)
Great Resources!
Dr. Fungus - http://www.doctorfungus.org/
UC Berkeley's Introduction to Fungi - http://www.ucmp.berkeley.edu/fungi/fungisy.html
OBJECTIVES:
Identify the various classes of fungi and major features among them.
Identify key representatives of each class.
MATERIALS NEEDED:
culture of Saccharomyces cerevesiae

phenol red sugar broths with durham tubes (lactose, sucrose, glucose)
prepared slides of Rhizopus
prepared slides of Penicillium and/or Aspergillus, and Candida albicans
prepared slides of dermatophytes (Microsporum, Trichophyton, Epidermophyton)
fresh cultures of fungi on agar plates (Rhizopus, Penicillium, Aspergillus)
Page 26
THE PROCEDURES:
1. Saccharomyces cerevesiae
Yeast reproduce asexually by budding, small daughter cells arising from the mother cell.
They will stay attached until disturbed, and then break off.
• Make a wet mount of the culture (SMALL inoculum) in a drop of lactophenol cotton
blue (10X and 40X). Use phase-contrast or brightfield microscopy.
• Make a smear of the yeast and simple stain with crystal violet. Use brightfield
microscopy.
• Look at prepared smears of mixed yeasts (Saccharomyces and Candida)
• Inoculate the three sugar broths (lactose, sucrose and dextrose) using the culture of S.
cerevisiae provided. Incubate at room temperature until the next laboratory session.
After incubation observe the tubes for fermentation of the sugars (a change in color of
the phenol red indicator from red to yellow) and also note if carbon dioxide gas was
produced by looking for bubbles in the inverted Durham tube.
2. Rhizopus prepared slides
If 2 different strains (called + and – strains) are placed together on a culture medium (or in
nature), the hypha will grow towards each other and conjugation will occur. This produces a
sexual spore called a zygospore—a diploid sexual spore.
• On 10X and 40X, identify hyphae, sporangia, and sporangiospores.
• Differentiate between the sexual zygospores and the sporangiospores on the slides.
3. Penicillium and Aspergillus On 10X and 40X, identify hyphae, conidia fruiting
structures, and the asexual conidiospores.
4. DEMOS only!
Dermatophyte genera: Microsporum, Trichophyton, Epdermophyton On
10X and 40X, identify various dermatophytes’ fruiting structures.
Page 27
QUESTIONS:
1. Record the shapes and features of the various fungi.
Saccharomyces Penicillium Aspergillus
Candida Rhizopus asexual spores Rhizopus sexual spores
2. 3. The mushrooms are in what class of fungi?
3. Is a zygospore a sexual spore OR an asexual spore? WHY?
4. Name a couple of ways in which the molds and yeasts differ.
5. By what major criterion are the fungi subcategorized into classes?
6. Differentiate hypha and mycelium
7. 6. Are the conidiospores of Penicllium and Asperillus arranged differently ?
8. Which one of those fungi is commonly known as brewer’s or baker’s yeast?

Page 28
Page 29
PROTOZOA
The protozoa are contained within the kingdom Protista along with the unicellular algae. The
classes of protozoa are categorized by a variety of factors: cell architecture, motility structure,
even hosts. They do not photosynthesize, rather being chemoheterotrophic like animals.
This means that they use chemicals for energy production and they get their carbon from the
same compounds, e.g. sugar. However, there are some pigmented protozoa and there are
even a few that seem to be crossover organisms, being claimed by the botanists because of
their photosynthetic ability. for example, the flagellate Euglena will photosynthesize in light (it
contains chlorophyll) or will switch over to regular aerobic respiration (chemoheterotrophism)
without light.
Many of the protozoa form a resistant, dormant structure called a cyst. Parasitic protozoa
are identified by the active feeding stage, called a trophozoite, in addition to the cyst stage,
both of which may be found in the feces.
For our purposes, there are only 4 groups of protozoa that will be covered here: these
groups are separated by motility and cell structure.
• Amebas (representative: Ameba proteus)
• Flagellates (representative: Trypanosoma, Euglena)
• Ciliates (representative: Paramecium)
• Apicomplexa (representative: Plasmodium)
Many protozoa are found in the gut of warm-blood animals and cold-blooded animals, as well
as in insects such as termites and cockroaches. In addition, there are quite a few
protozoans that live in blood. You will see some of these examples in lab.
Amebas move by cytoplasmic streaming, having no motility structure. You will likely see
some freshwater amebas in the pond water, some of which may have tests or 'shells' that
surround their cytoplasm. The flagellates have flagella or an undulating membrane for
motility. The ciliates have cilia. The Apicomplexa have a unique arrangement of
microtubules, called the apical complex (used in the takeover of the host cell), in the cell.
This last class has most of the human and animal pathogens in it.
EXTERNAL LINKS:
Pond Water Critters –

http://microscope-microscope.org/applications/pond-critters/pond-critters.htm
movies of protozoa at Molecular Expressions -
http://micro.magnet.fsu.edu/moviegallery/pondscum.html
OBJECTIVES:
Identify common species of protozoa.

Differentiate among the major classes of protozoa.
Identify different types of motility.
Page 30
MATERIALS NEEDED:
prepared slides:
• Trypanosoma
• Plasmodium
fresh specimens:
• Ameba
• Paramecium
• pond water
cover slips
pipets for specimens
THE PROCEDURES:
1. Preparation of wet mounts using live protozoa

Paramecium
Ameba
pond water
• Using the pipet provided, go down to the bottom of the container or in the algae and
dirt to get your specimen to make a wet mount slide. Do NOT stir the specimen: you
will get fewer that way.
• Place the drop on a microscope slide and cover with a cover slip. Be sure that you
have one cover slip only to place on top of the slide.
• Start with the 10X and go to 40X. Oil-immersion will magnify too much for most pond
water protozoa. Begin on brightfield microscopy.
• Once you have found your objects on brightfield, change over to darkfield and phase-
contrast for even better viewing.
2. Observation of prepared, bought slides of blood parasites
Trypanosoma
Plasmodium
Use brightfield microscopy. Start with the 10X objective lens, ending up on 100X
Trypanosoma will be easy to see: it is far larger than the red blood cells. However,
Plasmodium will be difficult since the parasite will be inside of the RBCs. These are
small protozoa, and so have to be viewed with 100X magnification.
Page 31
QUESTIONS:
1. Draw the organisms seen.

Paramecium Ameba
Trypanosoma Plasmodium
2. Where is the malarial parasite Plasmodium located---in the RBC or in the plasma outside
of the RBC?
3. By what major criterion are the protozoa subcategorized into classes?
4. What motility structure does Trypanosoma use?
5. In which class does Plasmodium reside?
6. What organisms are in the kingdom Protista?
7. Which of the organisms seen in lab are intracellular parasites?

Page 32
Page 33
SIMPLE STAINING &

SMEAR PREPARATION
In order to stain the bacterial specimen for microscopy one must first prepare the smear on
the slide. This basically involves 3 steps----transferring a liquid suspension of the bacterium
on the slide, drying the smear, and then heating slightly to firmly attach the smear to the slide.
Once this is done, the staining procedure begins. This simple stain procedure has already
been done, using material from your own mouth, in a previous lab---PREPARATION OF
SPECIMENS. Refer back to the discussion and procedure in that lab exercise.
CAUTION: Do NOT make your smear suspensions too thick: The dye will not penetrate
well, and there will be far too many bacterial cells to see individual shapes and arrangements.
One needs to be careful about thick smears when taking the specimen from an agar medium.
RECOMMENDATION: You may find it helpful to draw a circle (wax pencil is best) on the
opposite side of the slide where you will spread your smear. This will help you later in
locating the smear, sometimes a problem when moving the slide back and forth looking for
your bacteria. The wax pencil is better than a marker because it will not wash off easily from
the glass.
OBJECTIVES:
Prepare a smear from a bacterial specimen.

Prepare a simple stain of the smear.
Use the microscope to identify features (shape, arrangement, size) of the bacterium.
MATERIALS NEEDED:
TSB culture of Staphylococcus epidermidis

TSA plate of E. coli
dye rack for staining
dye kit with reagents
clean microscope slides
wax pencil
blotting paper
immersion oil
prepared, bought slides of stained bacteria (mixed shapes, Bacillus, etc.)
THE PROCEDURES:
1. First, make sure that you have a few clean slides. We recycle our microscope slides
and the smears have to be removed from the glass. Bon Ami, a cleansing powder
similar to Ajax or Comet (but not as abrasive on the glass), works well to remove the
smear and the dye from the glass.
Page 34
o At the sink, pick up the bottle of Bon Ami, shaking some on top of the smear on
the slide. Add a couple of drops of tap water and make a thick suspension
(consistency of toothpaste).
o Use your finger to spread the Bon Ami suspension over each side of the slide,
and then wash WELL with tap water.
o Dry the slide.
2. You will make smears using 2 different types of cultures, a broth and an agar culture.
o Label the 2 slides with the names of your bacteria.
o Shaking the culture first, aseptically transfer a drop of the TSB culture using an
inoculating loop to a clean slide.
o From the agar plate culture, remove a small inoculum (use only a small amount
of large colonies) with a loop and suspend it in a small drop of distilled water.
Mix the culture well into the water drop.
3. Label 2 slides---Staph and E. coli.
4. Spread the smear suspensions over the glass slide so that it forms a thin layer and it is
the size of nickel or larger. NO COVER SLIP USED!
5. Let the slide air-dry—totally. This step facilitates the fixing of the smear to the slide.
6. Heat-fix the dry smear by running the slide quickly through the flame a few times. If
your fingers get hot, you have heat-fixed TOO MUCH. The heat-fixing will coagulate
some of the protein material and cause the suspension to adhere to the glass slide.
7. A different dye will be used for each bacterium---
E. coli and crystal violet
Staphylococcus and safrinin
8. Staining the smear can be done 2 ways, depending on whether the dye is in a dropper
bottle or in a jar:
o Place the slide on the wire over the stain tray, and place the slide on the wire
mesh. Flood the smear with the dye: let sit for 1 minute.
o Place the slide, end up, into the jar of dye for 1 minute. Be SURE that the dye
is full in the jar (there are stock solution bottles in the cabinets to fill them).
9. Wash the slide WELL with distilled water. Blot the smear slide with your bibulous
paper pad.
10. Refer to the lab exercise on MICROSCOPY for help with the microscope.
11. Focus on the sample using the 10X lens (Be SURE that you are on brightfield
microscopy).
12. Focus on the smear using the 100X oil immersion lens. Be sure to read the directions
in the MICROSCOPY exercise so that you know how to move to the 100X objective
lens using oil.
13. Identify the various shapes and arrangements of bacteria using the fields below.
14. Look at the prepared slides of various bacteria. You will see different shapes and
arrangements.
15. Return dirty slides to your slide box. Return prepared slides to the trays on the bench.
Page 36
QUESTIONS:
1. Record your results of staining.
E. coli Staphylococcus
2. Since everything on the slide will be the same color in simple staining, what you
possibly tell about the organisms?
3. Are all bacteria the same size?
4. What names are given to the shapes seen in the 2 bacteria used in lab?
5. Why heat-fix the smear?
6. Why are basic dyes more useful than acidic dyes when staining bacteria?
7. What is the disadvantage of having a really thick smear when staining?

Page 37
THE GRAM STAIN

The gram stain, originally developed in 1884 by Christian Gram, is probably the most
important procedure in all of microbiology. It has to be one of the most repeated procedures
done in any lab. Gram was actually using dyes on human cells, and found that bacteria
preferentially bind some dyes. The Gram stain is a differential stain, as opposed to the
simple stain done recently. Differential stains use 2 or more dyes, producing different colored
cells depending on their chemical and physical properties.
Before staining, the specimen must be mounted and

fixed on the slides, as previously done in the simple
staining technique. Because of the 2 dyes used in
the procedure---crystal violet and safrinin---as well as
the decolorizer acetone-alcohol, bacteria will fall into
2 groups based on their gram reactivity. Gram
positive bacteria retain the crystal violet even through
the decolorizor step: gram negative bacteria do not
retain the crystal violet, are decolorized, and then
pick up the safrinin dye. Both gram + and – bind to
the crystal violet: the key step to their differentiation
is the decolorization.
Take a look at the accompanying diagram of the

stain procedure and its effects on the bacterial
color. During the crystal violet-iodine step, the
bound molecules within the peptidoglycan of the
gram + cell wall are held tightly. The acetone-alcohol
actually causes the peptidoglycan molecules
(arranged in a latticework) to shrink, thereby holding
the crystal violet-iodine even tighter. In the gram –
cell, the outer lipopolysaccharide layer of the wall is dissolved by the decolorizer agents, and
because the peptidoglycan layer is so thin in that group of bacteria, the crystal violet is
leached out of the wall.
Although there is a standard routine and set reagents used in this stain, each person has to
find a particular method that works best for them. The many variables that can affect this
stain are age of the culture, amount of decolorizer used, the time of decolorization, the type of
organism (acid-fast bacteria and spores do not stain well), thickness of the smear, and the
general care of the stainer.
The most common reasons for false gram reactions?

• Some bacterial species tend towards gram variable, and will show both colors
although most often gram +.
• Over decolorizing the smear, too long a time.
• Using old cultures (preferably, the cultures should be 18-48 hours old).
CAUTIONS:
Page 38
o Make sure that your smear is not too thick: otherwise, either the dyes cannot
penetrate or the cells will not be decolorized adequately.
o Be sure that you properly time the decolorization step.
o Flood the smear with decolorizing agent evenly over the smear.
o Practice will give you consistent results.
OBJECTIVES:
Become proficient at performing the gram stain consistently and accurately.

Differentiate among various shapes, sizes, arrangements, and gram reactions of bacteria.
MATERIALS NEEDED:
unknown bacteria on various culture media

Gram negative control: E. coli on TSA slants or plates
Gram positive control: Bacillus subtilus in TSB
prepared, bought slides of various gram stained bacteria
dye kit
clean microscope slides
dye stain rack and container
immersion oil
blotting paper
THE PROCEDURE: done individually
We have cultures of E. coli and Bacillus for your table to gram stain also. This will give you
gram + and gram - controls to check your procedure against. It is helpful to divide the slide
into 3 sections, making smears of the 2 control bacteria along with the unknown bacterium.
There are also prepared, gram stained slides of bacteria of different shapes and sizes of
bacteria to look at.
1. Make sure that you have all of your materials---dye kits, rack, slides, cultures, water---
available. You do not want to be scrambling, looking for materials, once you have
started the stain procedure.
2. Refer back to the exercise SIMPLE STAINING to read about cleaning microscope
slides and how to make smears.
3. Make the bacterial smear from broth, slant, or plate.
o If taken from a broth, use 1-2 loopfuls of the broth solution.
o If taken from a solid agar medium (plate or slant), suspend the inoculum in a
drop of water on the slide and mix it well.
o Spread the suspension on the slide so that it covers an area at least the size of
a nickel, preferably a quarter.
o You might think about marking the smear area with a surrounding wax pencil
mark so that you can find the smear under the microscope easily.
o Place a piece of tape on the side of the smear so you know which way is UP.
4. Let the slide air-dry totally before proceeding on with the procedure.
5. Heat-fix the slide by holding the slide at one end with your fingers and quickly moving
it back and forth a few times over the flame.
Page 39
6. STAIN procedure
o Place slide into container of crystal violet, leaving in for 1 minute. Wash WELL
with water.
o Place slide into container of Gram's iodine, leaving in for 1 minute. Wash WELL
with water.
o Flood acetone-alcohol quickly on the slide, and wash off within 5-10 seconds
(from beginning of decolorizer added). Wash WELL with water.
o Place slide into container of safrinin, leaving in for 1 minute. Wash WELL with
water.
o Blot dry with bibulous paper.
7. Focus on smear using low power lens, ending up on 100X oil immersion. Be sure that
you have a drop of oil on the slide before rotating your 100X objective lens into place.
Page 40
QUESTIONS:
1. Critiquing your gram stain technique:

o Are the cells well distributed on the slide?
o Are the cells stained uniformly and is the gram reaction correct?
o Is the arrangement of the bacterium consistent across the various fields of vision?
2. What are the gram reaction, shape, and arrangement of Bacillus?
What are the gram reaction, shape, and arrangement of E. coli?
3. What color is E. coli when gram stained? Name the dye that gives it this color.
4. To what cell structure do the 2 dyes bind?
5. 2. List at least 3 differences between gram positive and gram negative bacteria.
6. Would it be useful to perform a gram stain on a mixed culture? Why?
7. If a gram stain gives you some valuable information on features of the bacterium,
what would be the benefit of ever performing a simple stain?
Page 41
THE ENDOSPORE STAIN

Endospore production is a very important characteristic of some bacteria, allowing them to resist
adverse environmental conditions such as desiccation, chemical exposure, extreme heat, etc.
Bacterial endospores are the most resistant structures of all living organisms, and they can live
in this dormant dehydrated state for hundreds and hundreds of years (even some documented at
thousands of years). Endospores are not for reproduction: One spore forms inside of the
vegetative cell. When the spore germinates, one vegetative cell will be produced. The stimulus
for sporulation can vary—nutrient depletion, desiccation, chemicals, heat, etc.
As a spore forms inside of the vegetative cell, the spore wall chemically changes and thicken.
This sporulation process changes the spore’s stainability, making it increasingly resistant to the
staining dyes, and so a gimmick—steaming---enhances the primary dye’s penetration. The
primary dye malachite green is a relatively weakly binding dye to the cell wall and spore wall. In
fact, if washed well with water, the dye comes right out of the cell wall, however not from the
spore wall once the dye is locked in. That is why there does not need to be a decolorizer in this
stain: it is based on the binding of the malachite green and the permeability of the spore vs. cell
wall. Spores will be a light green: Vegetative cell walls will pick up the counterstain safranin.
This lab will employ the Schaeffer-Fulton Method: there are variations of the spore stain
method.
The identification of spores is also very important for the clinical microbiologist who is analyzing
a patient's body fluid or tissue since there are not that many spore-forming genera. In fact, there
are two major pathogenic spore-forming genera, Bacillus and Clostridium, together causing a
number of lethal diseases---botulism, gangrene, tetanus, and anthrax, to name a few. Some
spore-forming species produce spores only under adverse environmental condition, while a few
species easily produce spores without much prodding.
OBJECTIVES:
Learn to perform the spore stain.

Identify spores on a bacterial smear.
MATERIALS NEEDED:
dye kit
dye stain rack
hot plate
paper towel (cut the size of the slide)
Bacillus culture on an TSA plate
THE PROCEDURES:
1. Prepare the bacterial smear of Bacillus, air-dry, and heat-fix.

2. Put a beaker of water (a metal beaker) on the hot plate and boil until steam is coming up
from the water. Then turn the hot plate down so that the water is barely boiling.
Page 42
3. Place the wire stain rack over the beaker which now has steam coming up from the boiled
water.
4. Cut a small piece of paper towel and place it on top of the smear on the slide. The towel
will keep the dye from evaporating too quickly, thereby giving more contact time between
the dye and the bacterial walls.
5. Flood the smear with the primary dye, malachite green, and leave for 5 minutes. Keep the
paper towel moist with the malachite green. DO NOT let the dye dry on the towel.
6. Remove and discard the small paper towel piece in the trash.
7. Wash really WELL with water and move the slide and wire rack from the boiling water to
the regular stain tray to finish up the last step in the procedure.
8. Place the smear in the stain jar or flood the smear with the counterstain dye, safrinin,
and leave for 1 minute.
9. Wash WELL with water. Blot dry with bibulous paper.
QUESTIONS:
1. Draw some spores along with some vegetative cells for comparison.
2. Can you find any spores still within the vegetative cell?
If so, notice where the spore is in the cell, if there is one---terminal, central, subterminal.
Why might that information be helpful to a microbiologist?
3. What is the purpose of the steam in this stain?
4. Why does there not have to be a decolorizer in this stain?
5. If you mistakenly did this stain on an acid-fast Mycobacterium, what color do you think the
cells would come out to be?
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THE ACID-FAST STAIN

The ability of the bacteria to resist decolorization with ACID alcohol confers acid fastness to
the bacterium. Acid-fast bacteria, of which there are very few---the major genus
Mycobacterium, have a high concentration of mycolic acid, a lipid, in their walls. This
differential stain is very important for diagnoses of leprosy and tuberculosis, caused by 2
different species of the genus Mycobacterium (commonly called AFB in clinical situations).
The method used in this lab is the Ziehl-Neelsen method: (there are 2 different methods of
AF staining).
As in the spore stain, steam is used as a gimmick to get the carbol fuschin primary dye to go
into the wall. Once in, it will not come out: But the acid alcohol decolorizer WILL take it out
of the nonacid-fast wall since the primary dye does not bind strongly to the cell wall. Acid-fast
bacteria are hot pink or fuchsia. Nonacid-fast bacteria are light blue.
OBJECTIVES:
Learn to perform the acid-fast stain.

Differentiate between acid-fast and nonacid-fast bacteria.
MATERIALS NEEDED:
cultures of Mycobacterium smegmatis and E. coli

dye kit + dye stain rack
hot plate and beaker
paper towel (cut the size of the slide)
THE PROCEDURES:
1. Prepare the bacterial smear, air-dry, and heat-fix. You will make a mixed suspension of
Mycobacterium and E. coli on one slide.
2. Put a beaker of water (metal beaker) on the hot plate and boil until steam is coming up
from the water. Then turn the hot plate down so that the water is barely boiling.
3. Place the wire stain rack over the beaker which now has steam coming up from the water.
4. Cut a small piece of paper towel and place it on top of the smear on the slide. The towel
will keep the dye from evaporating too quickly, thereby giving more contact time between
the dye and the bacterial walls.
5. Flood the smear with the primary dye, carbol fuschin, and leave for 5 minutes. Keep the
paper towel moist with the carbol fuschin. DO NOT let the dye dry on the towel.
6. Remove the piece of paper and discard in the garbage. Wash the slide really WELL.
7. Add the decolorizer acid alcohol (1% HCl + ethanol) and decolorize for 15-20 seconds.
8. Wash WELL with water.
9. Flood the smear or place the slide into a jar with the counterstain dye, methylene blue,
and leave for 1 minute.
10. Rinse with water. Blot dry with bibulous paper.
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QUESTIONS:
1. What is chemically unique about the Mycobacterium genus that causes it to be acid-
fast?
2. How is this stain procedure similar to the spore stain procedure?
3. If you gram stain Mycobacterium, although gram +, is never becomes a dark blue-
purple but rather stays a light purple. Why?
4. What is the color of E. coli when acid-fast stained? Acid-fast or not acid-fast?
What was the color of E. coli when gram stained?

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THE CAPSULE STAIN

The capsule is a thick polysaccharide or polypeptide(or both) layer around the outside of the
cell. It is nonionic so that the dyes that we commonly use will not bind to it. Two dyes, one
acidic and one basic, are used to stain the background and the cell wall, respectively.
The capsule gives added protection to the bacteria, making it virtually impossible to be
phagocytosed by white blood cells.
OBJECTIVES:
Identify capsules around bacteria.
MATERIALS NEEDED:
prepared slides of encapsulated bacteria
THE PROCEDURES:
These stains are bought and ready to use. Although they have cover slips, you still use oil
when on 100X magnification.
Be sure to remove the oil with the lens paper.

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QUESTIONS:
1. Why does the capsule NOT take in any dye?
2. What is the function of the capsule?
3. The capsule is a type of negative stain. What does that mean?
4. What is phagocytosis?
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TRANSFER OF BACTERIA USING

ASEPTIC TECHNIQUE
GENERAL GUIDELINES:
Safety
• Wear a lab coat and have your goggles on!
• ALWAYS disinfect the tables BEFORE and AFTER lab.
• Wash your hands with soap both BEFORE and AFTER lab, and, in addition, when you have a
spill.
• Be sure to have nonessential materials (the ONLY essential thing is the lab handout and a
notebook to write in) off of the table.
• Place test tubes in racks when working at your table: never lay the tubes down--they leak.
• Do not dump ANY microbial suspension down the drain--only in the discard area.
• The gas should be turned all of the way on, so that the level is parallel with the rubber tubing.
THE LAST PERSON ON THE TABLE CHECKS TO MAKE SURE THAT ALL 4 GAS JETS
ARE OFF!
• If there is a spill:
o Tell your instructor about it.
o Flood the area of the spill with disinfectant and leave on for 10 minutes before using
paper towels to soak up.
Beginning the lab
• Label all test tubes and petri plates with your name (initials), your table #, date, exercise #,
and name of organism.
• You always pick up your microorganisms as a set for your table, sharing them between
the table members, and discard them after use.
• Use masking tape only to mark on the tubes: You can use tape or mark directly on the agar
plates.
Technique
• ALWAYS use the proper aseptic technique when transferring cultures from one medium to
another.
• Keep test tube caps and petri dish covers on media to reduce contamination (matters not
whether it is sterile media or already cultured).
• Check for contamination in or on media. Remove contaminated media and place in discard
area.
Finishing the lab
• When using microbial cultures, take one of each organism for the table, use it, and then
DISCARD properly.
o Tubes in test tube rack discard
o Agar plate in autoclave bag
• REMOVE ALL labels from test tubes when discarding.
• Be sure to put any unused media tubes BACK into the racks if unused: Replace unused agar
plates back into bags if not used.
By now you know what media is: it is the nutrient-rich material that provides food to the
microbes. There are 3 forms of media:
• An agar medium contains agar (1.5-2%) as a solidifying agent for isolation and
purification.
• A broth medium has no agar: it is for fast, luxuriant growth.
• A semi-solid has a reduced percentage of agar, and, therefore, has qualities of both
agar and broth..
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In addition to nutrients and growth factors, and perhaps agar, there are additives such as
NaCl salt, pH buffers, and pH indicators that allow biochemical reactions to be identified.
In this exercise you will learn how to subculture bacteria, using a variety of culture media as
your inocula sources and as your new culture media. Different species of bacteria will be
used so that you can become familiar with different growth patterns. You will also have a
mixed culture with 2 species in order to learn how to best separate and isolate bacterial
species. Streak plates are a great technique for separating mixed cultures into visibly
separate species, which can then be isolated and grown as pure cultures on fresh media.
Each colony represents a different clone of cells, each originating as a single mother cell.
All agar plates are incubated UPSIDE DOWN (exceptions will be pointed out occasionally).
WHY?
• It reduces bacterial contamination since the bacteria, even if they get into the plate in
between the lid and bottom, would have to go UP to get to the agar.
• It reduces the possibility of water condensation that may be on the lid dropping onto
the agar, causing fluid to run across the agar medium.
ALWAYS check agar plates carefully to make sure that there are no mold or bacterial
contaminants on the plate: if so, discard the plate in the autoclave bag. Do the same with
any tube media that you pick up. If contaminated, discard the tube. or plate
If you see water running on the agar plate, you can do 2 things:
• Place the agar plate upside down in the 37C incubator with the top cracked.
• Get another agar plate.
OBJECTIVES:
Subculture bacteria in/on sterile media of various forms.

Eliminate potential contamination of bacterial cultures by using aseptic technique.
Practice hand coordination required in good transfer techniques.
Identify different ways by which bacteria grow in culture—in agar deeps, on agar slants, on
agar plates, in broths.
MATERIALS NEEDED:
set of cultures for the table:

a TSA slant culture of Bacillus subtilus
a TSB culture of Staph epidermidis
a TSA plate of E. coli
sterile media: (per person)
1 TSB
1 TSA slant
1 TSA plate
1 TSA agar deep
THE PROCEDURES:
BE SURE TO READ OVER THE STEP-BY-STEP DESCRIPTION OF TRANSFER

TECHNIQUE BELOW BEFORE PERFORMING THE TRANSFERS.
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THE TRANSFERS: (performed individually)
1ST session:
1. Subculture a broth culture of Staph epidermidis into a TSA deep.
2. Subculture a slant culture of Bacillus subtilus into TSB.
3. Subculture a plate culture of E. coli onto a TSA slant and onto a TSA plate.
4. Incubate all newly-made cultures at 25 or 37 degrees C, wherever your instructor directs
you.
2nd session:
1. Check each culture for the presence of bacterial growth.
2. Use the interpretation section at the end to determine the bacterium’s growth
characteristics on a slant, in broth, and in deeps. Check the agar plate culture for growth.
ASEPTIC TECHNIQUE:
1. Have both the culture that you are taking the inoculum from and the new, sterile medium
in front of you. Be sure that the new medium is already labeled so you do not confuse the
various cultures. Be sure that you have all inoculating equipment handy.
2. Pick up both tubes in the hand not using the inoculation instrument.
3. Heat the inoculating wire of the loop or needle until red-hot, and be sure that the ENTIRE
wire is sterilized. You are now ready to pick the inoculum from the
bacterial culture.
• Be sure to COOL your inoculation instrument a few seconds
before picking the inoculum. If you hear a sizzle as it touches
the medium, it was TOO HOT.
• Use an inoculating needle for agar deeps and an inoculating
loop for the agar plate and the broths.
4. Keeping the sterile inoculation instrument in your hand, remove
both tube caps with your little finger.
5. Run the tops of the tubes through the heat to create an updraft (taking air contaminants
AWAY from the tube entrance).
6. Holding the tubes at a 45 degree angle, remove the inoculum and QUICKLY place the
inoculum into the new medium tube.
7. Sterilize the tops of the tubes again (to eliminate potential air contamination again) and
replace the caps.
8. Heat the inoculating wire of the loop or needle again before placing on the table.
9. Incubate the plates and tubes on the 25 degree C shelf. (room temperature) Look at the
section below in INTERPRETION to read your tube results.
TAKING THE INOCULUM
--FROM A BROTH CULTURE:

The inoculum is obtained by first shaking the culture a bit, and then
going into the broth with the loop. A film of broth culture can be seen
across the loop as you remove it from the tube.
--FROM AN AGAR SLANT CULTURE:

The inoculum is picked off of the top of the slant, like a scraping
motion.
--FROM AN AGAR PLATE CULTURE:

The plate cultures have isolated colonies of bacteria growing on
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them. Pick only one colony or a bit of a colony, if big, with your loop. Lift the lid of the plate
just a bit, in order to get your inoculating loop into it: DO NOT TAKE THE ENTIRE LID OFF.
INOCULATING THE NEW MEDIUM

--INTO A BROTH:
The inoculum is just knocked around in the broth, and against the sides of the tube in the
broth.
--ONTO AN AGAR SLANT:

Place the loop with the bacteria into the slant tube, all the way down to the bottom of the
slant. There are 2 ways to inoculate the slant:
If your goal is to identify the type of growth pattern, then just bring the loop straight up the
slant.
If your goal is to have a luxuriant culture, inoculate in a zig-zag pattern, starting at the bottom
of the slant. This increases the surface area of the culture.
--INTO AN AGAR DEEP:

Use the needle to inoculate the deeps or semi-solid agars. Stab the inoculum down to the
bottom of the deep in a clean, straight stroke.
--ONTO AN AGAR PLATE:
1. In the pure culture technique exercise, you will learn how to make a streak plate but for
right now just inoculate the agar plate with a zig-zag motion from top to bottom of the
plate.
2. While doing this, lift the lid just enough to insert the loop underneath: this will reduce
contamination.
3. When streaking the agar, keep the loop horizontal and only streak the surface of the
agar: DO NOT DIG INTO THE AGAR.
4. Replace the lid and invert the plate. Incubate the plate.
INTERPRETATION OF RESULTS:
AFTER INCUBATION, check the growth patterns of all tubes and plates.
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QUESTIONS:
1. Why streak from the bottom of the agar slant medium up to the top in a straight line,
rather than a back and forth wavy inoculation from side-to-side on the slant?
2. Of what use is it to know what kind of growth pattern on an agar slant or a broth
medium an organism has?
3. Why use an agar plate to grow a bacterium rather than an agar medium slant or a
broth medium?
4. What is the purpose of flaming the mouth of the tube?
5. How do you determine if an organism is growing in a broth medium?

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PURE CULTURE TECHNIQUES

Most specimens (from animal tissue, plant tissue, or environmental samples) will be mixed,
with a variety of bacteria (or other microorganisms). A single gram of feces, for example, has
over 1010 bacteria and that gram would have over 20 different bacterial species.
Particularly in a medical setting, where a patient’s body fluid or tissue (skin, blood, spinal
fluid, urine) is sent to the microbiology lab for analysis, the specimens will most often be
mixed. In order to identify the bacteria and run antibiotic susceptibilities on the causative
agent of the infection, the microbiologist must be working with a pure culture of the bacterium.
This exercise begins with a mixed culture of bacteria and will end with, hopefully, pure
cultures of the 2 bacteria. Luckily, the 2 bacteria being used look different from each other
when growing on agar plates. Two different types of agar plate isolation techniques will be
used---streak and pour. Each method has advantages and disadvantages, and particular
uses. The same mixed culture will be used for both methods. Another technique, called the
spread plate, is used commonly for counts, as is the pour plate technique. It uses pre-made
agar plates, with the fluid inoculum being placed on top of the agar medium. The inoculum is
then spread around on the plate with a bent glass rod. That procedure will not be done
today.
This also gives you a chance to see how agar plates are made. Some of the TSA plates are
already made: others will be made by you. This requires that liquefied agar medium (sitting
in a water bath) be kept above the solidification temperature. Agar’s solidification
temperature is 42 degrees C, but its liquefaction temperature is 100 degrees C. When
sterilized in an autoclave or boiled, the medium will stay liquid until the temperature gets to 42
C. When that happens, the medium will solidify very fast. If it solidifies on you before you
finish making your cultures, it has to be disposed of (if not used yet, it can be placed back in
the sterile media racks).
REMEMBER…..
ALWAYS check agar plates carefully to make sure that there are no mold or bacterial
contaminants on the plate: if so, discard the plate in the autoclave bag.
If you see water running on the agar plate, you can do 2 things:
• Place the agar plate upside down in the 37C incubator with the top cracked.
• Get another agar plate.
OBJECTIVES:
Compare different agar plate isolation techniques.

Differentiate among various colony morphologies.
MATERIALS NEEDED:
a culture of E. coli and Serratia marcscens mixed together in TSB

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water bath at 45 degrees C
per person: 2 TSA plates (+ 2 more plates for 2nd session)
per table: 3 TSA agar deeps
3 sterile Petri dishes
1 TSA agar plate
THE PROCEDURES:
Each student will make 2 streak plates from the mix, incubated at 25 C
Each table will make a set of pour plates from the mix, incubated at 25 C.
Each table will make an extra streak plate from the mix, incubated at 37 C.
POUR PLATE TECHNIQUE (performed by the table)
You will dilute the bacterial sample by transferring loopfuls of the specimen from media tube
to media tube, with fewer bacteria ending up in successive tubes. The solidified agar deeps
will need to be boiled and liquefied (agar medium will liquefy at 100 C), then cooled down
BEFORE inoculating with the bacterial samples. Otherwise your bacteria will be cooked.
After adding the bacterial samples, the agar is poured into sterile petri dishes and incubated.
1. Place 3 TSA deep tubes into a glass beaker of water. Be sure that the water just
covers the agar, no higher. Place the heater on high. Once at boiling temperature,
the agar will be liquefied. Using the test tube holder, remove the hot agar tubes to a
test tube rack, carry it over to the water bath, and place into the tube racks in the water
bath. The water bath is set at 45 degrees C. It will take about 15 minutes for the hot
agar medium to cool from 100 to 45 C.
2. Obtain sterile Petri dishes (make sure that cover stays on) and label as #1, #2, and #3
on the bottom of the dish.
3. Remove 1 tube of liquefied TSA from the water bath and take it to your table. DO
NOT carry all 3 tubes at once: the others will solidy before you can inoculate them.
4. Pick up a loopful of your inoculum from the mixed bacterial culture and transfer it
ASEPTICALLY into the first tube of liquefied TSA.
5. Mix this tube #1 by rotating it quickly between your 2 hands: If done adequately, there
will be good mixing of the bacteria into the medium.. DO NOT invert the tube of agar.
6. From tube #1, aseptically transfer a loopful of the medium aseptically into tube #2.
7. BE SURE THAT YOUR PETRI DISH IS SITTING RIGHT-SIDE UP. Pour the entire
contents of tube #1 into the Petri dish marked #1. Open the larger top of the dish
RIGHT BEFORE pouring the agar into the dish, and replace the top as soon as
finished. Do this quickly. Gently swirl the closed plate about 4 times in a large dinner
plate-sized circle to spread the bacterial out well in the agar.
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8. Mix tube #2. From this tube, aseptically transfer a loopful of the medium aseptically
into tube #3. Mix the tube well by rotating it between your hands quickly.
9. Pour the entire contents of tube #2 into the bottom of the Petri dish marked #2. Gently
swirl the closed plate in large circles o spread the bacterial out well in the agar.
10. Mix tube #3. Pour the entire contents of tube #3 into the bottom of the Petri dish
marked #3. Gently swirl the closed plate in large circles to spread the bacterial out
well in the agar.
11. Let the 3 plates solidify for at least 15 minutes before incubating. Invert the agar
plates when placing them at 25 C.
12. After incubation, check the 3 pour plates for colonies and their features in/on the
agar.
STREAK PLATE TECHNIQUE: (performed individually and as a table)

Subculture a mix of E. coli and Serratia marcescens on a TSA plate, using 2 different streak
techniques
1ST session: each student streak a plate from the mix, and the table does an extra plate
In this first technique, you will flame the loop between each plate section, thereby diluting the
sample as you move from section to section. Remember that you go into the mixed culture
tube ONLY ONCE, before the first section.
1. Until you become well-acquainted with this procedure, you might want to draw the 3
sections that you will streak inside of, on the back (bottom of plate containing agar
medium) with a sharpie pen.
2. Pick up a loopful of your inoculum from the mixed bacterial culture. Using a sterile agar
medium plate, streak a vertical line straight down. While doing this, lift the lid just
enough to insert the loop underneath: this will reduce contamination.
When streaking the agar, keep the loop horizontal and only streak the surface of the
agar: DO NOT DIG INTO THE AGAR.
3. Move the loop in a zig-zag pattern across the agar until 1/3 of the plate is covered,
finishing the first section. Remember to close the lid on the Petri dish between
streakings.
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4. Sterilize the loop in the flame and let it cool before continuing to spread the bacteria.
You can do this by 1) sticking the hot loop in the agar at the edge of the agar away
from the bacteria, or 2) just holding the loop for a few seconds while it cools.
5. Rotate the plate about 90 degrees and spread the bacteria from the first streak into a
second area using the same zig-zag spread technique. Lift the lid only enough to
effectively streak.
6. Sterilize the loop again. Rotate the plate about 90 degrees and spread the bacteria
from the second streak into the 3rd area in the same pattern. Replace the lid.
7. Sterilize the loop again. Invert the plate. Incubate the plate at 25 C.
8. Each table will also make an extra streak plate from the mix, incubating it at 37 C.
This plate will be compared to one incubated at 25 C.
Second streak technique:

This is identical to a procedure performed in an earlier lab.
Incubate the plate inverted at 25 C. Each student will
streak agar plates using these 2 techniques.
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2nd session:
1. Check the agar plate culture of the mixed culture for 2 very different colony types. E.
coli colonies will be rather transparent and large. Serratia marcescens colonies will be
pink/orange, about the same size as E. coli. Also, check your streak technique or get
your instructor to do so, for feedback.
2. Use your mixed culture from your agar plates to produce 2 pure cultures of the E. coli
and Serratia marcescens. Stay away from merging colonies or close colonies.
Subculturing in that situation will increase the chances of picking up another mixed
culture, consisting of the 2 species that were merged together. ALWAYS pick a well-
isolated colony when subculturing.
a. Pick a colony of each of the 2 different species of bacteria onto 2 new TSA agar
plates, using your best aseptic streak technique.
b. Incubate at 25 degrees C.
c. Check the plates for purity the next period.
INTERPRETATION OF RESULTS:
1. AFTER INCUBATION, check the growth patterns of all plates.

Compare the size, shape, and location of colonies on the 3 kinds of plates.
Where are the colonies in the pour plates? Why?
Why is there so much variation in colony sizes and shapes in the pour plates?
Which method is best for accurate counting?
Which method is the easiest to do?
Which method gives easy access to the colonies---for subculturing to another medium?
Which method has the greatest space between the colonies?
2. Compare the streak plates incubated at 25 C and 37 C. Check pigmentation.
3. Compare your 2 streak techniques.

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QUESTIONS:
1. When performing the streak technique, why do you cross over back the 2nd streak
section back into the 1st section, and from the 3rd section back across the 2nd
section?
2. Why use a streak plate to grow a bacterium rather than an agar medium slant or a
broth medium?
3. What is the purpose of flaming the mouth of the tube?
4. On the agar plate culture of E. coli and Serratia marcescens mixed together, were the
colonies in the first section of the plate the same size as those in the 3rd section?
Explain why this would happen.
5. Why are the colonies within the agar of a pour plate smaller than those on the surface
of the agar?
6. What difference was seen in the Serratia colonies grown at 25 C and 37 C? Why?
7. What procedure has been used to make this culture plate---pour, spread, or streak?
Does it have good isolation? Why?

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BACTERIAL COLONY MORPHOLOGY

Bacteria grow on solid media as colonies. A
colony is defined as a visible mass of
microorganisms all originating from a single
mother cell, therefore a colony constitutes a
clone of bacteria all genetically alike.
In the identification of bacteria and fungi

much weight is placed on how the organism
grows in or on media. This exercise will help
you identify the cultural characteristics of a
bacterium on an agar plate---called colony
morphology. Although one might not
necessarily see the importance of colonial
morphology at first, it really can be important
when identifying the bacterium. Features of
the colonies may help to pinpoint the identity
of the bacterium.
In the accompanying picture of a mixed culture, an agar plate that has been exposed to the
air and many different colony morphologies can be identified. Eight obviously different
colonies are numbered: some colony types recur in various areas of the plate (note # 3 and
# 4). Not only are pigment differences seen, but also size, edge, pattern, opacity, and shine.
Two circles have been drawn around merging colonies, where the species of the 2 colonies
are different. Trying to pick a bit of one of those adjacent colonies increases the chances of
picking up another mixed culture, consisting of the 2 species that were merged together.
ALWAYS pick a well-isolated colony when subculturing.
WHOLE SHAPE OF COLONY

SIZE OF COLONY (measure with a millimeter rule), less than 1mm = punctiform (pin-point).
EDGE/MARGIN OF COLONY: magnified edge shape
CHROMOGENESIS (pigmentation): white, buff, red, purple, etc.
Some pigments are water-soluble, others are not. If you take a large inoculum and
place it in a tube of water or saline, do you see color? Do you see any pigment if the
organism is growing in a broth medium?
OPACITY OF COLONY:
transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking
through frosted glass), iridescent (changing colors in reflected light)
ELEVATION OF COLONY (turn the plate on end to determine height)
SURFACE OF COLONY:
smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)
CONSISTENCY:
butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks
apart), mucoid
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Science Buddies, Interpreting Plates
http://www.sciencebuddies.org/mentoring/project_ideas/MicroBio_Interpreting_Plates.shtml
OBJECTIVES:
Describe features of colonies.

See variations in colonial morphology among various species of bacteria.
MATERIALS NEEDED:
agar plates of various bacteria (Pseudomonas, Chromobacterium, Micrococcus, Bacillus,

Streptomyces, Streptococcus, and Neisseria)
agar plates of E. coli and Serratia marscescens from last period
THE PROCEDURES:
1. Use a plate which has well-isolated colonies. Look at the largest colonies with the
naked eye to determine general shape and chromogenesis.
2. Use a dissecting/stereoscopic microscope for more detail. Place the plate RIGHTSIDE
UP on the stage, leaving the petri dish cover ON (Otherwise, your culture will become
contaminated.) There are 2 lenses on our scopes—10X and 20X: the black lens knob
is on the right side of the head of the microscope. The magnification is especially
helpful for the study of elevation, surface, opacity, size, and edge. There are 2 lights
on these microscopes that you might find helpful, either using one at a time, or both, or
even sometimes without them. Two small black rotating knobs on either side of the
base control the 2 lights, one light from above and one light from below the stage.
3. Or you may want to use the Quebec colony counter since it has a magnifying glass,
and a light behind the plate stage. Make sure that the dish is right-side up.
4. If you see water condensation on the lid cover, take a KimWipe and carefully remove
the water from the cover, then quickly replacing the cover on the dish.
5. In order to determine CONSISTENCY, you need to use your inoculating loop or needle
to pick up the colony and determine the consistency of the inoculum material as the
loop leaves the agar medium.
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QUESTIONS:
1. Could you identify a contaminant bacterium on your pure culture plates? How?
2. Define a clone.
3. Which organism’s colony is easy to pick up using a inoculation loop/needle? Which

one is difficult?
4. Are the sizes of the colonies on a single petri-plate similar?
5. Why might the colonies of a particular bacterium vary when streaking onto different
plate media?
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PIPETTING & DILUTIONS

Dilutions are used many times during the semester in the microbiology lab, for a variety of
purposes. Therefore, it is important that each person understand how to use the pipette, how
to read the pipette accurately, and how to determine what dilution was produced.
It is a common practice to determine bacterial counts for both liquid and solid specimens---
suspensions of E. coli in nutrient broth all the way to soil samples and hamburger meat. The
following protocol is a step-by-step procedure to working dilution problems, and includes
some practice problems at the end.
The purpose can be determination of bacterial, fungal, or viral counts (commonly called
colony-forming units, CFUs, for bacterial or fungal counts, or plaque-forming units, PFUs, for
viral counts).
USE OF THE PIPETTE
Be SURE that you know how to read the lines on the various sizes of pipettes.
Remember that the reading is taken at the bottom of the meniscus. (the reading on the pipet
in picture is at 8.1ml)
Place the end of the pipette straight into the opening on the pi-pump (green pi-pump for 5 and
10ml, blue pi-pump for 1ml).
Place the pipette tip into the solution; rotate the pi-pump wheel so that the fluid ascends.
Slowly move the wheel so that you correctly deliver the required amount.
© Peg Johnson
In this little exercise, no aseptic technique is required, but accuracy is all important. You will
be starting out with a methylene blue-colored water and diluting it in various ways. If done
correctly, the last tube of each of the 4 dilution sets will be the EXACT shade of blue. If not,
you have inaccurately diluted the samples---either pipetting inaccurately or adding water to
the tubes inaccurately.
OBJECTIVES:
Become proficient with the use of the pipette.
Identify different ways to dilute.
Learn how to solve a dilution problem.
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MATERIALS NEEDED:
flask of blue water

nonsterile pipettes - 1, 5, and 10ml
8 nonsterile test tubes
blue and green pi-pumps
THE PROCEDURE:
1. Fill a tube with methylene blue water.

2. Prepare the following dilution blanks with
tap water using a 10ml pipette and the
green pi-pump---4.5ml, 9ml, 9.9ml, 9.5ml,
4ml, 3ml, 12ml.
3. Make 4 sets of dilution tubes as seen in
accompanying diagram. Use the most
appropriate pipette size to transfer the
solution, slowly and accurately aliquot the
correct amount into the new tube, AND mix
the tube contents (either roll the tube
between your hands or pipet up and down
a few times).
• Dilution set 1: Transfer 0.5ml of blue

water into the 4.5ml of water, then 1ml
of tube 1 into the next tube of 9ml water.
• Dilution set 2: Transfer 1ml of blue
water into the 4ml of water, then 0.5ml
of tube 1 into the next tube of 9.5ml.
• Dilution set 3: Transfer 1ml of blue
water into the 3ml of water, then 0.5ml
of tube 1 into the next tube of 12ml.
• Dilution set 4: Transfer 0.1ml into the
9.9ml of water.
4. Interpretation of your dilution tubes---

a. Record the dilution values of your tubes in the LAB PREPORT SHEET below.
b. Check the last 4 tubes of each set against each other to make sure that they
are the same shade of blue.
SOLVING DILUTION PROBLEMS:
THE STANDARD FORMULA =

_________colony count on an agar plate_________________
total dilution of tube (used to make plate for colony count) X amount plated
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To work the problem, you need 3 values---a colony count from the pour or spread plates, a
dilution factor for the dilution tube from which the countable agar plate comes, and the
amount of the dilution that was plated on the agar plate.
STEP 1: Determine the appropriate plate for counting:

Look at all plates and find the one with 30-300 colonies (or plaques), preferably. Greater than
300 and less than 30 is a high degree of error. Air contaminants can contribute significantly to
a really low count and a high count can be confounded by error in counting too many small
colonies.
Use the total dilution for the tube from where the plate count was obtained.
If duplicate plates (with same amount plated) have been made from one dilution, average the
counts together.
STEP 2: Determine the total dilution for the dilution tubes:

Dilution = amount of specimen transferred divided by the [amount of specimen transferred +
amount already in tube].
Determine the dilution factor for each tube in the dilution series.
Multiply the individual dilution of the tube X previous total dilution
To calculate this dilution series:
Determine the dilution of each tube in the set.

dilution factor for a tube = amount of sample
amount of sample + amt. of diluent in tube
But after the first tube, each tube is a dilution of the previous dilution tube.
SO…..
total dilution factor = previous dilution of tube X dilution of next container
FOR THE ABOVE DILUTION SERIES:

0.5 ml added to 4.5ml = 0.5/5.0 = 5/50 = 1/10 for 1st tube
1ml added to 9ml = 1/10 for 2nd tube
previous dilution of 1/10 (1st tube) X 1/10 (2nd tube) = total dilution of 1/100
STEP 3: Determine the amount plated (the amount of dilution used to make the particular
pour plate or spread plate).
There is nothing to calculate here: the value will be stated in the procedure, or it will be given
in the problem.
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SOLVING ABOVE PROBLEM:

1. The countable plate is the one with 45 colonies.
2. The total dilution of the 2nd tube from which that pour plate was made = 1/102 X 1/10
(equals 10/100) = 1/103.
3. The amount used to make that pour plate = 0.1ml (convert to 1/10 - cannot multiply
fractions and decimals together).
45 colonies = 45 X 104 = 4.5 X 105 (scientific notation) OR 450,000/ml

1/103 X 1/10
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QUESTIONS:
1. What is the meniscus?
2. If you transfer 0.1ml of a sample into a 99.9ml saline blank, what is

the dilution?
3. How much fluid is IN the pipette at right?
4. For the dilution tubes with colored water, fill in the following values
SET 1 TOTAL DILUTION

tube 1 1/10
tube 2 1/100

tube 1 _______
tube 2 _______

tube 1 _______
tube 2 _______
SET 4 tube 1 DILUTION ________
5.
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6.
7.
Page 69
COUNTING BACTERIA
Many studies require the quantitative determination of bacterial populations. The two most
widely used methods for determining bacterial numbers are the standard, or viable, plate
count method and spectrophotometric (turbidimetric) analysis. Although the two
methods are somewhat similar in the results they yield, there are distinct differences. For
example, the standard plate count method is an indirect measurement of cell density and
reveals information related only to live bacteria. The spectrophotometric analysis is based on
turbidity and indirectly measures all bacteria (cell biomass), dead and alive.
The standard plate count method consists of diluting a sample with sterile saline or
phosphate buffer diluent until the bacteria are dilute enough to count accurately. That is, the
final plates in the series should have between 30 and 300 colonies. Fewer than 30 colonies
are not acceptable for statistical reasons (too few may not be representative of the sample),
and more than 300 colonies on a plate are likely to produce colonies too close to each other
to be distinguished as distinct colony-forming units (CFUs). The assumption is that each
viable bacterial cell is separate from all others and will develop into a single discrete colony
(CFU). Thus, the number of colonies should give the number of bacteria that can grow under
the incubation conditions employed. A wide series of dilutions (e.g., 10-4 to 10-10) is normally
plated because the exact number of bacteria is usually unknown. Greater accuracy is
achieved by plating duplicates or triplicates of each dilution, although we will not be doing that
in this exercise.
Increased turbidity in a culture is another index of bacterial growth and cell numbers
(biomass). By using a spectrophotometer, the amount of transmitted light decreases as the
cell population increases. The transmitted light is converted to electrical energy, and this is
indicated on a galvanometer. The reading, called absorbance or optical density, indirectly
reflects the number of bacteria. This method is faster than the standard plate count but is
limited because sensitivity is restricted to bacterial suspensions of 10 7 cells or greater. The
procedure for the spectrophotometer use is at the end of this exercise.
Why Is E. coli used in this exercise? When working with large numbers and a short time
frame, one of the most reliable microorganisms is one that has been used in previous
experiments, namely, Escherichia coli. E. coli has a generation time at 37C of 20 minutes.
Thus, it reproduces very rapidly and is easy to quantify (i.e., the number (biomass) of viable
E. coli cells in a bacterial culture can be easily determined by spectrophotometry).
OBJECTIVES:
Correlate absorbance value for a bacterial suspension with an accurate bacterial count.
Become proficient at dilutions.
Become proficient at performing a standard plate count and determining bacterial counts in a
sample.
MATERIALS NEEDED: per table (exercise performed by table)
24-hour 10ml nutrient broth culture of Escherichia coli

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4 sterile 99-ml saline blanks
1-ml pipettes with pi-pump
6 petri plates
6 agar pour tubes of nutrient agar (plate count agar)
48 to 50C water bath
boiling water bath
Bunsen burner
6 micro-cuvettes and rack
1 micro-cuvette holder
spectrophotometer
4 tubes of 5ml nutrient broths
4-5ml pipets and pi-pump
THE PROCEDURES:
STANDARD PLATE COUNT
BE SURE TO SAVE YOUR ORIGINAL TUBE OF E.COLI FOR THE NEXT SECTION!
START BOILING WATER TO LIQUEFY NUTRIENT AGAR DEEPS before starting this
section. When the deeps are liquefied, they can be placed into the water bath to cool so that
the pours can be made without killing the bacteria. Cooling takes about 20 minutes.
1. Label the bottom of six petri plates 1-6. Label four tubes of saline 10-2, 10-4, 10 -6, and
10-8.
2. Using aseptic technique, the initial dilution is made by transferring 1 ml of E. coli
sample to a 99ml sterile saline blank (figure below. This is a 1/100 or 10-2 dilution.
3. The 10-2 dilution is then shaken by grasping the tube between the palms of both hands
and rotating quickly to create a vortex. This serves to distribute the bacteria and break
up any clumps.
4. Immediately after the 10-2 dilution
has been shaken, uncap it and
aseptically transfer 1ml to a
second 99ml saline blank. Since
this is a 10-2 dilution, this second
blank represents a 10-4 dilution of
the original sample.
5. Shake the 10-4 dilution vigorously
and transfer 1ml to the third 99ml
blank. This third dilution
represents a 10-6 dilution of the
original sample. Repeat the
process once more to produce a
10-8 dilution.
6. Shake the 10-4 dilution again and aseptically transfer 1.0 ml to one petri plate and 0.1
ml to another petri plate. Do the same for the 10-6 and the 10-8 dilutions.
7. Remove one agar pour tube from the 48 to 50C water bath. Carefully remove the
cover from the 10-4 petri plate and aseptically pour the agar into it. The agar and
sample are immediately mixed gently moving the plate in a figure-eight motion or a
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circular motion while it rests on the tabletop. Repeat this process for the remaining
five plates.
8. After the pour plates have cooled and the agar has hardened, they are inverted and
incubated at 25C for 48 hours or 37C for 24 hours.
9. At the end of the incubation period, select all of the petri plates containing between 30
and 300 colonies. Plates with more than 300 colonies cannot be counted and are
designated too many to count (TMTC). Plates with fewer than 30 colonies are
designated too few to count (TFTC). Count the colonies on each plate. A Quebec
colony counter should be used.
10. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the
number of colonies by the dilution factor multiplied by the amount of specimen added
to liquefied agar.
number of colonies (CFUs) = # of bacteria/ml
dilution X amount plated
11. Record your results.
TURBIDIMETRY DETERMINATION OF BACTERIAL NUMBERS
THIS SECTION DOES NOT HAVE TO BE DONE ASEPTICALLY!
1. Put the ORIGINAL tube of E. coli and four tubes of the sterile
NB in a test-tube rack. Each tube of NB contains 5 ml of sterile
broth. Use four of these tubes (tubes 2 to 5) of broth to make
four serial dilutions of the culture (figure 2).
2. Transfer 5ml of E. coli to the first tube of NB, thoroughly
mixing the tube afterwards. Transfer 5ml from that tube to the
next tube, and so on until the last of the 4 tubes has 5ml
added to it. These tubes will be ½, 1/4, 1/8, and 1/16 dilutions.
3. The directions for spectrophotometer use are BELOW.
a. The wavelength is preset somewhere between 550-
600nm. DO NOT change it! (Instructor will set the
wavelength)
b. Standardize the spectrophotometer as directed.
c. Obtain the 6 micro-cuvettes. The cuvettes will look like either of the
2 shown to the right. The lined or etched sides of the cuvettes
face you, with the clear sides facing the light source. The micro-
cuvette must contain1ml for the spectrophotometer to read the
fluid, but you can guesstimate the amount by eyesight. Notice the
arrow on picture above showing where level of fluid must be.
d. The BLANK used to standardize the machine is sterile nutrient broth: it is called
the BLANK because it has a sample concentration equal to zero. Pipette 1ml of
the sterile NB into one of the micro-cuvettes. Place into the black cuvette holder
(red line towards you), close the cover and read. Save BLANK to re-standardize
the machine to infinity absorbance and zero absorbance before each reading
because the settings tend to drift.
e. Pipette 1ml of the original bacterial specimen into a second micro-cuvette. Place
in cuvette holder and read. When read, discard micro-cuvette into bleach
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container on your table. Next pipette the ½ dilution into the third cuvette and read
it. Repeat this with the ¼, 1/8, and 1/16 dilutions.
Things to remember :
o Place micro-cuvette with parallel lines facing you into cuvette holder.
o Close the hatch when reading the spectrophotometer so no light enters.
o Re-standardize between readings to account for drift.
o Mix the dilutions before pipetting into the micro-cuvette to read
absorbance.
o Read to the nearest thousandth (0.001) on the absorbance digital display.
4. Record your values, along with the dilutions that they came from. Using the plate count
data, calculate the colony-forming units per milliliter for each dilution.
DATA COLLECTION
dilutions absorbance (X) # of bacteria (Y)
original E. coli
1/2
1/4
1/8
1/16
1. Fill in your absorbance values for the 5 tubes read in the spectrophotometer.
2. Calculate the number of bacteria in the original tube of E. coli, and place that value in
the top right cell of the table.
3. Calculate the approximate numbers of bacteria in the ½, 1/4, 1/8, and 1/16 by halving
the number in the cell above.
4. Plot these 5 coordinates on a graph, using EXCEL software (it is available in the
computer labs). The DIRECTIONS on how to use the software is at end of exercise.
5. Here is an example of a graph.
YOU MUST HAVE EQUAL INTERVALS ALONG BOTH X AXIS AND Y AXIS.
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QUESTIONS:
1. DATA COLLECTION:
dilutions absorbance (X) # of bacteria (Y)
original E. coli
1/2
1/4
1/8
1/16
2. Why do a standard plate count when running turbidity values the first time?
3. If you have a graph for E. coli, can it also be used for another bacterium like Staph?
4. How is transmission different from absorbance?
5. Give the formula for determining bacterial counts.
6. Give the bacterial count per milliliter of E. coli suspension in the original culture tube.
Page 74
USE OF THE SPECTROPHOTOMETER
Light entering a cloudy solution will be absorbed. A clear solution will allow almost all of the
light through. The amount of absorbance can be determined by using a spectrophotometer,
which measures what fraction of the light passes through a given solution and indicates on
the absorbance display the amount of light absorbed compared to that absorbed by a clear
solution.
Inside, a light shines through a filter (which can be adjusted by controlling the wavelength of
light), then through the sample and onto a light-sensitive phototube. This produces an
electrical current. The absorbance meter measures how much light has been blocked by the
sample and thereby prevented from striking the phototube. A clear tube of water or other
clear solution is the BLANK and has zero absorbance. The amount of substance in the
solution is directly proportional to the absorbance reading. A graph of absorbance vs.
concentration will produce a straight line.
The following numbered steps

coincide with the numbers on the
spectrophotometer at right.
1. The wavelength and filter have already been set. DO NOT change it. (Instructor will
tell you what the wavelength should be so you can double check the displayed
setting.) The 1st white button (labeled #3) on the Display panel will toggle between
MODE and TRANSMITTANCE.
2. Pressing the MODE button will toggle to the TRANSMITTANCE mode. With the
cuvette chamber empty and the cover closed, use the power switch knob on the LEFT
to adjust the digital display to 0% Transmittance. .
3. Set the display mode to ABSORBANCE by pressing the MODE control key until the
appropriate red LED is lit.
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4. Fill a micro-cuvette with 1ml of
NB to serve as the BLANK. Wipe
it free of moisture and
fingerprints with a KIMWIPE and
insert it into the cuvette holder
with the "V" mark facing you (OR
we may have the cuvettes
which have parallel lines on
the sides---if so, the lined
sides will FACE you). Then
place into the cuvette chamber.
Close the cover.
5. With the right-hand knob, adjust
the display to read zero absorbance.
6. Remove the BLANK from the cuvette chamber, and insert the holder and micro-
cuvette with your sample into the chamber.
7. Read the absorbance value directly from the digital display.
8. It is necessary to reset the machine to infinity absorbance and zero absorbance before
each reading because the settings drift a bit. BOTH infinity and zero absorbances
must be re-set.
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Plotting Data Using Microsoft Excel
Prepared by Dr. Sara Perez-Ramos, modified by Jackie Reynolds
Plotting Best-Fit Lines of Absorbance vs. Numbers data
1. Double click on the Microsoft Excel icon to open the program. You will see a grid. Each box in the
grid is called a cell. Each cell has an “address” made up of a letter indicating the column the cell
is in and a number indicating the row the cell is in, e.g. the upper left cell is A1.
2. Double click on cell A1 to start entering information. Using the diagram on this next page as a
reference, enter the data of bacterial numbers and absorbance readings of your table.
3. Click on cell A2 with the left mouse button (LMB) and drag the mouse to highlight the area
containing all the data points (go down to cell B8). The screen should look like this:
4. Once the data is highlighted, look on the upper toolbar and click on “Insert”, then on “Chart”. A
box with graph options will appear. Select “XY(Scatter)” and click on “Next”. (Leave the chart
sub-type alone: the top chart should be highlighted.)
Your data will appear already plotted on an x-y axis system. Now you will learn how to set-up the
axes, write the title of the graph and write the legend. You will also command the computer to draw
best-fit lines and calculate the slope of each plot. You can also choose to make some cosmetic
touches (changing the size of fonts, colors of the plot etc.…) on your graph before printing.
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5. Find “Series” towards the top of the graph and click on it. “Series1” should appear highlighted. To
the right of this there is an empty space for you to write the NAME of this plot: click on this empty
box and type “Absorbance vs. Bacterial Numbers”.
6. Click on “Next”. Another box will appear with options to modify titles on the graph.
a. Make sure that in the window “Chart Title” your graph is titled (e.g. Using Turbidemetry to
Guesstimate Bacterial Numbers”).
b. Click on space below Value(X)Axis and type: Absorbance
c. Click on space below Value(Y)Axis and type: Bacterial Numbers (X 106)
d. Click on “Axes” (upper left of graph) and make sure that Value(X)Axis and Value(Y)
Axis have a √ .
e. Click on “Gridlines” and add lines. Click on “Major Gridlines” on the x-axis and y-axis.)
f. Click on “Legend” and choose the location of the legend (I chose bottom).
7. Click on “Next” click on the button titled Place chart as object in sheet 1 (NOT as new sheet).
Click on “Finish”.
Your graph will appear on the screen as shown below. You can alter its size by clicking the LMB on
the black squares around the graph and dragging the mouse with the LMB pressed down.
8. Now you are going to add the line that goes through the data points (trendline). Click on the
graph to make sure that the black squares around the graph are there.
Move the pointer to any of the data points of a particular series (any ∆, for example). As you
do this, information about this data point will appear on the screen. Press the RIGHT mouse
button. A box with choices appears. Select and click on “Add Trendline”. Another box will
appear. Select Linear (should already be highlighted). Before clicking OK, go to “Options” on
the top of the box. Click on “Display equation on chart”. Click on “OK”. The graph will show a
best-fit line. On the graph you should see an equation which gives you the value of the
slope for the line).
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THE GRAPH SHOULD LOOK LIKE THE PICTURE ABOVE, BUT WITH A LINE AND THE SLOPE
FORMULA.
9. Cosmetic changes for graphs:
a. Changing the size of the font used for the equation: Click on the equation to be changed.
Click the RIGHT mouse button. Select and click on “Format Data Labels”. Click on “Font” and
reduce the font size (I chose 8). Click on “OK”.
b. Changing the position of the equation: Click on the equation and drag that box to the desired
position.
c. Changing the size of the axis label: Click on the desired label to change (e.g. the x axis label
that says “Time in seconds”). Click the RIGHT mouse button. Select and click on “Format
Axis Title”. Click on “Font” and reduce the font size (I chose 10). Click on “OK”. Do the same
for the other axis.
You may also want to alter the size of the numbers on the scale. Click the RIGHT mouse button
on top of the scale numbers. Click on “Format Axis”, then set the font to the desired size (I chose
8). Do this for the scale on the other axis also.
d. To edit the Legend box: Click on the Legend box. Click on The legend box until you see
black squares around its box. Press the RIGHT mouse button, select and click on “Clear”.
You can also alter the size of the font for the legend items. Click on top of 1/2x [enzyme](for
example) with the RIGHT mouse button until black squares around it appear. Select “Format
Legend”, and change the font size as desired (I chose 10). Do this for all the items in the
legend.
e. To change the color of the graph background: Press the RIGHT mouse button while the
cursor is on the gray area. Select and click on “Format Plot Area”. You may want to choose
a white background if your printer is black and white.
10. Go to the top toolbar. Click on “File”, “Print Preview” and check if the graph is ready to print. If it
is ready, click on “Print” and “OK”. If it’s not ready try more cosmetic changes as desired, until
you are satisfied with the product.
11. Before proceeding any further, you want to be sure that all your work is saved. Insert your 3½”
diskette in the computer. Go to “File”, “Save As” and choose A: 3½” drive as your destination.
Name the file: EXAMPLE: turbidimetry.xls.
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ENVIRONMENTAL CONDITIONS &

BACTERIA GROWTH
Many environmental conditions can affect microbial growth---temperature, pH, osmotic
pressure, radiation, and barometric pressure. PH is the value determined by the
concentration of hydrogen ions in a liquid, represented by the formula:
[H+] = moles of H+/liter
The value represents a base 10 logarithm, and pH therefore defines a logarithmic scale of
acidity. The difference in numbers on the pH scale (0 to 14) is 10-fold. That is, pH 8 is 10X
more basic than pH 7 and pH 3 is 100X more acidic than pH 5. Water naturally dissociates
into H+ and OH− ions with equal concentrations of 1×10−7 M, hence a pH of 7 is neutral. Less
than 7 is acidic (the more extreme the number, the greater the H ion), while higher than 7 is
basic (the more extreme, the less the H ion). Most bacteria live within the pH range of 4-9.
Acidophiles and alkalinophiles are the extreme microorganism groups. Fungi often can live
at lower pH conditions, e.g. citrus fruit decay.
Like your human cells, most bacteria optimally grow in isotonic conditions. However, many
microbes, particularly fungi, can tolerate hypertonic conditions produced by high salt or high
sugar concentrations. The flow of water is controlled by the osmotic pressure of the
environment, and that is determined by the concentration of solute molecules. Osmophilic
microorganisms tend to be fungi. Those organisms that require high salt are called
halophilic, some requiring concentrations of 15-20% NaCl. Many bacteria can tolerate high
salt concentrations (halotolerant), although they do not really require the salt to grow.
Hypertonic environmental conditions cause the cells to dehydrate as water osmoses out of
the cell. In the following exercise, you will look at the effect of various salt concentrations on
bacterial growth. Generally, the degree of inhibition of growth will depend on the type of
dissolved particles (salt, sugar, etc.), its concentration, and the type of microorganism.
Radiation is often used to control microbial growth (rooms, foods, packaged products, etc.).
The killing ability of radiation is due to mutations in the DNA produced by the radiation. Two
major forms of electromagnetic radiation can be mutagenic—ionizing radiation (X-rays,
gamma) and ultraviolet. Ionizing is the more potent since it is much shorter wavelength,
causing electrons to be pulled off of DNA molecules and oxidizing it. Ultraviolet radiation
exerts it effect by causing adjacent pyrimidine nitrogenous bases (commonly thymine bases)
to bond with each other, meaning that the
strand cannot effectively attach to its
complementary strand. These thymine
dimers affect the replication of the DNA in
the dividing cell. There are different kinds
of ultraviolet, the most germicidal being
UV-C (because it penetrates the best).
Ionizing radiation has much greater kill
ability because of its penetration (short wavelength). Ultraviolet radiation does not generally
penetrate clothes, glass, or plastic. Obviously you will have to remove the tops of the plastic
petri dishes when exposing the bacteria to the UV. Cells have repair mechanisms that
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counteract these mutations, but if the mutagenic agent is producing the mutations faster than
can be fixed, the cell will not live. The cell repairs the mutation in different ways:
1. excising the incorrect section of the DNA with enzymes
2. producing an enzyme that uses energy of visible light to split the thymine dimmers
The latter method is called photoreactivation.
OBJECTIVES:
Identify the effects of ultraviolet radiation on bacterial growth, and determine how the damage
can be reversed.
Identify the effects of pH on bacterial growth.
Identify the effects of osmotic pressure on bacterial growth.
MATERIALS NEEDED: per table
OSMOTIC PRESSURE exercise

1 TSA plate each of 0.5% (regular TSA), 5%, and 10% NaCl concentrations
PH exercise
1 TSB pH 3, 7, and 10 for each organism
UV radiation exercise
4 X 6 index cards
5 TSA plates
8 sterile cotton swabs
brown paper bag
UV lamp (bulb should be positioned 18 inches above the table top)
TSB cultures (same density of cells in each) of Pseudomonas aeruginosa, E. coli,
Micrococcus luteus, Staph aureus, Streptococcus lactis, Alkaligenes fecalis, Serratia
marcescens, Bacillus subtilus, and a spore suspension of Bacillus subtilus (for UV study only)
THE PROCEDURES:
OSMOTIC PRESSURE (each table uses all 3 organisms) different inoculation patterns
1. Each table will obtain one TSA plate of each salt concentration and divide the plate
into 3 sections.
2. Inoculate a section of each agar plate with a loopful of your organism (E. coli, regular
Bacillus, and Staph on the sections of the plate), trying to use the same amount of
inoculum on each plate and inoculated the same way for consistency.
3. Incubate the plates at room temperature.
4. INTERPRETATION: During the next lab period you will determine
the effect of salt on the growth of your bacterium. Quantify the amount of growth by
comparing the density of the organisms on the plates.
S no growth
+1 very light growth
+2 medium growth
+3 heavy growth
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ULTRAVIOLET RADIATION (each table will use1 organism)
1. Label your 8 plates with the name of the bacterium, exposure time, and whether it will
be used for photoreaction or has a lid.
2. Dip the swab in your assigned culture. Use the swab to smear the culture all over the
ENTIRE plate of agar. Streak back and forth in a DENSE zig-zag pattern repeatedly
to cover every bit of agar.
3. Take your plates to the UV lamp and place on the table top (18 inches below the bulb).
4. Do NOT remove the tops of the petri dishes until READY to expose to UV. You should
be able to get all of the plates directly under the lamp.
5. Marking the time on your watch or a clock as time ZERO), remove the petri dish cover
and expose the plate to the UV for the assigned amount of time. Do the same with all
of the other agar plates, except the 10 minute exposure (see directions below).
exposure times for plates:
10 seconds
60 seconds (2 plates—1 exposed, 1 covered with lid)
5 minutes
10 minutes (Place the index card over ½ of the agar
plate at a right angle to the swab lines, on top of the
petri dish with cover on it, Remove the plate cover
when ready to start timing)
WARNING: Do not look directly into the UV light as it can cause damage to your
eyes. Do not expose your direct skin to the UV.
6. Remove the each plate from the UV, remove the card, and cover the plate with the
Petri dish lid.
7. QUICKLY place each plate into a paper bag, a total of 6 plates. Two plates will be left
out for photoreactivation—a 60 second and a 5 minute. Place those 2 plates (with lids
now on) under a regular lamp for 10-15 minutes.
8. Incubate all plates at room temperature 25 degrees C for 24 hours.
PH (each table will use 1 organism)
1. Each table will obtain 3TSB tubes of pH 3, 7, and 10. Your table will use Alcaligenes
faecalis, Streptococcus lactis, Bacillus subtilus or Escherichia coli.
2. Inoculate each TSB with a loopful of your organism.
3. INTERPRETATION: During the next lab period you will determine the effect of pH on
the growth of your bacterium. SHAKE each broth and hold all tubes in one hand up to
the light. Quantify the growth by comparing the densities.
S no growth
+1 very light turbidity
+2 medium turbidity
+3 heavy turbidity
Page 82
QUESTIONS:
1. Record your results from the osmotic pressure exercise, using -, +1, +2, or +3 to
quantify.
species 0.5% NaCl 5% NaCl 10% NaCl
Bacillus
E. coli
Staph
2. Record the results from your pH exercise, using -, +1, +2, or +3 to quantify. Include
data on the other organisms from the class results.
species pH 3 pH 7 pH 10
Alcaligenes faecalis
E. coli
Strep lactis
Bacillus subtilus
3. What do you call an organism that prefers acidic environments?
4. Which of the organisms grew best in alkaline pH?
Which bacterium was the most halophilic?
5. Record your data from the UV exercise. Include data on the other organism from the
class results.
UV EXPOSURE TIME
species 10 sec 60 sec 5 min. 10 min. 15 min.
Page 83
EFFECT OF TEMPERATURE ON
BACTERIA GROWTH
Bacteria and fungi can grow across a large spectrum of environmental conditions. Even
though the bacterium may grow well in the human body at 37 C at pH 7 conditions, it may
also be able to withstand out-of-the-ordinary pH, temperature, and osmotic pressure.
Basically, there are 3 large groups of microorganisms with respect to their temperature
preference---mesophiles, thermophiles, and psychrophiles. Animal pathogens are
mesophilic, growing well in the range of 20-45C. Psychrophiles have an optimal range below
mesophiles (even below freezing), while thermophiles grow at temperatures over 45C.
Interestingly, many organisms grow differently in different temperatures, and you have seen
evidence of this already in lab. In an earlier exercise, you should have noticed that the
orange-pigmented Serratia marcescens pigments at 25C but not at 37C. The prodigiosin
pigment, a waste product produced during certain metabolic pathways, is temperature-
dependent. Also, some fungi will change structure at these 2 temperatures. For example,
Candida albicans, the cause of thrush and yeast infections, will produce unicellular yeast
cells at 37C, but at 25C will produce chlamydiospores and hyphal filaments. Although
endospore-forming bacteria such as Bacillus can endure very high temperatures, these
bacteria do not optimally grow in those temperatures, hence the term thermoduric. The spore
wall composition and its dehydrated state make it very resistant to harsh environmental
conditions.
OBJECTIVES:
Recognize the effects of temperature on bacterial growth and spore resistance.

Identify the temperature group based on bacterial growth pattern.
Optimal growth - 5 TSB tubes (3ml) per organism

Resistance to Extreme Temperature - 8 TSB for E. coli and Bacillus spore suspension
1ml pipets and pi-pumps
2 TSA plates
TSB cultures (same density of cells in each) of Pseudomonas, E. coli, Micrococcus luteus,
Bacillus stearothermophilus, Bacillus subtilus
spore suspension of Bacillus subtilus (for extreme temperature section ONLY)
PROCEDURE
1. OPTIMAL GROWTH TEMPERATURES – each table uses 1 bacterium at all temperatures

a. Inoculate 5-3ml TSB tubes with a loopful of the test organisms--- Bacillus
stearothermophilus, Pseudomonas, E. coli, or Micrococcus. You will use 5 tubes
per organism. Be sure to label each tube with temperature and the name of the
organism.
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b. Incubate each tube at a different temperature:
10oC (fridge)
25oC (room temperature shelves)
30oC (small yellow incubator)
37oC
45oC (small metal incubator)
c. INTERPRETATION: Determine the optimal temperature at which your bacterium
grows. To do this, you need to quantify the amount of growth by comparing the
turbidity (AFTER shaking the contents) in all 5 tubes.
S no growth
+1 very light turbidity
+2 medium turbidity
+3 heavy turbidity
2. RESISTANCE OF CELLS AND SPORES TO EXTREME TEMPERATURES

a. Inoculate 4 tubes of TSB broth each with a 0.1ml inoculum from the E. coli culture
and another 4 tubes with the Bacillus spore suspension. Label the tubes as
control, 50, 70, and 100, plus the type of culture on each tube.
b. Place each tube in the water bath equilibrated to one of the temperatures, or in a
boiling water bath. Incubate for exactly 10 minutes. Be sure that the water is deep
enough so that the broth of the tube will be totally immersed.
c. After 10 minutes exposure to the temperature, streak out a loopful of each of the
cultures from each temperature on a separate TS plate and incubate at room
temperature.
control 50C control 50C Use a zig-zag or a straight line

inoculation, but be
70C 100C 70C 100C CONSISTENT between
sections.
E. coli spores of Bacillus
d. INTERPRETATION: Determine the effects of various temperatures on a

vegetative cell suspension and the spore suspension. To do this, you need to
quantify the amount of growth by comparing the amount of growth on the agar
plate.
S no growth
+1 very light growth
+2 medium growth
+3 heavy growth
Page 85
QUESTIONS:
1. Fill out the table below by PREDICTING where each type of organism would optimally
grow. Use a + sign to denote growth.
Organism 10 °C 25 °C 30 °C 37 °C 45 °C
Psychrophile
Mesophile
Thermophile
Hyperthermophile
2. Record your results by adding 0, +1, +2, or +3 for growth.
Species 10 °C 25 °C 30 °C 37 °C 45 °C Classification based on

Optimal temperature
E. coli
Micrococcus
Pseudomonas
Bacillus
stearothermophilus
3. Record the results of the extreme temperature exercise---- 0, +1, +2, or +3 for growth.
Temperature E. coli cells Bacillus spores

Control
50
70
100C
4. What is the difference between a thermophile and a hyperthermophile?
5. How does extremely high temperature kill vegetative cells?

Page 86
Page 87
EVALUATION OF
ANTIMICROBIAL CHEMICALS
Antiseptics and disinfectants are chemicals which kill or inhibit growth, but not 100% kill.
Sterilizing agents have a 100% kill. However, the chemicals that you are familiar with are not
sterilizing agents. This lab will give you the chance to evaluate your household chemicals.
Antiseptics are used on tissues, whereas disinfectants are for inanimate objects (utensils,
tables, floors, etc.). Generally, disinfectants are harsher on human tissue, although they are
not always more effective than antiseptics.
You will need to bring your own antiseptic or disinfectant chemicals for this lab. Be
sure to write down the active ingredient in the chemical: it will be listed on the label.
In the second exercise, we will look at the effectiveness of a disinfectant or antiseptic against
mixed bacterial flora in your mouth. A glass rod will simulate a thermometer. Four different
“thermometers” will be exposed to a disinfectant or antiseptic for a certain amount of time.
OBJECTIVES:
Compare the activity of antiseptic and disinfectant chemicals.

Identify which categories of chemicals are most effective.
Compare kill ability of a chemical at different exposure times.
4 sterile glass rods ("thermometers")

1 TSA plate
4 test tubes (3 for water, and 1 tube for the antiseptic)
A small beaker with tap water
large bowl of bleach (to discard “thermometers”)
TSB cultures of Staphylococcus epidermidis and E. coli
2 TSA plates
sterile paper disks
4 tiny beakers
antiseptic and disinfectant chemicals (4-5 per table)
forceps and beakers of ethanol
THE PROCEDURE: Antiseptic Action on Oral Bacteria
Each table will test one particular antiseptic against oral normal flora.
ALL glass rods from mouth or from bacterial culture go into a bleach container at your table.
1. Prepare materials for the exercise:

a. Divide the TSA plate into 4 quadrants with a marker. Label the quadrants as
follows: control, 15 seconds, 30 seconds, and “1 minute.” Be sure to include the name
of the chemical your table is using as well as the specimen type.
b. Fill 3 tubes with 5 ml of deionized water
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c. Fill 1 tube with your chosen antiseptic.
2. Place the 4 glass rods into your mouth for 3 minutes. BELOW is a schematic of this
activity.
3. After 3 minutes, place the 1st rod in your first tube of sterile water and stir it around for a
couple of seconds. THIS IS THE CONTROL—no exposure to a chemical.
4. Remove the rod from the water, let the excess water drip off (you do NOT want the fluid to
run from one quadrant to another quadrant), and streak the tip of the rod on the control
sector of the TSA plate. Be careful that the inoculum does not enter the other
sector of the plate.
5. Remove the other glass rods and expose each to the same chemical for the designated
time period. As you did with rod #1, stir rods 2-4 in its own tube of sterile water, let
excess water drip off the tip, and then streak a quadrant of the TSA plate with the rod
tip.
• Place rod #2 into your tube of chemical for 15 seconds.
• Place rod #3 into your tube of chemical for 30 seconds.
• Place rod #4 into your tube of chemical for 1 minute.
6. Incubate the TSA plate at 37C until the next lab period.
Page 89
THE PROCEDURE: Comparison of Antimicrobial Chemicals
Inoculate the plates of TSA with the bacterial species by swabbing the cultures well over the
entire surface of the plate. Each table should have 2 agar plates with the 2 species of
bacteria.
Pick 4 chemicals to test, pouring a small amount of each liquid into a tiny beaker.
Label the bottom of the agar plates with the names of the chemicals being evaluated.
Using alcohol-flamed forceps, pick up a sterile paper disk and dip it halfway into the
chemical, then place the disk on the inoculated media.
Repeat the procedure with the other chemicals. The 2 organisms will be tested with the same
chemicals.
Incubate the plates at room temperature, 25oC.
INTERPRETATION:
Antiseptic Action on Oral Bacteria

Look at each quadrant of your TSA plate and determine whether there is growth or no growth.
Use the following quantitative criteria to determine effectiveness of the chemical at different
exposure times----no growth = — , little growth = +1, much growth = +2
Comparison of Antimicrobial Chemicals

Measure each zone of inhibition around the paper discs. Be sure that you use the millimeter
measurement on the metric ruler. The paper disc is about 6mm diameter.
If there is no zone around the disk, or if the zone size is very small---less than 10mm—record
as 0.
Record the results for the entire class in the table on this handout.
QUESTIONS:
1. Record data from your own chemical and specimens as well as other tables’ data.
no growth = — , little growth = +1, much growth = +2
other chemicals used?
time 70% EtOH 3% H2O2 mouthwash A mouthwash B
control
15 seconds
30 seconds
1 minute
control
15 seconds
30 seconds
1 minute
control
15 seconds
30 seconds
1 minute
Page 90
2. Record the results for the various chemicals used in this exercise.
Name of chemical Active ingredient ZONE DIAMETER (millimeters)
Staph E. coli
3. List the 3 chemicals used in lab that had the greatest killing ability.
4. To which chemical is this bacterium most resistant?
5. How long an exposure time does one need for kill of the bacteria?
Page 91
KIRBY-BAUER TEST FOR ANTIBIOTIC

SUSCEPTIBILITY
A true antibiotic is an antimicrobial chemical produced by microorganisms against other
microorganisms. Mankind has made very good use of these antimicrobials in its fight against
infectious disease. Many drugs are now completely synthetic or the natural drug is
manipulated to change its structure somewhat, the latter called semisynthetics. Bacteria
respond in different ways to antibiotics and chemosynthetic drugs, even within the same
species. For example, Staphylococcus aureus is a common normal flora bacterium found in
the body. If one isolated this bacterium from 5 different people, the 5 isolates would likely be
different strains, that is, slight genetically different. It is also likely that if antibiotic sensitivity
tests were run on these isolates, the results would vary against the different antibiotics used.
The Kirby-Bauer test for antibiotic susceptibility, called the disc diffusion test, is a standard
that has been used for years. It has been superseded in clinical labs by automated tests.
But the K-B is still used in some labs, or used with certain bacteria that automation does not
work well with.
The basics are easy: The bacterium is swabbed on the agar and
the antibiotic discs are placed on top. The antibiotic diffuses from
the disc into the agar in decreasing amounts the further it is away
from the disc. If the organism is killed or inhibited by the
concentration of the antibiotic, there will be NO growth in the
immediate area around the disc: This is called the zone of
inhibition. The zone sizes are looked up on a standardized chart
to give a result of sensitive, resistant, or intermediate. Many charts
have a corresponding column that also gives the MIC (minimal inhibitory concentration) for
that drug. The MIC is currently the standard test run for antibiotic sensitivity testing because
it produces more pertinent information on minimal dosages.
The Mueller-Hinton medium being used for the Kirby-Bauer test is very high in protein.
OBJECTIVES:
Determine the susceptibility of various bacterial species to various antibiotics and synthetic
agents.
4 Mueller-Hinton agar plates

24 hr old cultures of Staph, E. coli, Bacillus subtilus, Strep fecalis
sterile swabs
antibiotics
ethanol + forceps
Pseudomonas aeruginosa Kirby-Bauer plate for demo
guidelines chart for interpretation of antibiotic susceptibility
Page 92
THE PROCEDURES:
1. Swab a Mueller-Hinton plate with
each of the bacteria. Dip a sterile
swab into the broth and express
any excess moisture by pressing
the swab against the side of the
tube.
2. Swab the surface of the agar
completely (you do not want to
leave any unswabbed agar
areas at all). In the pictures
above and below, you can see what happens when the plate is not swabbed correctly with
even coverage of the bacterium over the entire agar.
3. After completely swabbing the plate, turn it 90 degrees and repeat the swabbing process.
(It is not necessary to re-moisten the swab.) Run the swab around the circumference of
the plate before discarding it in the discard bag.
4. Allow the surface to dry for about
5 minutes before placing
antibiotic disks on the agar.
5. THE ANTIBIOTIC DISKS:
o The antibiotic dispensers
have 8 antibiotic cartridges
in them. If you do not see
8 disks come out onto
your agar plate, you will
have to manually remove
the antibiotic from a free
cartridge (see line below).
o Each free antibiotic cartridge should have a little metal arm that allows you to
dispense the disc right onto the agar. Even so, sometimes the discs pop out and
fall in a place on the agar that you do not want it to be. Just quickly pick up the
disc and move it to the appropriate place with the sterile forceps.
o Lightly touch each disc with your sterile inoculating loop to make sure that it is in
good contact with the agar surface. Incubate upside down and incubate at 37o C.
INTERPRETATION:
1. Place the metric ruler across the zone of inhibition, at the
widest diameter, and measure from one edge of the zone to
the other edge. HOLDING THE PLATE UP TO THE LIGHT
MIGHT HELP.
2. The disc diameter will actually be part of that number.
3. If there is NO zone at all, report it as 0---even though the disc
itself is around 7 mm.
4. Zone diameter is reported in millimeters, looked up on the lab
chart, and result reported as S (sensitive), R (resistant), or I
(intermediate).
5. Record the results for everyone on your table in the table
below.
Page 93
QUESTIONS:
1. Record the results for the 5 bacteria with all of the antibiotics.
ANTIBIOTICS Strep E. coli Staph Bacillus Pseudomonas

Zone S, R, Zone S, R, Zone S, R, Zone S, R, Zone S, R,
Dia. or I? Dia. or I? Dia. or I? Dia. or I? Dia. or I?
zone diameter in millimeter units Use chart for sensitive, resistant, or intermediate
2. The larger the zone size, the more ____________ the bacterium is to that antibiotic.
3. What measurement units are used to measure the zone sizes?
4. Methicillin and ampicillin are semisynthetic drugs. What class are these drugs in?
What does semisynthetic mean?
5. How does Ps. aerugenosa compare in its sensitivity to the other four bacteria?
Page 94
Page 95
THE SURGICAL HANDSCRUB

The purposes of a surgical handscrub (even though hands are protected by gloves)
• Remove debris and transient microorganisms from the nails, hands, and forearms
• Reduce the resident microbial count to a minimum, and
• Inhibit rapid rebound growth of microorganisms
The standard procedure (from INFECTION CONTROL TODAY online)

• Wash hands and arms with antimicrobial soap.
• Clean areas under the nail with a nail file, stick, or brush.
• Scrub each side of each finger, between the fingers, and the back and front of the
hand with the antiseptic for 3-5 minutes. Accepted antimicrobial agents include
alcohols, chlorhexidine, iodine compounds and iodophors, phenolics, Triclosan, and
quaternary ammonium compounds
• Scrub arms, keeping the hand higher than the arm at all times.
• Wash each side of the arm to three inches above the elbow for one minute.
• Repeat the process on the other hand and arm, keeping hands above elbows at all
times. If the hand touches anything except the brush at any time, the scrub must be
lengthened by one minute for the area that has been contaminated.
• Rinse hands and arms by passing them through the water in one direction only, from
fingertips to elbow. Do not move the arm back and forth through the water.
You will be using a modified procedure for this lab, not the procedure listed above. In
addition to testing a couple of antiseptic chemicals, you will also learn about the tenacity of
your normal flora and how difficult it is to rid yourself of them. Chloroxylenol (also called
PCMX) is a halogenated phenolic compound, while Povodone is an iodophor, a combination
of iodine and a surface-active agent that frees elemental iodine in solution.
There are a variety of microorganisms on the skin, both bacteria (Staphylococcus,

Micrococcus, Corynebacterium rods) and fungi (yeasts such as Pitysporum). Resident
normal flora are deeply entrenched in ecological niches, such as pores and ducts. The
transient normal flora come and go, traded between people when shaking hands or when you
open the door of the bathroom.
OBJECTIVES:
Compare the killing power of different aseptic chemicals.

Understand the standard scrub procedure.
6 BHI (brain heart infusion) agar plates (3 for left hand/chemical, 3 for right hand/handsoap)
chloroxylenol (Ultradex/PCMX) OR an iodine (Povodone Iodine) surgical scrub brush
1 regular scrub brush
Pump handsoap will be at the sinks
sterile paper towels
Page 96
THE PROCEDURE:
One person on the table is being scrubbed, henceforth called the “surgeon.” The “assistant”
will help with the scrubbing and drying of hands.
1. FIRST, label the 2 sets of BHI agar plates. Both sets will be labeled “A”, “B”, and “C”,
but one set will be LEFT hand (iodine or chloroxylenol) and the other set will be RIGHT
hand (handsoap).
• A plates – before anything done to the hand
• B plates – after using a scrub brush with water for 15 seconds
• C plates – after using the designated chemical with scrub brush for 2 minutes
2. Make a control plate---no action performed on the hands. Use your middle 3 fingers
on your left hand to press onto the BHI plate. This will be the “LEFT A” plate.
3. At the sink, the ASSISTANT will thoroughly scrub the left hand of the SURGEON with
water ONLY, up to the wrist (NO hand soap), for 15 seconds.
The SURGEON will rinse the left hand in tap water, and the ASSISTANT will dry that
hand. Do not touch anything else!
4. Use the 3 middle fingers on the left hand to press onto plate “LEFT B”.
5. The ASSISTANT will now use the iodine or chloroxylenol scrub brush over the entire
left hand of the SURGEON for 2 minutes.
6. The ASSISTANT will rinse the SURGEON’s left hand in tap water. The ASSISTANT
will use a sterile paper towel to dry the left hand of the SURGEON. Do not touch
anything else!
7. Use the 3 middle fingers on the left hand to press onto plate “LEFT C”.
8. NOW, the same thing will be done in steps 2-7 with the RIGHT hand of the
SURGEON. And in step 5, rather than use a chemical you will be using a pump
handsoap.
9. Incubate all BHI agar plates at room temperature.
RESULTS
Record results as follows: more than 50 colonies = heavy growth = +2

5- 50 colonies = light growth = +1
less than 5 colonies = sterile (practically)= –
chemical plate A plate B Which chemical plate C

used?
Left hand
Right hand
Page 97
QUESTIONS:
1. Which normal flora---residents or transients---were the first to be killed during the first
scrub?
2. Can one easily get rid of resident normal flora?
3. Are normal hands washings with antimicrobial soap effective to remove microbes?
Page 98
Page 99
BACTERIOPHAGES
Bacteriophages are viruses which infect bacteria. PHAGE (as in phagocytosis) means "to
eat", and generally refers to a virus. Most bacteria have phages that are able to parasitize
them. In fact, the ability to be infected with a known phage type is used to identify some
strains of bacteria (like Staph), known as phage typing . As the virus infects bacterial cells
that it has been mixed with, the lytic infection destroys the bacteria. The bacteria have been
poured into what is called a bacterial lawn on the agar plate. As the surrounding cells are
infected and killed by the released viruses, a clear spot on the agar---in the bacterial lawn---
develops, called a plaque. The plaques can be counted and the number of virus particles or
virions in the original specimen, can be quantitated as viruses/ml of plaque-forming units/ml.
In this lab, 2 kinds of bacteriophages will be used---T4 and phi 174 viruses. Their host
bacteria are 2 different strains of E. coli, so these bacteriophages are called coliphages. The
purpose of using 2 different viruses is to show the specificity of a virus for its host, even for
these little bacterial viruses.
The procedure is really very easy.

• The phage specimen is diluted.
• Bacteria and phage are mixed together in tubes of soft agar. The mix is incubated in
the water bath.
• After incubation the mix is added to the soft agar and poured over the tryptone agar
plates.
OBJECTIVES:
Learn how to culture viruses in a host cell.

Quantitate viruses in an specimen.
Identify viral plaques in a bacterial lawn.
MATERIALS NEEDED:
per table
10-3 dilution of the bacteriophage

1ml pipettes and pi-pump
5 - 9ml saline for dilutions of bacteriophages
50oC water bath
1 strain of E. coli (B or C)in TSB and 1 type of virus (T4 and phi 174)
6 TSA plates
6 - 4 ml soft agar tubes (kept in water bath the entire time)
Page 100
THE PROCEDURE:
The 4 parts of this exercise:
1. Make dilutions of the virus
2. Add E. coli to the soft agar.
3. Add the viral dilutions to the E. coli-soft agar mix.
4. Pour the soft agar (virus + bacterium) onto pre-made agar plates.
Be SURE to mix the dilutions well.

Change pipette s between dilutions.
Each table will use a different combination of a phage and an E. coli host.
• T4 and E. coli B
• phi 174 and E. coli C
1. Set up 5 saline (0.85% NaCl) dilution tubes labeled 10-4, 10-5, 10-6, 10-7, and 10 -8. You will
be making 1/10 dilutions.
2. Starting with the 10-3 dilution you received, transfer 1ml to the dilution tube marked
10-4 and mix.
3. Make 4 more dilutions out to 10-8.
4. Take your E. coli over to the water bath, and inoculate 0.3ml into 6 (LABELLED 10-3 to
10-8 ) soft agar tubes. Keep these soft agars in the water bath to keep from solidifying.
5. Now take your 6 viral dilutions (10-3 to 10-8) in a rack over to the water bath, and
transfer 0.1ml of each dilution into 1 soft agar. Mix these well.
6. Remove 1 soft agar at a time and pour it onto the TSA agar plates, gently rotating the
plate WELL so as to distribute the phage-bacteria all over the agar. Although you
may not have the ability use a sterilizing burner, use your BEST aseptic technique.
7. Allow the plates to harden and incubate at 37oC right side up.
Page 101
INTERPRETATION
1. Lay the 6 plates right side up, from lowest dilution towards highest dilution.
2. Pick each plate up, hold it up to the light, and determine which one has between 30-
300 plaques (you can also use the Quebec colony counters---good backlighting!)
3. Get an accurate count of that plate. Fill in the formula for viral counts.
# viruses/ml = P F U s
dilution of tube X amount plated
4. Calculate the number of viruses per ml. of original specimen.

Page 102
QUESTIONS:
1. Why are viral counts expressed as plaque forming units (pfu)?
2. Why was E. coli added to the soft agar overlays?
3. Why did the two phages not grow on both E. coli strains?
4. Give the plaque count/ml for your viral specimen.

Page 103
IDENTIFICATION OF
UNKNOWN BACTERIA
It is virtually impossible to identify bacteria based on physical characteristics alone. This is
due to the fact that there are only a few basic shapes and physical features commonly seen
in the prokaryotic world. Instead, biochemical testing has been used to make bacterial
identification down to the “species” level. These schemes are based on creating and
matching biochemical profiles of the production of enzymes, acids and gases by isolated pure
cultures of a given microorganism. Identification schemes and flow charts can be found in
reference texts such as “Bergey’s Manual of Determinative Bacteriology” or “The
Prokaryotes”.
Each group of students will receive a pure culture of an unknown bacterium belonging
to the Family Enterobacteriaceae. It is the responsibility of the group to maintain stock
cultures of the organism provided. Working stock cultures will be used to inoculate the
various biochemical test media over the next several weeks and should be fresh and free
from contaminants. A reserve stock culture should be made and after incubation and
comparison with the original slant, kept with the original slant in the refrigerator.
It is critically important that aseptic techniques are used during transfers and
inoculations to prevent contamination of your cultures. If contamination is suspected, you will
be able to fall back to your reserve stock. If you fail to maintain a reserve stock you will not
be able to recover your organism if disaster strikes. The instructor will not provide a new
culture for you to start with in the middle of the unknown exercises.
OBJECTIVES:
1. Maintain a pure culture of the unknown organism provided

2. Determine physical characteristics of the organism provided
3. Inoculate various biochemical tests and be able to read and understand the
significance of each test, whether positive or negative.
4. Use the information generated by testing, along with the given reference flow chart
and identification texts, to deduce the Genus and Species designation of the unknown
organism provided.
5. Hand in a report of the testing performed (Unknown identification sheet), a journal of
how you arrived at the identification you indicated and a TSA plate containing the
unknown organism streaked to demonstrate isolated colonies. Each student will hand
in their own separate report even though you have performed the work together as a
group. Remember it is import to keep your own journal and not to plagiarize other
students in the group.
MATERIALS NEEDED:
TSA plates
TSA slants
TSB broths
Clean glass slides
Gram stain reagents
Oxidase strips and reagent
Page 104
Various biochemical media (distributed in sets during subsequent lab periods)
THE PROCEDURES:
SEQUENCE OF EVENTS FOR UNKNOWN IDENTIFICATION

1st session Get unknown Æ inoculate TSA slant, TSA plate, do gram stain
2nd session run oxygen requirements, colony morphology, catalase & oxidase tests
3rd session start biochemical tests
4th session more biochemical tests
5th session more biochemical tests
6th session 2 streak plates---1 to turn in for grade, other for API20 test next period
7th session run API20
8th session finish reading all test, prepare to turn in unknown identification
1st Session
1. Label two slants (one for reserve and the other as the working stock) and inoculate
using an inoculating from the original culture you have been given.
2. Inoculate a TSB broth from the original slant
3. Streak a TSA plate for isolated colonies using the 3 section method, using the original
agar slant culture.
4. Incubate all cultures at 25 c or 37 C as directed by your instructor.
5. Gram stain your unknown organism using the TSB broth culture. Note the shape,
arrangement and Gram reaction of your organism.
6. Place the original stock slant labeled with your group names and instructor in the
refrigerator.
2nd Session
1. Observe your new slants looking carefully for signs of contamination. Compare to the
original slant noting the color (note pigmentation), texture, opacity and odor.
2. Describe the colony morphology displayed by isolated colonies observed on the
streaked plate (refer back to the Colony Morphology experiment).
3. Record the oxygen requirements of your unknown organism using the reference
organisms as a guide.
4. Run the oxidase and catalase tests (exercises follow)
5. In order to confirm the oxygen requirements of your unknown you will perform the
oxygen requirements exercise using the known organisms provided and your unknown
organism. (see Oxygen Requirements exercise).
6. Prepare a new working stock TSA slant from the previous working stock.
7. Incubate all cultures as directed by your instructor.
3rd Session
1. Observe the biochemical reactions and record the results on your unknown
identification sheet. Make any additional notes in your journal.
2. Inoculate the first series of biochemical tests using your working TSA slant culture.
These tests are the HIGHEST PRIORITY TEST results, the test reactions most
important in the identification of your organism.
Nitrate broth
SIM tube (H2S, indole reaction and motility, see IMViC tests)
Simmons Citrate agar (see IMViC tests)
Page 105
Phenylalanine deaminase agar (Decarboxylation & Deamination of Amino acids)
Decarbolyxase broths (Decarboxylation and Deamination of Amino acids)
3. Prepare a new working TSA slant for the next day.
4. Incubate all cultures as directed by your instructor.
4th Session
2. Use your fresh working stock slant to inoculate the next series of biochemical tests:
3. Use your fresh working TSA slant to inoculate the next set of biochemical tests
Hydrolytic tests (refer to the section on each specific test in your lab manual)
Starch plate
Casein plate
Lipid plate (same as lecithinase test)
Urea broth
Gelatin broth
4. Prepare a fresh working stock TSA slant in case any additional tests are required next
day to finalize identification of the unknown.
5th Session
2. Use your fresh working TSA slant to inoculate the next set of biochemical tests
Oxidation-fermentation tests: (read sections on each specific tests first)
Phenol red sugar broths with Durham tubes (gas production)
- lactose
- glucose
- mannitol
- sucrose
6th Session
1. Read and record final tests results according to each separate procedure in the
laboratory manual. Review the resources available in the laboratory (Bergey’s Manual
and other reference textbooks). If you are still unsure of the identity of your unknown,
inoculate other tests as indicated in the reference texts for next class.
2. Streak fresh TSA plates for isolated colonies. One plate is to be used to inoculate
a biochemical test strip called an API20e. A second plate should contain the organism
streaked to show isolated colonies and will be handed in with the reports.
7th session
Inoculate the API20e as described in the appropriate exercise.
8th Session
1. Double check your own results with the identification made using the API20e test strip.
Do they match? If they don’t you will have to try to make the best match that you can
with the information you have collected.
2. Hand in your Unknown identification sheet (each student), your journal (each student)
and the streaked plate of the unknown (1 per group)
Page 106
QUESTIONS:
1. Why is biochemical testing used to identify bacteria to the species level?
2. What other methods are available for identification of bacteria to the species level?
3. Are there methods for identifying bacteria below the species level? What are some
examples of this and how useful are they?
4. Why is it important to be able to isolate, purify and identify specific bacteria?

GRAM - BACTERIA
Aerobe Facultative anaerobe Page 107
These are key tests for ID, but oxidase

oxidase NOT the only test differences
among organisms. This lists the
predominant test reaction (there - +
indoie
- may be a species which is the
opposite reaction. For the key to
+ work for you, be sure that you are
accurately interpreting the test. - +
H2S
motility
- + Chromobacterium Aeromonas
Nitrate + -
reduction
Phenylalanine Phenylalanine
Burkholderia Acinetobacter deaminase deaminase
- + + -
+ -
- VP citrate Proteus
citrate
Some sugars +
motility +
used Escherichia - +
ornithine Morganella Providencia

+ - motile nonmotile lysine
Providencia
- +
Pseudomonas Branhamella
DNAse - +
Alcaligenes Neisseria Klebsiella

+ -
Citrobacter Salmonella
Serratia Enterobacter
Page 108
Page 109
MOTILITY TESTS
There are a variety of ways to determine motility of a bacterium—biochemical tests as well as
microscopic analysis. Microscopy is the most accurate way to determine motility, assuming
that you have a fresh culture of bacteria. Not only can motility be identified, but also the
organization and number of flagella.
Motile bacteria move about with structures called flagella (a few exceptional bacteria move
with the help of axial filaments, which cannot be seen in the microscope). Nonmotile bacteria
without flagella are called atrichous.
Categories of flagellation:
• monotrichous = single flagellum
• peritrichous = flagella all around
• amphitrichous = flagella at both ends
• lophotrichous = tuft of many flagella at
one end or both ends
Motility can be identified in a couple of different ways:

- the hanging drop wet mount
- motility agar media (SIM and tetrazolium motility agars used later)
Looking at living bacteria are not as easy as one would think. First of all, living bacteria have
no color, and they are small: therefore, they are really difficult to see, even with the oil
immersion lens. Second, all bacteria have some vibrational movement, even nonmotile ones.
This Brownian movement is caused by water molecules bouncing around in the solution,
knocking up against each other and the microorganisms. Kinetic energy inherent to all
molecules causes this kind of movement. On the other hand, those bacteria with flagella will
be very apparently moving about the field of vision, although perhaps not all of the bacteria
will be moving. Some cells will "run" straight across the field, others will "tumble" across the
field in a slower motion.
The keys to a good hanging drop slide are 1) use a small drop of bacterial suspension, but do
not let it dry out, and 2) use a young culture of bacteria.
Another way to determine motility---TTC motility agar with tetrazolium---will be used in lab.
The tetrazolium makes the motility agar much easier to read for motility. The tetrazolium is a
colorless salt which becomes red when reduced, occuring as a result of bacterial metabolism.
NOTE: Strict aerobes may not grow well or at all in this medium.
OBJECTIVES:
Differentiate between Brownian movement and true motility.

Identify flagella on bacterial cells.
Differentiate among different types of flagellation.
Identify motility using different methods.
Page 110
prepared flagella stains

(mixed flagella slides - amphitrichous, atrichous; Proteus slides - peritrichous)
fresh TSB cultures of Klebsiella, E. coli, and Pseudomonas (less than 24 hours old optimally)
2 Motility agar deeps with tetrazolium dye
cover slips
depression microscope slides
vasoline jelly in syringes
THE PROCEDURES: all procedures done as a table
Microscopy
1. wet mount/hanging drop
a. Place a drop of the bacterial culture (optimally from a young broth culture) in the
middle of a cover slip. Makes 2 hanging drop slides---Pseudomonas and
Klebsiella.
b. Place a thin, surrounding line of petroleum jelly all around the edge of the cover
slide.
c. Turn the depression slide upside-down (depressed area facing down) and gently
touch the cover slide. The jelly holds the cover slip to the slide and also keeps the
suspension from drying out.
d. Now flip the entire microscope slide/cover slip combination over. It should look like
the diagram below.
2. prepared flagella stains

• These stains are bought and ready to use. Although they have cover slips, you still
use oil when on 100X magnification. Be sure to remove the oil with the lens paper.
• You will have to move around the slide to find the best field of vision. Often, the
flagella will break off and you may not see many in some areas of the slide.
Biochemical media
1. Inoculate E.coli and Staph cultures into tubes of motility agar with tetrazolium dye
with a NEEDLE, all the way to the bottom.
2. Incubate at 30 or 37 degrees C.
3. Look for the spread of the inoculum away from the inoculation line.
Hold the tube up to the light and look at the stab line to determine motility. If NONMOTILE,
you will see the intact straight stab line. If MOTILE, the original stab line will diffuse out into
the medium as the bacteria spread throughout. BUT with the added, helpful colored dye
tetrazolium which turns red as a result of the bacteria metabolizing.
Page 111
QUESTIONS:
1. Make drawings from the 2 prepared slides..
peritrichous amphitrichous
Proteus bacterium
2. Draw the 2 tubes of TTC media with the growth patterns of the 2 bacteria.
Staph Pseudomonas
3. What designation does one used for a bacterium without flagella?
4. What is the function of tetrazolium?
5. How do you tell if the organism is motile?
6. Why is petroleum jelly used to make this slide?
7. What is the cause of Brownian movement?
8. What feature of the bacterial culture will increase the probability of true motility?
Page 112
Page 113
OXYGEN REQUIREMENTS & CULTURING

ANAEROBIC BACTERIA
An excellent way to determine the oxygen needs of a bacterium is to grow it in different
oxygen environments---atmospheric oxygen of 22%, no oxygen at all (GasPak jar), and
reduced oxygen at less than 10% (candle jar)--and compare the quantity of growth. By
studying growth in different environments, one can determine whether the organism is a
facultative anaerobe, an anaerobe, an aerobe or a microaerophile.
The candle jar at right has 3-5% CO2 and 8-10% O2 (0.3% and 21% in the atmosphere,
respectively). This is a handy way to determine if you have an aerobe which is
microaerophilic, since they grow optimally under reduced (but present) oxygen conditions
as in the candle jar. Many microaerophilic bacteria will grow poorly at 22% O2, whereas
some will not grow at all (e.g. Neisseria gonorrhoea). Possibly the by-products of aerobic
respiration, superoxide radicals and hydrogen
peroxide, make it difficult for the microaerophiles
to do well in 22% O2. Some microaerophiles are
actually capnophilic (requiring elevated CO2
levels to grow). Strict aerobes may not grow well
in a candle jar, depending on the species. The
Gram + genus Bacillus and Gram – genus
Pseudomonas include aerobic bacillus-shaped
bacteria.
On the left is a GasPak jar, with a gas generator

envelope inside. The environment is 0% O2,.
The newer anaerobic system (seen at left)

consists of a plastic container (for the agar
plates) and a paper gas generating sachet.
The paper sachet contains ascorbic acid and
activated carbon which reacts on exposure to
air. Oxygen is rapidly absorbed and CO2 is
produced. When the paper sachet is placed in
a sealed plastic pouch, this reaction will create
ideal atmospheric conditions for the growth of
anaerobes. Because a GasPak jar looks the
same, whether it has oxygen inside or not, an
indicator strip, containing methylene, is
included in the jar. Methylene blue is blue
when oxidized, colorless when reduced. The
carbon within the puch reacts with free oxygen
in the jar, producing 10-15% CO2.
Page 114
Quite a few human pathogens are strict anaerobes, exemplified by the bacillus-shaped
genera---Gram – Bacteroides, Bacillus (anthracis), and Gram + Clostridium (tetani,
botulinum).
Aerotolerants are anaerobes that can grow in the presence of O2 (compared to the strict
anaerobes which would likely die), but they do not use it. And last, but very common, are the
facultative anaerobes which prefer to use O2 when present but will grow without it.
Another way to culture and grow anaerobes is the use of reduced media---media
without oxygen. Thioglycollate broth has a reducing agent in it---the chemical
thioglycollate---which binds any free oxygen within the medium. You will also
notice that these tubes have screw caps, allowing a tight closure, to reduce
oxygen entry. However, some oxygen will be in the tube between the cap and
the broth and there is no way to get rid of it. So there will be some diffusion of
oxygen into the top portion of the broth, and that is where any aerobic bacteria
may grow. An indicator, resazurin, in the medium will be a light pink in the area
of higher oxygen. Where the bacterial growth is located in the tube indicates
whether it is an anaerobe, facultative anaerobe, or an aerobe.
growth is indicated
by gray area
CAUTIONARY NOTES:
• Do not shake the thioglycollate broth. Oxygen will permeate the broth then this
medium sits around for a while. Check for the pink color: if so, boil the broth for 5
minutes (removes the oxygen).
• Ambient air is 22% O2. All of our incubators are ambient air incubators: O2 comes in
from the outside atmosphere.
• Before the anaerobic jar is opened, the methylene blue strip should be checked to
make sure that it is COLORLESS (verifies anaerobiosis).
OBJECTIVES:
Identify the 3 major categories of microbes based on oxygen requirements.

Learn different ways to culture anaerobic bacteria.
Identify the oxygen requirements of your unknown bacterium.
Page 115
MATERIALS NEEDED:
1 thioglycollate broth per bacterium – 4 total

2 TSA plates (divide into 4 sections)
GasPak jar for entire lab
GasPak envelope for the jar
methylene blue indicator strip for the jar
cultures: Clostridium species, E. coli, and Micrococcus luteus, AND your unknown
THE PROCEDURES: per table
Thioglycollate broth
1. The thioglycollate broth should be either boiled first before inoculation OR recently
made so that the oxygen content is very low. Your instructor will tell you what to do.
2. You will use 1 thioglycollate broth for each organism.
3. Inoculate a tube of thioglycollate broth: make sure that the loop or needle goes
down to the BOTTOM of the broth (do not get metal holder in the sterile broth).
4. Incubate at 37 degrees C
TSA plates in 2 different oxygen environments

1. Label 2 plates for the table--- ambient air and GasPak anaerobic jar.
2. Divide the plates into sections, one for each bacterium.
3. Inoculate the section by streaking a straight line or a a slight zig-zag. HOWEVER, be
sure that you inoculate all plates using the same technique.
4. Place your plates, upside-down, in the correct location---ambient air 37C incubator or
GasPak anaerobic jar.
5. Your instructor will make sure that the jar has a methylene blue indicator strip inside.
He or she will also place the jar in the 37 C incubators
INTERPRETATION: after incubation
TSA plates
• Compare the presence/absence of growth, as well as the quantity of growth on the 2
plates. Quantify the growth as – (no growth), +1 (slight growth), or +2 (good growth).
• Determine whether each organism is aerobic, anaerobic, or facultatively anaerobic.
• Record your results in the LAB REPORT SHEET.
• Record the results of your unknown bacterium in your journal.
Thioglycollate broth
• Determine WHERE the most amount of growth occurs in the column of liquid---the top,
the bottom, top to bottom. DO NOT SHAKE IT!
• Record your results in the LAB REPORT SHEET.
• Record the results of your unknown bacterium in your journal.
Page 116
QUESTIONS:
1. Why should you boil thioglycollate broth if it is not freshly made?
2. Why might one use a candle jar for incubation of a bacterium?
3. Differentiate between an aerotolerant and a facultative anaerobe.
4. Data from TSA plates in different environments. Record as -, +1, or +2 growth.
5. Data from thioglycollate broths. Shade in where the

growth is located for the 3 bacteria.
Bacillus E. coli Clostridium

6. Which of the following bacteria---A, B, or C—is the strict anaerobe?
Page 117
CARBOHYDRATE UTILIZATION
Sugars are very important tests for a lot of microbes. Unfortunately, there are a few problems
related to sugars. Whereas most biochemical media is stable, allowing you to inoculate one
day and read a couple of days later, sugar is NOT. The problem is that there are other
nutrients in the media that can be used by the bacteria, like proteins. Although the sugar is
the primary nutrient used (if the organism uses that particular sugar), when the microbe runs
out of it, protein or other nutrients will be attacked. This can cause changes in the color of
the medium because there is a pH indicator added to detect acid production. When proteins
are used, alkaline by-products are produced and the medium can change colors. If you let
these sugar tests go for more than a day, you risk the possibility that the color will have
changed and you may call the test result negative rather than positive. Therefore, it is a
good idea to run into the lab and read these sugar tests somewhere between 8-12 hours, if
at all possible. There are no reagents that have to be added since the pH indicator is added
to the medium already.
You are going to see 2 different ways to run sugar tests: phenol red sugar broths and the
sugar disc methods. Some of the sugars come in phenol red broth, already with sugar in it
lactose, glucose (dextrose), and sucrose. Or, there are a few sugars that do not come as a
disc form nor do we have pre-made phenol red broth with the sugar. For the sugars maltose,
arabinose, and some others, we will add the sugar to the phenol broth as asked for. Please
just ask if you need either of these sugars
OBJECTIVES:
Learn different test procedures for determining carbohydrate use.
Identify different end products of sugar use.
MATERIALS NEEDED:
phenol red sugar broths (lactose, sucrose, glucose/dextrose)
TSB
small sterile tubes
1ml pipettes
pi-pump
sugar discs
ethanol
forceps
THE PROCEDURES:
for PHENOL RED BROTH: The broth can have various sugars added to it. It has a small upside
down tube called a Durham tube that collects CO2 gas.
1. Inoculate the phenol red glucose broth with your unknown bacteria.
2. Inoculate the organism into the tube with the disc and incubate at 25 or 37 degrees C.
Page 118
for SUGAR DISCS: You will need some forceps and the ethanol to maintain sterility of the sugar
discs when transferring them. There are at least a couple of dozen sugars that we have in
the refrigerator, but today you are using sucrose.
1. Obtain a tube of TSB broth and aliquot 0.5ml into a sterile tube.
2. Sterilely transfer a sugar disc into the new tube (using either STERILE forceps dipped
in alcohol and flamed OR dispensed with the metal holder on the sugar cartridge).
3. Inoculate the organism into the tube with the disc and incubate at 30 or 37 degrees C.
4. Record the results of your bacterial unknown in your journal.
If you do not find the sugar that you need for identification, PLEASE ASK.
INTERPRETATION:
PHENOL RED: You are looking for a change in the phenol red indicator to yellow, for acid (A)
production. In addition, there may be CO2 gas (G). The results can be reported as A/G, A/-, -
/G, or -/-. You may notice an alkaline change to a very red color, indicating basic end
products, but we don't care about basic reactions.
SUGAR DISCS: As above, you are looking for the yellow acid color. There is no way to
determine gas production with this method.
BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER
TABLES IN LAB.
QUESTIONS:
1. The pH indicator used in most sugars is:
2. Why should sugars always be read within 12 hours, IF AT ALL POSSIBLE?
3. What is the purpose of the durhm tube?
4. Sugar, when catabolized, is broken down to:

Page 119
CASEIN HYDROLYSIS
The enzyme caseinase is secreted out of the cells (an exoenzyme) into the surrounding
media, catalyzing the breakdown of milk protein, called casein, into small peptides and
individual amino acids which are then taken up by the organism for energy use or as building
material. The hydrolysis reaction causes the milk agar, normally the opacity of real milk, to
clear around the growth area as the casein protein is converted into soluble and transparent
end products—small chains of amino acids, dipeptides, and polypeptides.
OBJECTIVES:
Identify the reactions associated with growth on skim milk agar.
MATERIALS NEEDED:
1 skim milk agar plate per table
THE PROCEDURE:
1. Run this test using your unknown bacterium.

2. Inoculate the organism on the plate either a straight line or a zig-zag.
INTERPRETATION:
Hold the plate up to the light to see the zones. Positive reactions may be recorded as strong
+ or weak + reactions.
There is no reagent or indicator in the agar. A zone of clearing around the growth area
identifies the presence of the enzyme caseinase.
TABLES IN LAB.
Page 120
QUESTIONS:
1. What is the difference in a weak + and a strong + reaction on the agar plate?
2. The substrate for this enzyme is _____.
3. What is an exoenzyme?
Page 121
CATALASE TEST
Catalase is an enzyme that splits hydrogen peroxide into water and oxygen. Hydrogen
peroxide is a by-product of respiration and is lethal if it accumulates in the cell. Catalase is
an enzyme that can degrade the hydrogen peroxide in the cell before it can do any cell
damage. It splits the H2O2 to free oxygen (bubbles) and water.
IMPORTANT POINTS:
• Use a culture growing on an agar plate or agar slant to run the slide catalase test.
• You can drop H2O2 directly on an agar plate or slant, but it may kill the culture.
OBJECTIVES:
Test for the enzyme catalase on your unknown isolates.
MATERIALS NEEDED:
3% hydrogen peroxide
THE PROCEDURE:
1. Pick the inoculum from a plate culture or slant culture and place it on a slide.
2. Add one drop of H2O2 and record results.
INTERPRETATION:
Usually immediately you will see a reaction if there is one, most often lots of bubbles. Slight
bubbles indicate a positive reaction also.
TABLES IN LAB.
Page 122
QUESTIONS:
1. What are the bubbles that you see in a + reaction?
2. Name the reagent used for this test.

Page 123
DNASE PRODUCTION
Deoxyribonuclease (DNAse) agar actually has DNA in it. Deoxyribonuclease is an enzyme

excreted from the cell which will break the DNA down into smaller molecules. There is no
indicator in the medium, but HCl reagent is used to precipitate out the undestroyed DNA.
Around the growth area, if the bacterium has made DNAse and destroyed the DNA, there will
be clear zones seen. The rest of the plate with the remaining DNA will have turned an
opaque white.
OBJECTIVES:
Identify the presence of DNAse enzyme.
MATERIALS NEEDED:
1 DNAse agar per table

1 N Hydrochloric acid
culture of Staphylococcus aureus
THE PROCEDURE:
1. Divide the DNAse agar plate into 2 parts. On 1 side you will inoculate your unknown
organism, and the other side will have the + control, Staphylococcus aureus.
2. Inoculate one DNAse agar plate with the unknown organism---a straight line
inoculation.
4. AFTER INCUBATION: Flood the plate with HCl and wait for 1 minute.
NOTE; The lab may be using DNAse with methyl green indicator instead of regular
DNAse agar. The indicator methyl green is used rather than the HCL reagent.
INTERPRETATION:
The interpretation of the reaction requires a reagent added to the culture after it grows, HCl.
Within a minute after addition of the reagent, a clear zone (large or small is +) around the
growth area. BE SURE THAT THE PLATE IS SITTING ON THE BLACK TABLE TOP: The
black will enhance the zone identification.
If using the DNAse with methyl green, you will notice a light tan zone around the growth
area.
BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS.

Page 124

QUESTIONS:
1. When HCl is added to this agar, what happens to remaining/uninoculated DNA in the
agar?
2. Why look at the DNAse agar against a black background?
3. What is the purpose of the methyl green in this medium?

Page 125
DECARBOXYLATION & DEAMINATION

OF Amino Acids
These are about 20 amino acids, and most of them

can be used by one bacterium or another. Many of
the biochemical tests are based on protein and
amino acid use. In this lab you will look at 2
different amino acid tests, plus I have added a 3rd
that you may want to run at a later time.
There are 3 decarboxylase enzymes we can test

for---arginine decarboxylase, ornithine
decarboxylase, and lysine decarboxylase. These
enzymes break the bond holding the carboxylic (-
COOH) group to the rest of the amino acid. As a
result, the end product is a basic chemical which causes the pH to go up, changing the
indicator brom cresol purple to turn purple.
The deaminases do the opposite, knocking off the amino groups, and producing chemicals
which are acidic. We run one deaminase test---phenylalanine deaminase---which uses FeCl3
as the reagent, reacting with the phenylpyruvic acid that results from the breakdown of
phenylalanine.
NOTE: The decarboxylase test needs to be anaerobic (assuming your unknown is NOT a
strict aerobe), so you overlay the broths with a layer of sterile mineral oil.
A base broth without amino acid is run on each organism as an inoculated control. Since the
medium has sugar in it, you are making sure that the organism uses the sugar, turning the
indicator yellow. This is NOT a reaction reaction that you record.
OBJECTIVES:
Learn the various methods for determining deamination and decarboxylation.
MATERIALS NEEDED:
Moeller base broth, no amino acid added

Moeller ornithine decarboxylase broth
Moeller arginine decarboxylase broth
Moeller lysine decarboxylase broth
phenylalanine agar slants
mineral oil
pipette and pi-pump
AFTER INCUBATION: FeCl3 reagent
Page 126
THE PROCEDURE:
1. Inoculate the decarboxylase broth (along with a base without amino acid) with the
unknown bacterium and overlay with a layer of mineral oil (NOT immersion oil). The
layer should be 1/4-1/2 inch deep.
2. Inoculate the phenylalanine deaminase slant as you would a normal slant.
3. Incubate at organism's optimal temperature, 25 or 37 degrees C, for a couple of days.
4. AFTER INCUBATION:
o The decarboxylase broths are read as is, no reagent added (it already contains
a ph indicator, Bromcresol purple).
o 6-8 drops of ferric chloride are added to the phenylalanine agar slant, washing
down the slant.
INTERPRETATION:
ARGININE, LYSINE, ORNITHINE DECARBOXYLASE BROTHS: In a basic pH, as a result

of the decarboxylation process, the bromcresol purple pH indicator will be a purple or
purple-gray (yellow is acid, but is a negative reaction).
PHENYLALANINE DEAMINASE: The FeCl3 reacts with the acid produced as a result of
deamination, turning the slant an avocado green.
TABLES IN LAB.
Page 127
QUESTIONS:
1. In the presence of the enzyme decarboxylase, the amine side chain of the amino acid
molecule will be cleaved off---TRUE or FALSE?.
2. Why add mineral oil to the decaroxylase amino acid broths?
3. What is the amino acid in the deamination agar?
4. Name the pH indicator in the
Arginine decarboxylase test –
Phenylalanine deaminase test -

Page 128
Page 129
GELATIN HYDROLYSIS
Gelatin is a protein that is solid at room temperature. If the bacterium makes the enzyme
gelatinase (which optimally is produced at 25C, not 37C), the gelatin is hydrolyzed and
becomes a liquid. There is no indicator or reagent added, simply solidification or liquefaction.
OBJECTIVES:
Learn the method for determining gelatin hydrolysis and the reactions produced.
MATERIALS NEEDED:
1 nutrient gelatin deep for unknown
THE PROCEDURE:
1. Stab the gelatin deep with a needle all the way to the bottom (being careful NOT to get
the metal holder into the agar).
2. Incubate at 25°C for a couple days, up to a week. Keep the medium for a week if
negative.
INTERPRETATION:
The tube is placed on ice for about 15 minutes, or in the fridge for about 30 minutes, to
determine liquefaction. If the tube has been incubated at 37C it will be liquid when taken out
of the incubator, so remember to place it on ice before calling the reaction. The liquefaction
can be complete throughout the tube or perhaps partial.
If negative, keep the medium at 25C for a week to make a final call of negative.
TABLES IN LAB.
Page 130
QUESTIONS:
1. Why place this medium on ice to test for a positive reaction?
2. This gelatinase enzyme is temperature-sensitive, and preferably works best at what

temperature?
Page 131
IMViC TESTS: Indole, Methyl red,

Voges-Proskauer, Citrate + and H2S
These 4 IMViC tests (actually 6 tests if you include motility and H2S) constitute, perhaps, the
most critical tests used for identification of bacteria after the gram stain. The test results from
these 6 tests should carry more weight than almost any other tests, certainly higher priority
than sugar results since they are more stable reactions.
SIM deep is a multi-test medium comprising 3 tests: sulfide (H2S gas), indole production, and
motility. You have already tested for motility via the hanging drop slide and TTC, and here is
an additional way to determine it (although not as good as TTC). The amino acid tryptophan
can be converted by the enzyme tryptophanase into an end product called indole. This
chemical is identified when it reacts with Kovac's reagent.
The MRVP tests are run together in the same broth and then split into 2 tubes when ready to
be tested for the end products. The methyl red test determines the use of glucose, with the
subsequent production of acid, tested for by the pH indicator methyl red. The Voges-
Proskauer test also determines glucose use, but for a different end product---not acid but a
neutral product called acetoin (or acetylmethylcarbinol). The VP is really important for
identification of many bacteria, but it is a picky test, and MUST be done exactly right.
The citrate test identifies the use of citrate as a sole carbon source, since there are no other
nutrients in this medium. The basic end products will cause the brom thymol blue indicator in
the medium to turn from forest green to royal blue.
OBJECTIVES:
Understand the importance of these 4 IMViC tests.

Determine the various reactions for these media: MRVP broth, SIM, Simmon citrate.
MATERIALS NEEDED:
1 SIM deep per unknown
1 MRVP broth per unknown
1 Simmon citrate agar slants per unknown
AFTER INCUBATION: reagents
• Kovac's reagent
• Barritt's reagents A (alpha-naphthol) and B (KOH)
• methyl red reagent
Page 132
THE PROCEDURES:
Indole test, H2S, and motility

1. Inoculate into a tube of SIM media with a NEEDLE, all the way to the bottom.
3. AFTER INCUBATION: After reading both motility and H2S, add 6-8 drops of Kovac's
reagent.
Methyl red and Voges-Proskauer tests

1. Inoculate into the MRVP broth.
3. AFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube: run
the MR test in the tube with 2/3, and the VP test in the open tube with 1/3.
• for methyl red: Add 6-8 drops of methyl red reagent.

• for Voges-Proskauer: Add 12 drops of Barritt's A, mix, 4 drops of Barritt's B, mix.
• Let sit, undisturbed, for at least 15 minutes.
Citrate test
1. Streak up the slant with the inoculum.
INTERPRETATION:
SIM: Indole test, H2S, and motility
1. Look for H2S, a black precipitate within the agar deep.

2. Hold the tube up to the light and look at the stab line to determine motility. If
NONMOTILE, you will see the intact straight stab line. If MOTILE, the original stab line
will diffuse out into the medium as the bacteria spread throughout. In fact, you may
not even be able to see a stab line at all: it may be turbid throughout the medium.
FOR THAT REASON, you might want to compare the inoculated SIM with an
uninoculated one.
3. Lastly, you will add the Kovac's reagent to detect the presence of indole produced.
Within just a few seconds you can see the hot pink color of indole presence.
Methyl red
Within just a few seconds after adding methyl red reagent, you can see the red-pink
color of acid presence from glucose use.
Voges-Proskauer tests
The reagents MUST be added in the correct order, in the correct amounts, and the
tube must sit undisturbed, and open to the air (no cap) for at least 30 minutes (45
minutes is even better) as the light pink color intensifies at the top of the tube (the
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reagents react with acetoin). DO NOT shake the tube AFTER sitting it down for the
waiting period.
Citrate test
The alkaline by-products of citrate use will cause the pH indicator to turn royal blue.
BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS OF ALL OF THESE TESTS BY
VISITING OTHER TABLES IN LAB.
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QUESTIONS:
1. What are the reagents used in the indole test?
methyl red test?
Voges-Proskauer test?
2. The purpose of the citrate in the Simmon citrate medium is to determine if the organism
can used citrate as the sole ___________ source.
3. Why determine motility and H2S before adding Kovac's reagent?
4. What is so picky about the Voges-Proskaeur test?
5. The end product identified in the VP test is a neutral compound called ______.
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LIPID HYDROLYSIS
The enzyme lipase is excreted out of the cells (an exoenzyme) into the surrounding media,
catalyzing the breakdown of tributyrin, a vegetable oil, into fatty acids which can then be
taken up by the organism. This reaction causes the agar, normally a light blue opaque color
to clear around the growth area.
OBJECTIVES:
Identify the presence of the enzyme lipase.
MATERIALS NEEDED:
1 tributyrin agar plate with methylene blue/student
THE PROCEDURE:
1. Run this test using your unknown bacterium.

2. Inoculate the organisms on the plate either a straight line or a zig-zag.
INTERPRETATION:
Hold the plate up to the light to see the zones well.
Record positive reactions as weak + or strong +.

There is already an indicator, methylene blue, incorporated into the agar. A zone of clearing
around the growth area identifies the presence of the enzyme lipase.
TABLES IN LAB.
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QUESTIONS:
1. What is the substrate in this medium?
2. Name the indicator in this medium.
3. What kind of molecule is tributyrin?

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LITMUS MILK USE
Litmus is a multi-purposed and sometimes confusing medium because there are many
reactions that you can get with it. The indicator is litmus, the same one that is used with pink
litmus and blue litmus papers in a chemistry lab to test whether a solution is acid or base. It
starts out as a light lavender color.
The 2 nutrients in milk are casein protein and lactose sugar. A bacterium can use one of
these, both of these, or neither of these, hence a variety of end results. The use of sugar will
produce acid as an end product, sometimes to the extent of coagulating the milk protein and
causing curdling, also called a curd. The use of the protein casein produces alkaline end
products. The total breakdown of protein will cause peptonization, and a clearing of the
medium, almost looking like plasma.
NOTE: It is a good idea to incubate this medium for a week before making a final decision on
the reaction.
OBJECTIVES:
Identify the reactions associated with use of sugar and protein in milk.
MATERIALS NEEDED:
1 litmus milk per unknown
PROCEDURE:
1. Inoculate the tube of litmus milk with a loopful your unknown bacterium.
2. Incubate for up to 1 week, at 30 or 37 degrees C.
4. The instructor will inoculate a set of controls showing other reactions in the litmus milk-
--Bacillus, Alkaligenes, Strep lactis.
INTERPRETATION:
BE SURE to compare your reaction against an uninoculated control from the refrigerator.
The major + reactions:
basic reaction - blue
acidic reaction - pink
peptonization (proteolysis) – clearing of medium
acid clot/curd - solidified whitish material
TABLES IN LAB.
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QUESTIONS:
1. You are testing the bacterium's ability to use ________ and ________ in this medium.
2. What does a basic reaction look like on this medium?
3. The production of acid means that the bacterium is using the _____ in this medium,
producing acid as a by-product.
4. Would a curd be an acidic or an alkaline reaction?

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NITRATE TEST
Nitrate (NO3) reduction can be performed by many bacteria. Nitrites (NO2), ammonia (NH3),
and N2 gas can be the end products of denitrifcation. In the absence of oxygen, various
nitrogen compounds may be hydrogen acceptors: depending on which N compound is the
acceptor, the end product varies (see table below).
OBJECTIVES:
Identify the different ways that nitrate can be reduced by bacteria.
MATERIALS NEEDED:
1 nitrate broth per unknown

AFTER INCUBATION:
nitrate reagents A (sulfanilic acid) and B (naphthylamine)
wooden sticks for zinc
zinc powder
THE PROCEDURE:
1. Inoculate the nitrate broth with your bacterial unknown.

2. Incubate at the optimal temperature, 25 or 37C, for your organisms.
3. AFTER INCUBATION: Look for N2 gas first before adding reagents.
o Add 6-8 drops of nitrite reagent A.
o Add the same number of drops of nitrite reagent B.
o You should see a reaction within a minute or less.
o If you have not seen either nitrite or N2 gas, you need to add a bit of powdered
zinc.
o A bit of zinc is about the amount that sticks to the end of a wood stick.
INTERPRETATION:
There are various ways that a bacterium can utilize nitrate as the final electron acceptor in
anaerobic respiration. The first obvious product of reduction to look for is reduction to N2 gas,
called denitrification, within the Durham tube.
If there is no nitrogen gas, there are still a couple of possible interpretations---nitrate

reduction to nitrite (NO2), reduction to ammonia, or no reduction of nitrate at all.
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Color after
REACTION N2 gas Color after adding zinc
adding reagents
NO3 to NO2 none red -- (not added)
NO3 to N2 yes no color no color
NO3 to ammonia none no color no color
NO3 - no reaction none no color pink-red
TABLES IN LAB.
QUESTIONS:
1. Name the 2 major end products of nitrate reduction.
2. WHY add zinc powder? WHEN do you add it?
3. How do the definitions of nitrate reduction and denitrification differ?
4. Is nitrate reduction an aerobic pathway or an anaerobic pathway?

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OXIDASE TEST
The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox
+) and Enterobacteriaceae (ox -). The enzyme cytochrome oxidase is involved with the
reduction of oxygen at the end of the electron transport chain. The colorless reagent will
detect the presence of the enzyme oxidase and, reacting with oxygen, turn a color.
A couple of key points when doing this test:
• We keep the oxidase reagent either frozen or unopened in tubes until needed. If old
reagent is sitting out on the bench and is PURPLE, ask for a new tube from the
instructor
• Use a young culture, preferably less than 24 hrs old.
• Use a culture growing on an agar plate or agar slant.
• Use FRESH reagent, less than a couple of hours old (it is taken out of the freezer).
• Pick your inoculum, not with a metal loop (reagent may react with the metal), but with
a wooden stick.
• Read the reaction within 20 seconds (NOT after), usually it will change in less than 15
seconds. The oxygen will change the reagent color as time passes, so it must be read
quickly.
OBJECTIVES:
Test for the enzyme oxidase on your unknown isolates.
MATERIALS NEEDED:
oxidase reagent (Tetramethyl-p-phenylenediamine)
wooden rods
THE PROCEDURE:
1. Pick a good-sized amount of inoculum from a plate culture or slant culture and place it
on a piece of filter paper.
2. Add one drop of the reagent (if it is dark blue, it is old and should not be used).
3. TIME the reaction: a positive reaction will occur within 20 seconds. DO NOT READ
the reaction after 20 seconds.
INTERPRETATION:
A positive reaction will usually occur within 10-15 seconds, and will be a bluish-purple color
that progressively becomes more purple.
BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS.

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QUESTIONS:
1. Why do you have to read this reaction within 30 seconds?
2. Why does the oxidase reagent need to be fresh?

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STARCH HYDROLYSIS
The enzyme amylase is secreted out of the cells (an exoenzyme) into the surrounding media,
catalyzing the breakdown of starch into smaller sugars which can then be absorbed by the cells
for use.
Iodine reacts with starch, producing a deep purple color. As starch is catabolized and
converted to sugars, there will be less and less starch to react with the iodine. Strong amylase
producers may convert all of the starch in the agar to sugars, while weak amylase producers
may convert the starch surrounding the growth areas only.
OBJECTIVES:
Identify the reactions associated with growth on a starch agar plate.
MATERIALS NEEDED:
Gram's iodine reagent (AFTER incubation)

1 starch agar plate
THE PROCEDURE:
1. Make a single line streak of the unknown bacterium across the plate.
2. Incubate at either 25 or 37 degrees C.
3. AFTER INCUBATION & GROWTH: Flood the plate with iodine
INTERPRETATION:
Placing the agar plate on a white piece of paper or background will REALLY help you to
distinguish the zones.
In the presence of the enzyme amylase and subsequent starch hydrolysis around the growth
area, there will be a yellow/gold zone AROUND the growth.
In the absence of amylase, the starch will not have been degraded: the medium will be purple.
BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES
IN LAB.
Page 144
QUESTIONS:
1. Starch hydrolysis will result in a zone around the bacterial growth that is the color _____.
WHY?
2. The enzyme that does this is called ________.
3. What kind of molecule is starch?

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UREA HYDROLYSIS
Urea (a protein compound) can be broken down with the help of the enzyme urease,
producing the alkaline product of ammonia. That causes the phenol red pH indicator phenol
red to turn a beautiful shade of hot pink.
OBJECTIVES:
Understand the reactions of bacteria in urea broth.
MATERIALS NEEDED:
1 urea broth/per unknown
THE PROCEDURE:
1. Inoculate a tube of urea broth with your unknown bacterium.

2. Incubate until next period at optimal temperature, 25 or 37 degrees C.
INTERPRETATION:
The alkaline reaction turns the pH indicator to hot pink. A yellowish color is still a negative
reaction, although acidic.
TABLES IN LAB.
Page 146
QUESTIONS:
1. Is the end product being tested for an acidic, basic, OR neutral chemical? Name it.
2. Name the indicator in this medium.
3. What type of organic compound is urea?

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API 20E
This API-20E test strip (from bioMerieux, Inc.) is used to identify the enteric gram negative
rods (although API makes a variety of other test strips for yeast, Staph, anaerobes, etc.) 20
separate test compartments are on the strip, all dehydrated. A bacterial suspension is used to
rehydrate each of the wells. Some of the wells will have color changes due to pH differences:
others produce end products that have to be identified with reagents. A profile number is
determined from the sequence of + and - test results, then looked up in a codebook having a
correlation between numbers and bacterial species. This particular strip is for enteric
(intestinal) bacteria only, e.g. E. coli.
OBJECTIVE:
Learn how to perform and interpret the miniaturized, multi-test technique for bacterial
identification.
MATERIALS NEEDED:
agar plates of bacterial species

0.85% NaCl solutions (5ml)
sterile Pasteur pipettes + bulbs
sterile mineral oil
API 20E test strip (for oxidase - gram negative rods)
API test strip incubation chamber AFTER INCUBATION: 10% FeCl3, Barrett's reagents A
and B, Kovac's reagent
PROCEDURE:
Prepare a suspension of the bacteria in the saline tube
1. Inoculate a large colony (2-3mm diameter)of the bacterium (pure culture) into the
0.85% NaCl solution, making sure that the suspension is homogenous and without
clumps of floating nbacteria.
2. Use a McFarland barium sulfate standard #3 to quantitate the suspension.
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Inoculate the API strip
1. Holding the strip at a slight angle up from the table top, you will
now inoculate the bacterial suspension into each well with the
sterile pipette.
2. Touch the end of the pipette to the side of the cupule, allowing
capillary action to draw the fluid into the well as you slowly
squeeze the bulb. This should eliminate any bubbles forming in
the wells. Each well should be filled up to the neck (see diagram).
3. CIT, VP, and GEL have boxes around their
names. These test wells will be filled all the
way up to the top of the well.
4. LDC, ODC, ADH, H2S, and URE are filled as
described in step B, but they will then be filled
up to the top with sterile mineral oil.
Incubate the strip in its chamber
1. The bottom of the incubation chamber has

small indented wells in the bottom: fill it with
water just enough to fill these indentations.
2. Place the strip into this bottom. There should not be so much water that it slops onto
the API strip.
3. Place the top of the incubation chamber
over the bottom, and label it.
4. Place the strip at 37o C for 18-24 hours.
INTERPRETATION:
1. Add the proper reagents to the

compartments:
o 1 drop of Kovac's to the IND (read within a couple of minutes)
o 1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes)
o 1 drop of FeCl3 to TDA
2. Read all other tests as described (chart below) without reagents.
3. Record results on the diagram handed out to you in lab (1, 2, or 4 points for + reaction,
0 points for - reaction). The oxidase test reaction should be negative, and is added as
the last test result.
4. Three test reactions are added together at a time to give a 7-digit number, which can
then be looked up in the ONLINE codebook at
http://industry.biomerieux-usa.com/industry/food/api/apiweb.htm
A more specific 9-digit number can be obtained with a few

more tests:, but NOT DURING THIS LAB.
Page 149
(pictures from the API 20E documentation, from BioMerieux)
READING THE API 20

TESTS SUBSTRATE REACTION TESTED - RESULTS + RESULTS
ONPG ONPG beta-galactosidase colorless yellow
ADH arginine arginine dihydrolase yellow red/orange
LDC lysine lysine decarboxylase yellow red/orange
ODC ornithine ornithine decarboxylase yellow red/orange
CIT citrate citrate utilization pale green/yellow blue-green/blue
H2S Na thiosulfate H2S production colorless/gray black deposit
URE urea urea hydrolysis yellow red/orange
TDA tryptophan deaminase yellow brown-red
IND tryptophan indole production yellow red (2 min.)
VP Na pyruvate acetoin production colorless pink/red (10 min.)
GEL charcoal gelatin gelatinase no diffusion of black black diffuse
GLU glucose fermentation/oxidation blue/blue-green yellow
MAN mannitol fermentation/oxidation blue/blue-green yellow
INO inositol fermentation/oxidation blue/blue-green yellow
SOR sorbitol fermentation/oxidation blue/blue-green yellow
RHA rhamnose fermentation/oxidation blue/blue-green yellow
SAC sucrose fermentation/oxidation blue/blue-green yellow
MEL melibiose fermentation/oxidation blue/blue-green yellow
AMY amygdalin fermentation/oxidation blue/blue-green yellow
ARA arabinose fermentation/oxidation blue/blue-green yellow
OX oxidase oxidase colorless/yellow violet
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QUESTIONS:
1. What is the purpose of the water in the tray?
2. What is the function of the mineral oil?
3. What are the advantages of this test (compared to regular biochemical tube media)?
4. What are the disadvantages of this test?
5. Did your table’s unknown bacterium correctly identify?

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GENUS STAPHYLOCOCCUS:
Isolation and Identification
Staphylococcus is a genus of Gram +, non-spore-forming cocci (0.8 – 1.0 µm dia.) belonging
to the family Micrococcaceae that are often found as normal human microbiota of the skin
and nasal cavity. There are five organisms to consider as potential human pathogens in this
genus: S. aureus, S. epidermidis, S. saprophiticus S. haemolyticus, and S. hominis but the
first three are the most common isolates. S. aureus is often considered to be the most
problematic of the three pathogens and is distinguished from the other two by being the only
one able to coagulate plasma. S. aureus is able to cause many superficial pyogenic (pus-
forming) infections of the dermis and underlying tissues as well as serious systemic
infections. It can produce a range of toxins including enterotoxins (food poisoning),
cytotoxins (general systemic toxins), and toxic shock superantigens. The other coagulase-
negative staphylococci (S. epidermidis and S. saprophiticus) are much less frequently found
as pathogens but are occasionally associated with endocarditis, prosthetic joint infections,
wound infections and infections of cerebrospinal fluid.
This exercise gives you the opportunity to use selective media, in this case based on high
sodium chloride (MSA and SM110 are both selective media for the isolation of
Staphylococci- 7.5% NaCl). A selective medium has an inhibitory agent which favors the
growth of certain bacteria by inhibiting others. MSA contains an additional indicator for
monitoring mannitol fermentation, which makes it a differential media also. In a previous
exercise, you learned about halophilic bacteria. Staphylococcus is not halophilic, but rather
haloduric (salt tolerant), in that it can live in or endure high NaCl concentrations (but does not
require the salt). The high salt content in SM110 and MSA inhibits other common skin
microorganisms. The other media being used in this exercise are for differentiating
pathogenic Staphylococcus from nonpathogenic, and for identification of the species.
Not only salt resistant, Staphylococcus is always facultatively anaerobic. When stained, it will
be seen in small clusters (staphylo = cluster). Staphylococcus is usually either beta
hemolytic or not hemolytic at all (called gamma hemolysis).
Pathogenic Staphylococci can produce a variety of virulence factors, including toxins,

coagulase, leucocidins, and hydrolytic enzymes that can damage host tissues.
OBJECTIVES:
Obtain presumptive staphylococci from nasal specimens

Isolate and identify different staphylococci species using selective and differential agar
MATERIALS NEEDED:
Sterile swabs
Sterile saline solution
2 Mannitol salt agar (MSA) plates (only 1 used for 1st session)
Containers of alcohol + forceps
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Columbia naladixic acid blood agar plates (CNA)
Staphylococcus medium 110 (SM110) agar plates
DNase agar plates
Novobiocin (5 microgram) antibiotic disks
Metric rulers
Rabbit plasma (frozen) for coagulase test
Staphylococcus identification tables
Optional media for identification: arginine and ornithine decarboxylase broths
urea broth
VP broth
SCHEMATIC OF IDENTIFICATION PROCEDURE

1st period Æ 2nd period Æ 3rd period Æ 4th period Æ 5th period
MSA from CNA and MSA gram stain coagulase finish test reading
Nasal catalase optional tests prepare report
SM110 TSA (Staphyloslide)
DNAse
THE PROCEDURES:
Be sure to keep a list of all test results for your isolates.
1st Session
1. Two people on the table will perform nasal swabs, each
specimen streaked onto a mannitol salt agar plate.
2. Aseptically remove a cotton tipped swab from its wrapper
and dip into a tube of sterile saline. Remove excess saline
by pressing against the tube wall and rotating the swab.
3. Use the moistened swab to collect bacteria from the anterior
opening of the nostrils.
4. Using a DENSE zig-zag inoculation, inoculate a MSA plate and incubate at 37°C or
room temperature (if over the weekend).
2nd Session
1. Your aim is to isolate a Staph from your MSA plate, using salt resistance as a key
indicator. Staphylococcus is salt resistant, although not the only genus of bacterium
that will grow in high salt. Another characteristic you should look for is any colony
around which the medium has turned yellow (identifying the fermenters of mannitol, of
which some species of Staph are). Identify 2 colonies from the 2 MSA plates that fit
this Staph profile.
2. Divide a Columbia naladixic acid (CNA) agar plate and another MSA plate in half. You
will use a dense zig-zag inoculation (see above diagram) to streak the 2 potential
Staph colonies on opposite sides of the CNA and MSA plates. Be SURE that you
have only 1 colony on the loop for this transfer.
3. Place the forceps into the alcohol and then sterilize the forceps by running them
through the flame quickly. The alcohol will catch on fire and when evaporated from the
forceps, they will then be sterile. You may now pick a novobiocin disc from the holder
to place on the opposite sides of the CNA plate.
4. Incubate plates at 37°C or room temperature (if over the weekend).
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3rd Session
1. Observe the MSA plates noting IF colonies grow, meaning that they are salt resistant.
If growth is present, then the second question is whether the bacterium uses the sugar
mannitol. If the medium surrounding the colonies has changed from red to yellow,
mannitol has been fermented and the phenol red pH indicator in the medium has
changed colors as a result of the acid from sugar breakdown.
2. On the CNA plate, you are checking for 2 reactions---sensitivity to novobiocin antibiotic
AND hemolysis of blood.
a. Novobiocin sensitivity - A zone 16 mm or larger indicates sensitivity
b. Hemolytic reactions
• alpha (α) hemolysis – green zone around colony, caused by leaking
hemoglobin converted to biliverdin, called a partial hemolysis
• beta (β) hemolysis – complete clearing around colony caused by breakdown of
RBCs by streptolysin enzymes
• gamma (γ) hemolysis - no hemolysins, no zone
Staphylococcus species are either beta hemolytic or gamma (not hemolytic).
Staph aureus produces alpha toxin which typically causes wide zones of
beta (complete) hemolysis.
3. Gram stain the 2 colonies (taken from CNA plate) to verify that you have gram + cocci
in clusters. If your isolate is not this profile, that will be the end of the isolate’s use in
this exercise.
4. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase +
(all species of Staph are +). If your isolate is not this profile, that will be the end of the
isolate’s use in this exercise.
a. You will carry through the rest of this exercise with your table’s 2 Staph
isolates..
b. IF NEITHER OF YOUR TABLE’S ISOLATES ARE STAPHYLOCOCCUS, ask
your instructor to give you an unknown Staph to work with for the rest of this
exercise, or you can “borrow” another table’s isolate. You will need to do a
gram stain and catalase test as if it were your own isolation from your nasal
cavity.
5. Using your Staphylococcus isolates, streak 2 agar plates---SM110 and DNAse (divide
each in half).
6. Incubate plates at 37°C or room temperature (if over the weekend).
4th Session
1. Check DNase plates for hydrolysis. Refer back to that DNAse exercise.
2. Check the SM110 for growth and for pigment. Nutrients and vitamins in this medium
enhance the pigmentation of the pathogenic Staphylococcus, those colonies becoming
a yellow-orange colony.
3. What you do with your Staph isolate is now determined by its reactions on SM110 and
DNAse.
• If DNAse- and SM110 –,
• If DNAse + and SM110 +, run the coagulase test on the isolate
4. Presumptive S. aureus can be confirmed using the coagulase test. Since there are 2
kinds of coagulase enzyme—bound and free---there are 2 different tests that can be
used to identify these enzymes. Both of the enzymes activate fibrinogen in plasma, in
different ways. The TUBE method is the definitive test of the 2, the most reliable.
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• THE SLIDE AGGLUTINATION TEST
Make a 1 inch diameter circle on a clean glass slide using a wax pencil. Place two
drops of thawed rabbit plasma into the circle and using a wooden pick or a clean loop.
Add a single colony and emulsify it in the plasma. Fibrin threads form between the
cells, causing them to agglutinate, or clump. There will a visible clumping of cells
within 10-15 seconds. This test is for the bound coagulase enzyme.
• THE TUBE COAGULATION TEST
Inoculate a tube with a ½ ml of rabbit plasma with the bacterial inoculum. Place at
37C and check at the next lab period. To interpret, tip the tube at an angle, looking
for solidification of the plasma. Any degree of coagulation is considered a positive
test for the free coagulase enzyme.
5. There are enough tests that have been performed to identify your Staphylococcus
isolates, assuming that your species are common ones. The following table has those
3 species listed with the corresponding test results. Try to identify your isolates.
6. If you have Staph aureus, subculture the bacterium onto a TSA plate to run the
Staphyloslide test coming up in lab. Incubate at 37°C or room temperature.
7. If your isolate does not match the profile of the 3 species of Staphylococcus
shown in this table, you can run the following media (we have used all of those
in other exercises). Inoculate the following media, read your reactions the NEXT lab
period, and refer to the Staph identification tables available in lab.
• Urea broth
• Voges-Proskauer broth (see MRVP in IMViC exercise)
• Ornithine decarboxylase (see Decarboxylation and Deamination exercise)
• Arginine decarboxylase (see Decarboxylation and Deamination exercise)
Differentiation of S. aureus from other common staphylococci microbiota

S. aureus S. epidermidis S. saprophiticus
Alpha toxin (β-hemolysis) + (most strains) - -
Acid from mannitol + - + (most strains)
Coagulase reaction + - -
Pigment production (SM110) Usually golden Usually white Usually white
DNase production + - -
Sensitivity to novobiocin sensitive sensitive resistant
8. Your table will write up this exercise—1 report for the table. Word or another
word processing program is the best procedure because you can embed pictures into
the document.
For both isolates, include:
• All test results
• A flow chart/dichotomous key for identification
• A diary/journal of this activity (include visuals of the test results, e.g. digital
camera)
Page 155
GENUS STREPTOCOCCUS:
Isolation and Identification
Streptococci are part of normal human microbiota, found on the skin, in the upper respiratory
tract, digestive tract and oral cavity. Streptococci are Gram-positive cocci which grow
primarily in pairs and in chains of cells. Streptococci are often grouped by hemolytic reaction
on blood agar and by serological typing using the Lancefield classification system. The most
significant pathogens are Streptococcus pyogenes (Group A), Streptococcus agalactiae
(Group B) and Streptococcus pneumoniae. Some streptococci (formerly group D) now
placed in the genus Enterococcus are of medical significance because they are often difficult
to treat because of antibiotic resistance. Hemolysis is a very important feature used to identify
the streptococci, so it is important that you understand the categories of hemolysis.
• alpha (α) hemolysis – green zone around colony, caused by leaking hemoglobin
converted to biliverdin
• beta (β) hemolysis – complete clearing around colony caused by breakdown of RBCs
by streptolysin enzymes
• gamma (γ) hemolysis
The common oral Streps fall into 4 categories:

• Mitis—Strep mitis, gordonii, sanguis, oralis---associated with dental plaque
• Mutans—Strep mutans, sobrinus—associated with plaque and dental caries
• Salivarius—Strep salivarius, vestibularis—covering mucosal surfaces
• Anginosus/milleri—Strep anginosus, intermedius—in gingival crevices
The most common species of Streptococci in the oral cavity are the ‘viridans’ group---the
alpha hemolytic species. Streptococcus mutans is the leading cause of dental caries in
humans, and it is one of the organisms that we will try to identify. Because there are so many
species of different bacteria genera in the mouth (more than 200 species have been cultured
and identified), you will be using a selectively differential medium called mitis-salivarius agar.
M-S agar has sugars, particularly sucrose, that give a luxuriant growth of the Strep species,
and the dye trypan blue produce different color. Tellurite and crystal violet dye inhibit gram –
bacteria as well as most gram +.
The pygenic Streptococci group contain the pathogenic species, ones causing strep throat,
toxic shock, impetigo, newborn infections, and necrotizing fasciitis. These species will be
beta hemolytic.
The group D Streptococci are resistant to extreme temperatures, and are a hardier group
than most of the other Streps.
As with the Staphylococci, Streptococci have a wide variety of virulence factors---enzymes

such as hyaluronidase (spreading factor for necrotizing fasciitis), leucocidin, and
streptolysins, as well as exotoxins.
Streptococcus is facultatively anaerobic or aerotolerant, but generally favoring high carbon

dioxide levels (hence, the use of the candle jar for incubation). The genus is always catalase
negative. When stained, it will be seen in pairs or chains that vary from a few to dozens.
Page 156
Streptococcus can be beta, alpha, or gamma hemolytic. Streptococci, like staphylococci,
cause a wide range of superficial skin infections along with more serious diseases like
pneumonia, meningitis, erysipelas and necrotizing fasciitis.
OBJECTIVES:
Obtain presumptive streptococci from throat swabs and attempt to isolate from blood agar
plates
Using isolates to identify different types of hemolysis
Perform testing to narrow down the identification of the streptococcal isolate
MATERIALS NEEDED:
Sterile cotton sawbs

Tongue depressers
CNA Blood agar plates containing naladixic acid
Containers of alcohol + forceps
Blood agar plate (5% sheep red blood cells)
mitis-salivarius agar plate (M-S)
Sensitivity disks (Bacitracin, SXT, Optochin)
3 Bile esculin slants
3-6.5% NaCl broth
TSB
SCHEMATIC OF IDENTIFICATION PROCEDURE

1st period Æ 2nd period Æ 3rd period Æ 4th period
Throat culture - blood agar blood agar gram stain 10 and 45 C incubation
Tooth scraping – M-S agar catalase
Bile esculin
6.5% NaCl
THE PROCEDURES:
Be sure to keep a list of all test results for your isolates.
1st Session
1. Two specimen types will be taken---a throat specimen and a specimen from the tooth-
gum area.
a. The subject should tilt the head back. Use a tongue depressor to push the
tongue down so a sterile swab can be used to obtain a sample from the back of
the throat (pharynx). Avoid swabbing the cheeks, tongue or teeth.
b. Try to get back back in the area of the molars or molar/premolars to get the
tooth culture. Run the cotton swab along the gum-tooth line..
2. Cover one third of a Columbia naladixic acid blood agar (CNA) plate with the throat
swab Then use a sterile loop to make a standard ISOLATION STREAK PLATE, using
3 or 4 sections). The procedure is identical to a streak plate procedure, with the
exception of using a cotton swab for the first section and then switching over to the
Page 157
inoculating loop. It is imperative that you get good isolation of colonies for this
exercise.
3. Using the swab from the tooth-gum area, streak a mitis-salivarius agar plate. Use the
same technique described above in step 2.
4. Incubate the plates in a candle jar at 37°C. The reduced oxygen atmosphere will
encourage good hemolytic reactions and provide more luxuriant growth of
Streptococcus.
2nd Session
1. Check the throat culture for hemolytic zones. It is not likely that you will find a beta
hemolytic since those species are pathogenic.
• alpha (α) hemolysis – green zone around colony, partial hemolysis
• beta (β) hemolysis – complete clearing around colony
• gamma (γ) hemolysis - no hemolysins, no zone
2. IF YOU HAVEA BETA HEMOLYTIC COLONY, subculture the colony onto a new
blood agar plate using proper islation streak technique. In addition, place a bacitracin
disc on the first section of the plate (using alcohol-flamed forceps).
3. Check the mitis-salivarius agar plate for different kinds of colonies.
• Strep mitis tiny blue
• Strep salivarius large, light blue “gumdrop”, mucoid colonies
• Enterococcus fecalis blue/black
• Strep mutans raised pale blue, granular “frosted glass”
• Strep sanguis smooth, hard colonies embedded in agar
4. Identify 2 different colony types on the M-S agar to streak to a blood agar plate. The
BAP will be divided in half—with each of your 2 table’s isolates streaked in a dense
zig-zag pattern. Do not get the 2 isolates too close to each other on the plate.
5. On each side of the BAP, on top of each streaked isolate, aseptically place an
optochin disc and an SXT disc (using alcohol-flamed forceps). Space the 2 discs out
enough so they are not too close together.
3rd Session
FOR COMMON ORAL STREPTOCOCCI
1. Gram stain the 2 colonies (taken from BAP) to verify that you have gram + cocci in
pairs or chains. If your isolate is not this profile, that will be the end of the isolate’s use
in this exercise.
2. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase -
(all species of Strep are -). If your isolate is not this profile, that will be the end of the
3. Check the growth of your Strep isolates on the blood agar plate.
• Identify the type of hemolysis (alpha or gamma)
• Determine the sensitivity of the isolates to the 2 discs
o ANY zone around the SXT disc means sensitivity
o A zone around the optochin disc must be at least 14mm. Strep
pneumonia is sensitive to optochin, and it is normal flora in many people.
4. Use the BAP to streak the surface of a bile esculin slant and to inoculate a tube of
6.5% sodium chloride broth.
5. IN ADDITION, each table will be given a group D Strep as a + control for bile esculin
and 6.5% NaCl. Inoculate this bacterium into those 2 media along with your own
isolates.
Page 158
6. Incubate the tests at 37°C.
IF YOU HAVE A BETA-HEMOLYTIC STREPTOCOCCUS

1. Gram stain the culture to verify that you have gram + cocci in pairs or chains. If your
isolate is not this profile, that will be the end of the isolate’s use in this exercise.
2. Check the sensitivity of the isolate to bacitracin. Any zone of inhibition shows
sensitivity. The group A Strep are sensitive to bacitracin.
3. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase -
(all species of Strep are -). If your isolate is not this profile, that will be the end of the
4. Use the BAP to streak the surface of a bile esculin slant and to inoculate a tube of
6.5% sodium chloride broth. Be sure to place the plate into a candle jar.
5. Incubate the tests at 37°C.
4th Session
1. Read the NaCl and bile esculin tests.
a. If the bacterium is resistant to high salt, it will grow and the NaCl medium will be
cloudy. This is a + test.
b. Bile esculin interpretation: Growth on the medium means that the bacterium is
resistant to 40% bile. In addition, those species that use the sugar esculin will
turn the medium dark brown/black (the color of black coffee). So if your slant is
dark brown/black, it means that it uses esculin as well as being resistant to bile.
These are both + test results.
2. If your isolate is + for bile resistance and esculin use, as well as resistant to high salt,
you likely have a group D Enterococcus. In this case, you will inoculate 2 tubes of
TSB and place the 2 tubes at 10C and 45C until the next lab period. Enterococcus will
grow at these temperatures.
3. Read any tests that were run last lab session.
4. If you had a beta hemolytic Strep that fits the Strep profile (correct gram stain and
catalase result), you will use this same culture for the rapid Strep test that is upcoming
in lab. Refrigerate the culture until that lab.
5. Use the chart below to presumptively identify your streptococci.
Organism Group Hemolysis Bacitracin SXT Optochin Bile: esculin 6.5% NaCl 10/45C
growth
S. pyogenes A beta S R R - : - -
S. agalactiae B beta R R R - : - -
E.faecalis/faecium D alpha or gamma R R R + : + + +
S. bovis D alpha, gamma R S R + : + -
S. mitis viridans Usually α -- R S R - : - -
S. salivarius Salivarius, (can be + if
S. mutans mutans, sobrinus testing
can be γ esculin alone)
S. pneumoniae alpha -R/S S S - : - -
R =resistant S =sensitive + =growth - =no growth
We have esculin sugar (without bile) available as a test. If you want to run esculin sugar alone, ask your
instructor to get you a disc. Add ½ ml of BHI broth to a sterile test tube, aseptically add the esculin disc, and
inoculate. Incubate the test til the next period. Look for a dark brown/black color change.
Page 159
QUESTIONS:
1. Why is a bacitracin (A) disk so important in strepotococcal identification? What are the
consequences of not making a diagnosis of bacitracin sensitivity?
2. Name two autoimmune diseases associated with the group A beta streptococci.
3. Why is the optochin test important for use on alpha streptococci?
4. Do a search using Google on the web, using the terms “viridans” and “color.” There is
a group of Strep called the Viridans group. What category of hemolysis?
5. What does a hemolysin do?

Page 160
Page 161
SEROLOGICAL TESTING-
LATEX AGGLUTINATION
The surface of microorganisms is covered with physical features that can be recognized by
the immune system. Any feature that can elicit an immune response is called an antigen.
The immune system makes proteins called immunoglobulins or antibodies which bind to
an antigen to either directly neutralize the antigen or cause it to be cleared more efficiently by
other components of the immune system. Antibodies linked to large particles such as latex
beads can be used to agglutinate microorganisms using specific reactive antibodies. Such
agglutination is directly observable to the naked eye as a clumping reaction.
This exercise involves 2 kinds of serological tests---one for the identification of an unknown
antigen and the other for the identification of an unknown antibody.
In the Monospot test, a known Epstein-Barr viral antigen is used in the test, for the
identification of patient antibody to this virus (which causes infectious mononucleosis,
Burkitt's lymphoma, and nasopharyngeal cancer). The interesting thing about this test is that
you are identifying NONSPECIFIC antibody rather than antibody against the Epstein-Barr
virus, the cause of infectious mononucleosis. This odd antibody, called ‘heterophile'
antibody reacts against sheep and bovine RBCs. The term heterophile antibody refers to
antibodies having the capacity to react with certain antigens, which are very different from the
ones inducing the antibody formation. The latex particles in the MONO-LATEX REAGENT
LATEX are coated with a suspension of mononucleosis antigen obtained from red blood cells
from cattle. If antibodies are present in the serum or plasma and mixed with the latex, a
specific reaction will result in a visible agglutination of the latex particles. The patient serum,
a NEGATIVE control, and a POSITIVE control are each placed on the glass slide, then
adding the reagent LATEX (plastic beads covered with antigen).
Another way to use serological testing is for the identification of the antigen itself,
using a known specific antibody for that antigen: a latex-conjugated antibody (Staphyloslide
kit) can be used to confirm the presence of Staphylococcus aureus. This is a good
confirmation of S. aureus that has been presumptively identified by biochemical and physical
testing. It differentiates Staphylococci which possess clumping factor and/or Protein A, Blue
latex powder pre-coated with the antibody to Staph aureus is mixed with a Staph culture. If
the specific antigen of Staph aureus is present, it will react with the latex-antibody, producing
a noticeable agglutination (which is more easily seen with the blue of the latex).
OBJECTIVES:
1. To make a serological identification using Staphylococcus previously isolated and

identified by biochemical testing.
2. To identify non-specific antibodies (heterophile antibodies) against Epstein-Barr virus.
MATERIALS NEEDED:
Staphylococcus strain isolated in the Staphylococcus experiment

Page 162
BD Staphyloslide kit
Monospot test kit for Epstein-Barr virus
Clearview Exact Strep II dipstick kit
cultures of Staph aureus and Strep group A to use as + controls
THE PROCEDURES:
Serological typing of Staphylococcus aureus
1. Mix the latex reagent by shaking; expel any latex from the dropper for complete mixing.
2. Dispense 1 drop of Test Latex (contains Staph antibody) onto one of the circles on the
reaction card and add 1 drop of Control Latex onto another circle.
3. Using a microbiological loop pick up and smear 5 suspect colonies (either a suspect
Staph from your body OR a known Staph aureus supplied from the lab) onto the Test
Latex-containing circle and mix this into the Test Latex reagent. Spread to cover the
circle. Be sure you have a homogenous bacterial suspension. Clumps will make the
reaction difficult to identify, and possibly give a false + reaction.
4. Repeat step 3 for the Control Latex, using the same bacterial culture.
5. Pick up and hand rock the card for up to 20 sec and observe for agglutination under
normal lighting conditions. Read macroscopically
6. Rock the slide back and forth for no longer than one minute and look for blue
agglutination. Clumping is a positive reaction for the identification of S. aureus.
7. Dispose of the reaction card in an appropriate biohazard container.
Serological identification of beta-hemolytic Streptococcus group A
This test is based on the same antigen-antibody reaction as the Staph

Page 163
There will be available cultures of Strep group A so that you can see what a + result
looks like. If you isolated a suspect group A Strep from your throat, you may use that culture.
Monospot test
1. Place a free-falling drop of NEGATIVE control serum on the glass slide

2. Place a free-falling drop of POSITIVE control serum on the glass slide in a different
well. Since we don't have patient serum, you will use the positive control serum as the
patient's serum.
3. Add 1 free-falling drop of the reagent LATEX to each well. Do not touch the reagent to
any of the drops already on the glass slide.
4. Mix the contents of each circle with a stirrer (different stirrers for each circle), covering
the entire surface of the circle.
5. Rock the slide in a circular motion for 2 minutes (40 rocks per minute). The test must
be read at 2 minutes.
6. Observe for agglutination. A light held directly over the slide will improve the readability
of the test.
Heterophile antibodies have also been identified with Burkitt's lymphoma,

cytomegalovirus, leukemia, viral hepatitis, and rheumatoid arthritis.
Page 164
QUESTIONS:
1. What feature of antibodies results in a positive test causing clumping?
2. Why are the antibodies attached to latex particles for this type of test?
3. If antibodies are regarded as being antigen-specific how can you explain the
occurrence of heterophile antibodies in mononucleosis?
4. In the Staphyloslide test, are you testing for a patient’s antibodies to Staph OR the
antigen itself?
BIOLOGY 2420 BIOCHEMICAL TESTS – FAQs Page 165
PURPOSE NAME OF MEDIUM INDICATOR or + REACTION - REACTION

NAME OF TEST REAGENT (visible reaction) (visible reaction)
bile resistance tolerates bile any growth no growth

bile esculin slant iron
esculin hydrolysis uses esculin sugar dark coffee slant no color change
casein hydrolysis
catalase no specific medium
coagulase test
citrate test
deaminase (phenylalanine)
decarboxylase (orn, lys,

arginine)
DNAse
gelatin hydrolysis
H2S
indole test
lipid hydrolysis methylene blue
methyl red test
salt resistance (7.5%) mannitol salt agar
mannitol use
Page 166
litmus milk
(different reactions)
TTC motility agar
nitrate reduction
oxidase test
ONPG test
phenol red sugar broths
starch hydrolysis
sugar fermentation
(various discs)
urea hydrolysis
voges-proskauer
Page 167
GLOSSARY OF TERMS
AEROBIC
requires oxygen (opposite of anaerobic)
AGAR
powder added to media for solidification
AIR-DRY
drying of slide suspension in air before heat fixing and staining
AGGLUTINATION
the reaction between antigen and specific antibody
ALIQUOT
dispense an amount of liquid using a pipette
ANALOG
similar structure, but not identical
ANTIBODY
specific, protective protein produced by the immune system in response to an antigen
ANTIGEN
foreign, non-self immunogenic material that elicits an immune response
ASEPTIC TECHNIQUE
procedure to guarantee sterility and to reduce contamination
ATRICHOUS
without flagella, nonmotile
AUTOCLAVE
moist heat method of sterilization using pressure
AXIAL FILAMENT
a structure for motility used by the Spirochaete bacteria
BIBULOUS PAPER
absorbant paper used to blot dry slides after staining
BHI
brain heart infusion, a really good enrichment medium
BRIGHTFIELD MICROSCOPY
full light directed towards the specimen, used for stained and live specimens
BROTH
medium without agar
BROWNIAN MOVEMENT
vibrations of an object seen in a microscope, not true motility
CANDLE JAR
candle burns in a closed container producing a carbon dioxide incubator, containing 2-
10% O2 and around 10% CO2
CFU
colony-forming units, i.e. colonies
CHEMICALLY-DEFINED MEDIUM
a synthetic medium, comprised of known ingredients and known amounts
CHITIN
a complex polysaccharide molecule found in walls of fungi and exoskeletons of
shrimps and crabs
CHROMATOGRAPHICALLY
migration and separation of molecules through an absorbant material like cotton, over time
Page 168
CNA
Columbia naladixic acid medium, selective (for Gram +) and differential medium
COAGULATION
clotting of blood, plasma
COLIFORMS
gram - rods which ferment lactose, nonsporeforming
COLONY
a visible mass of bacteria growing on solidifed medium, a clone
COMPLEX MEDIUM
medium with some unknown ingredients or amounts, i.e. blood agar
CONDENSER
collects all available light rays for direction up to the stage opening
COUNTERSTAIN
the 2nd dye added to a smear, taken in after the wall is decolorized, e.g. safrinin,
methylene blue
DARKFIELD MICROSCOPY
special condenser blocks most light rays, only reflected light bounces off specimen
into lens producing a black field of vision; used for wet mounts
DECOLORIZER
the reagent used to remove the primary dye from the cell wall in a differential stain,
e.g. acid alcohol, acetone-alcohol
DIFFERENTIAL STAIN
uses 2 or more dyes which allow differentiation between different bacterial groups or
structures
DIAPHRAGM
acts as an iris within the condenser which opens and closes for sensitive light control
DIPLOID
A full complement of chromosomes (e.g. 46 or 23 pairs for human)
DISACCHARIDE
2 sugar molecule, e.g. lactose or sucrose
EMB
Eosin methylene blue medium, selective (for Gram -) and differential medium
EXOENZYME
enzyme excreted away from the cell
FACULTATIVE ANAEROBE
uses oxygen when present but can either ferment or anaerobically respire without it
FASTIDIOUS
hard-to-grow bacteria, requiring grow factors or particular nutrients
FECAL COLIFORMS
gram - rods which ferment lactose, nonsporeforming, GI flora in animals, in feces
FLAGELLA
a structure for motility
FLAGELLATION
differential category of flagella placement around the cell
GENUS
category of organisms with like features and closely related, divided into species
HALOPHILIC
salt-tolerant or salt-loving (salt-requiring)
Page 169
HAPLOID
Half the normal number of chromosomes
HEAT-FIX
use of the flame to 1)coagulate proteins of the suspension, causing adherence to
slide, and 2) kill the microbes
HELMINTH
parasitic worm
HYPHA
A fungal filament (plural, hyphae)
IMMUNOASSAY
test which identifies antibody in patient based on use of a known antigen, or identifies
the antigen based on the use of a known antibody
IMViC
acronym = indole, methyl red, Voges-Proskauer, citrate
INOCULUM
a small sample of the microorganism
MIC
minimal inhibitory concentration of antibiotic that inhibits a bacterium
MICROAEROPHILIC
likes a reduced oxygen concentration
MICROTUBULES
cell organelles involved in structure, framework, skeleton of the cell
MONOSACCHARIDE
simple sugar, e.g. glucose
MYCELIUM
A visible mass of hyphal filaments
NA/NB
nutrient agar or nutrient broth
NONIONIC
no electrical charge
OBLIGATE AEROBE
requires oxygen to grow
OBLIGATE ANAEROBE
does not use oxygen to grow, may even be killed by it
OIL-IMMERSION LENS
100X objective lens, to be used always with immersion oil
PALLINDROME
a word or a group of letter that is read frontwards and backwards the same, e.g. radar
PARFOCAL
feature of microscope which allows rotation of lenses with only minor focus movement,
lenses alignment
PATHOGENIC
disease-causing
PCA
plate count agar medium, general all-purpose enrichment
PFU
plaque-forming units produced by bacterial viruses when infecting host bacterial cells
PHASE-CONTRAST MICROSCOPY
special condenser below stage and in objectives change speed of light rays,
enhancing density differences inside and outside of cells; used for wet mounts
Page 170
PHAGE TYPING
test to identify a bacterium based on which known virus infects it
PHENOTYPE
expression of a gene as a trait
PLAQUE
destruction of the bacterial lawn by a bacteriophage as the lytic infection progresses
PLATE COUNT AGAR
variation of nutrient agar, for optimizing counts of bacteria in samples
POUR PLATE
procedure where liquified agar has been poured into a petri dish after being mixed with
bacteria
PRIMARY DYE
the 1st dye used in a differential stain, e.g. malachite green, crystal violet, carbol
fuschin
RESIDENT FLORA
Microbes firmly entrenched in niches in/on the body
REVOLVING NOSEPIECE
rotating turret attached to the 3 lenses
RESAZURIN
oxygen indicator in thioglycollate broth, pink when oxygenated
SEROLOGY
the analysis of substances—antigen or antibody--in blood serum
SPECIES
a subdivision of a genus, almost identical organisms, a clone
SPREAD PLATE
procedure where pre-made agar plates have a sample of bacterium placed on top of
the agar and spread via a glass rod
STREAK PLATE
procedure where a bacterial specimen is placed on a pre-made plate and diluted out
using flame and multiple sections.
SYMBIOTIC/SYMBIOSIS
a relationship between 2 or more organisms
THERMAL DEATH TIME
minimal amount of time to completely sterilize a specimen at a certain temperature
TRANSIENT FLORA
Microbes easily dislodged from the body, come-and-go
TSB/TSA
trypticase soy broth or trypticase soy agar
ZONE OF INHIBITION
area of no bacterial growth around a chemical on a disc, indicates sensitivity

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RICHLAND COLLEGE

Study guide for lab quizzes/practicals

Table of Biochemical Tests---FAQs and Study Guide 165

PURE CULTURE TECHNIQUES

SIMPLE STAINING & SMEAR PREPARATION

MOTILITY (flagella stain, SIM, TTC motility, hanging drops)

CAPSULE STAINS interpretation

PIPETTING & DILUTIONS

COUNTING BACTERIA - STANDARD PLATE COUNT & TURBIDEMETRY

ENVIRONMENTAL INFLUENCES: pH, osmotic pressure

EFFECTS OF TEMPERATURE ON GROWTH

KIRBY-BAUER TEST FOR ANTIBIOTIC SENSITIVITIES

BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA

SEROLOGICAL LATEX SLIDE AGGLUTINATION: STAPH, STREP, and MONOSPOT

MATERIALS: glo-germ powder

1. Collect materials for exercise---1 plate of talcum powder per student.

USE OF THE MICROSCOPE

HANDLING THE MICROSCOPE:

Identify the parts of the microscope and their functions.

BEFORE PUTTING A SLIDE ON THE MICROSCOPE STAGE:

The Nikon lenses are PARFOCAL---the objectives are

TO MOVE INTO OIL IMMERSION, 100X MAGNIFICATION:

USING DARKFIELD AND PHASE-CONTRAST:

PLACING MICROSCOPES BACK INTO THE CABINETS:

Light off center?

• Not focusing on the specimen, but rather dirt on slide.

PART OF MICROSCOPE FUNCTION

1. coarse adjustment knob general focus, particularly for 10X lens

LABORATORY REPORT SHEET

1. Which objective lens is also called the oil-immersion lens?

SHAPES & ARRANGEMENTS OF BACTERIA

cocci (coccus = singular)

chain pair cluster tetrad

bacilli (bacillus = singular)

For example: Streptococcus (strepto = chain) is found as a chain of cocci.

Prepare wet mounts using live samples.

Prepare a wet mount of pond water.

Look at a bought prepared bacterial smear.

WASH YOUR HANDS AFTER WORKING WITH MICROORGANISMS AND STAINS!

Draw your results seen in the microscope (using 100X lens)

from the mouth from the bought prepared smear

1. What is a simple stain?

3. Draw a bacillus-shaped bacterium.

4. What would happen if you forgot to heat-fix the smear?

5. Why stain bacteria rather than looking at wet mounts of them?

These ectoparasite vectors are in the Kingdom

Dept. of Medical Entomology, Univ. of Sydney

Learn about various vectors of infectious diseases.

prepared slides of ectoparasites

flea louse bedbug

4. Why is malaria not common in the United States?

FEATURES OF HELMINTHIC GROUPS:

trematodes (flukes) – flat, leaf-shaped, unsegmented, separate sexes

cestodes (tapeworms) – flat, segmented, hermaphroditic

nematodes (roundworms) – unsegmented, separate sexes

Excellent Resource on Parasites at Centers for Disease Control!

Laboratory Identification of Parasites of Public Health Concern

Differentiate between flatworms and roundworms.