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Microbiology lab-2014
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Microbiology lab-2014
GENERAL DIRECTIVES
1. Attendance in lab is mandatory. Please be on time.
2. Shoes and appropriate dress must be worn at all times. Secure long hair.
3. Wear a lab coat—they are easier to sterilize than your clothing, should you spill a culture or
staining reagents.
4. Leave outerwear, backpacks, and any other extraneous materials in the lockers outside of the
lab.
5. Be careful with Bunsen burners—keep them away from microscopes, paper, ethanol, and
watch your hair. Never leave the flame unattended.
6. Always place used pipettes, swabs, and other materials in the biohazard bags provided so
that they can be autoclaved and disposed of properly. Do NOT throw trash in the autoclave
bag.
7. No eating or drinking in lab.
8. Never lick your fingers, or put your fingers in your mouth.
9. Treat every organism as a potential pathogen.
10. Treat spilled cultures with disinfectant before cleaning them up. Cover the spill with a paper
towel. Spray the paper towel with disinfectant until the towel is soaking wet. Let this sit for
10 minutes. Wearing gloves, pick up the paper towels and discard in the autoclave bag. Ask
the instructor or T.A. for help as soon as the spill occurs.
11. Remember to wipe the oil off the lenses before putting the microscope away.
12. No radios, MP3 players, or CD players in the lab.
13. No use of cell phones or texting in the lab.
14. Notify the T.A. or instructor of any accident, no matter how minor.
15. At the beginning of each lab period, clean your bench with disinfectant. Clean it again at the
end of lab.
16. WASH YOUR HANDS before beginning the lab exercises. WASH YOUR HANDS before
leaving the lab, even if it’s only for a break.
Material you MUST have to work in the microbiology lab:
A lab coat
Safety classes are recommended when working with the flame.
A thin tipped permanent, preferably black, marker for labelling.
A note book to record your results. Any type is acceptable. Do not waste your money.
A USB key to save your pictures
Optional but strongly recommended:
Do not wear contact lenses in the lab. They can be quite hazardous.
Notify the instructor of any safety or medical concerns so that appropriate accommodations can
be taken. For example, allergies, diabetes, hypoglycemia, epilepsy, exposed wounds, color
blindness, etc..
Notify the instructor of any special needs you may require so that appropriate accommodations
can be taken. For example, if you write your exams with SASS.
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Microbiology Lab-2014
Table of contents
Page
Grading scheme
4
3MT Presentations
5
Schedule
6
Lab 1: Dilutions and concentrations/Microscopy
7
Lab 2: Microbial growth in the lab
19
Lab 3: Counting bacteria
31
Lab 4: Growth media and soil bacteria
47
Lab 5: Bacterial diagnostics – Gram positive bacteria
58
Lab 6: Control of microbial growth
78
Lab 7: Water and food microbiology
90
Lab 8: Bacterial diagnostics –Gram negative bacteria
105
Lab 9: Immunological diagnostic/Epidemiology
117
Metric Units
127
Growth media composition
128
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Microbiology Lab-2014
Grading Scheme
Quiz
2 bonus points for 100% on 4/8 quizzes
Assignments
20%
Reports
10%
Midterm Exam
25%
3MT presentations
10%
Practical Exam
 In lab
 Job Interview
5%
5%
Final Exam
25%
Quizzes: At the beginning of each lab, a 10 minute quiz consisting of one to two questions will
be given. Your performance on these cannot have a negative impact on your final grade.
However, if you obtain 100% on at least 4 of the 8 quizzes you will be granted 2 bonus points on
your final grade.
Assignments: This course includes 5 assignments on the theory of the experiments performed
and the analysis of the results obtained. These assignments may be submitted either individually
or in groups of two (you and your lab partner). A 10% /day penalty will be imposed on late
assignments. (Weekends will be considered as one day) All assignments are due on the indicated
date (See schedule on page 6) before you leave the lab for the day.
Reports: This lab includes two formal lab reports on the identification of bacterial unknowns.
See the assignments document for the general requirements and directives for these reports. As
with the assignments, reports may be done individually or in groups of two.
Midterm and Final exams: A midterm and a final theoretical exam will be given at the dates
specified in the schedule on page 6 during lab hours. These exams will be given over a 1.5 hour
period. You are allowed a single one sided cheat sheet (8 1/2 X 11 in.), scrap paper, and
calculators.
3MT presentations: Each student will be asked to give a 3 minute presentation on a current
topic in microbiology. You are free to choose any topic of interest within the field. Presentations
will be evaluated by each of your teaching assistants as well as your peers. Consequently, you
must be present for all the presentations. Check the rules for these presentations on page 5.
Practical exam: This exam consists of two parts. The in lab component will require that you
come to the lab in order to accomplish a set of tasks commonly used within the lab. You shall be
evaluated on the following tasks: Gram Staining, streaking for single colonies, sterile technique,
dilutions and spreading of a broth culture. You will have 1 hour to accomplish these tasks. The
second part will consist of a mock 10 minute job interview for a position in the field of
microbiology. A job posting will be made available about one week before the interview date.
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Microbiology Lab-2014
3 MT Presentations
Rules

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

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A single static PowerPoint slide is permitted (no slide transitions, animations or
'movement' of any description, the slide is to be presented from the beginning of the
presentation).
No additional electronic media (e.g. sound and video files) are permitted.
No additional props (e.g. costumes, musical instruments, laboratory equipment) are
permitted.
Presentations are limited to 3 minutes maximum and presenters exceeding 3 minutes will
be stopped and penalized.
Presentations are to be oral without reading (e.g. no poems or songs).
Presentations are considered to have commenced when a presenter starts their
presentation through movement or speech.
Judging Criteria
COMPREHENSION & CONTENT:
 Did the presentation provide an understanding of the background to the topic being
addressed and its significance?
 Did the presentation clearly describe the key points of the topic including conclusions
and outcomes?
 Did the presentation follow a clear and logical sequence?
 Was the topic, key facts and significance communicated in a language appropriate to a
non-specialist audience?
 Did the presenter spend adequate time on each element of their presentation - or did they
elaborate for too long on one aspect or was the presentation rushed?
ENGAGEMENT:
 Did the presentation make the audience want to know more?
 Was the presenter careful not to trivialise or generalise the topic?
 Did the presenter convey enthusiasm for the topic?
 Did the presenter capture and maintain their audience's attention?
 Did the PowerPoint slide enhance the presentation - was it clear, legible, and concise?
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Microbiology Lab-2014
Schedule
Date
Due dates
Lab 1
Sept. 8 & 10
Lab 2
Sept. 15 & 17
Lab 3
Sept. 22 & 24
Lab 4
Sept. 29 & Oct. 1
3MT presentations
Oct.6
Midterm exam
Oct. 8
Study break
Oct. 13 - 17
Lab 5
Oct. 20 & 22
Assignment 3 due Oct. 22
Lab 6
Oct. 27 & 29
Report 1: Identification of Gram positive
unknowns due Oct. 29
Lab 7
Nov. 3 & 5
Assignment 4 due Nov. 5
Lab 8
Nov. 10 & 12
Lab 9
Nov. 17 & 19
Practical exam
 In lab
 Interviews
Nov. 24
Nov. 26
Final exam
Monday Dec. 1st
Assignment 1 due Sept. 17
Assignment 2 due Oct. 4
Assignment 5 due Nov. 19
Report 2: A potato epidemic due Nov. 26
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Microbiology Lab-2014
LAB #1
DILUTIONS AND CONCENTRATIONS
Day 1
One very important property of solutions that must be addressed is concentration. Concentration
generally refers to the amount of solute contained in a certain amount of solution. To deal
with concentration you must keep in mind the distinctions between solute, solvent and solution.
Because varying amounts of solute can be dissolved in a solution, concentration is a variable
property and we often need to have a numerical way of indicating how concentrated a solution
happens to be. Over the years a variety of ways have been developed for calculating and
expressing the concentration of solutions.
That can be done with percentages using measurements of weight (mass) or volume or both. It
can also be done using measurements that more closely relate to ways that chemicals react with
one another (moles).
In the pages that follow, several concentration types will be presented. They include volume
percent, weight percent, weight/volume percent, molarity (the workhorse of chemical
concentrations), and weight/volume.
You will get experience with more than one way of establishing the concentration of solutions.
You can prepare a solution from scratch and measure each of the components that go into the
solution. You can prepare a solution by diluting an existing solution.
PERCENTAGE
The use of percentages is a common way of expressing the concentration of a solution. It is a
straightforward approach that refers to the amount of a component Per 100. Percentages can be
calculated using volumes as well as weights, or even both together. One way of expressing
concentrations, with which you might be familiar, is by volume percent. Another is by weight
percent. Still another is a hybrid called weight/volume percent.
Volume percent is usually used when the solution is made by mixing two liquids.
For example, rubbing alcohol is generally 70% by
volume isopropyl alcohol. That means that 100 mL of
solution contains 70 mL of isopropyl alcohol. That
also means that a liter (or 1000 mL) of this solution
has 700 mL of isopropyl alcohol plus enough water to
bring it up a total volume of 1 liter, or 1000 mL.
Volume percent =
volume of solute
volume of solution
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x 100
Microbiology Lab-2014
Weight Percent is a way of expressing the concentration of a solution as the weight of solute/
weight of solution.
Weight percent =
weight of solute
x 100
weight of solution
As an example, let's consider a 12% by
12 g NaCl
12 % NaCl solution =
weight sodium chloride solution. Such a
100 g solution
solution would have 12 grams of sodium
chloride for every 100 grams of solution.
To make such a solution, you could weigh
out 12 grams of sodium chloride, and then
add 88 grams of water, so that the total
12 g NaCl
mass for the solution is 100 grams. Since (12 g NaCl + 88 g water) = 12% NaCl solution
mass is conserved, the masses of the
components of the solution, the solute and
the solvent, will add up to the total mass of
the solution.
To calculate the mass percent or weight percent of a solution, you must divide the mass of the
solute by the mass of the solution (both the solute and the solvent together) and then multiply by
100 to change it into percent.
Percentage weight/volume is a variation which expresses the amount of solute in grams but
measures the amount of solution in milliliters. An example would be a 5 % (w/v) NaCl solution.
It contains 5 g of NaCl for every 100 mL of solution.
Volume percent =
weight of solute (in g)
x 100
volume of solution (in mL)
This is the most common way that percentage solutions are expressed in this lab course.
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Microbiology Lab-2014
MOLARITY
Another way of expressing concentrations is called
molarity. Molarity is the number of moles of solute
moles of solute
dissolved in one litre of solution. The units, Molarity =
litre of solution
therefore are moles per litre, specifically it's moles
of solute per litre of solution.
Rather than writing out moles per liter, these units are abbreviated as M. So when you see M it
stands for molarity, and it means moles per liter (not just moles). You must be very careful to
distinguish between moles and molarity. "Moles" measures the amount or quantity of material
you have; "molarity" measures the concentration of that material. So when you're given a
problem or some information that says the concentration of the solution is 0.1 M that means that
it has 0.1 moles for every liter of solution; it does not mean that it is 0.1 moles.
WEIGHT/VOLUME
This means of expressing concentrations is very similar to that of percentages and is one of the
most popular ways used by biologists. In contrast to percent, the concentration is expressed as a
mass per any volume the user wishes to use. Most commonly, these concentrations are expressed
per one measuring unit. For example per 1 mL, 1 µL or 1L, etc. Essentially these expressions
represent the mass of solute present in a given amount of solution. For example a solution at a
concentration of 1mg/mL contains 1mg of solute in 1 mL of solution.
RATIOS
All the ways described above to express concentrations are done as a function of the total volume
of the solution which is the volume of the solvent and that of the solute. A common method used
by many microbiologists and chemists to express concentrations are ratios. In this case, the
relationship between the solvent and the solute is expressed independently of one another. For
example, we could say that the ratio between a solute and its solvent is 2:1. This indicates that
for two parts of the solute there is one part of solvent. Thus three parts total of solution.
DILUTIONS
The preparation of dilutions is essential in all fields of science as well as in everyday life.
Dilutions are used to precisely reduce the concentration of elements, either chemical or alive,
within a solution. For example, if you wished to reduce the concentration of fat in 3.5% milk to
0.35% you would have to perform a 10-fold dilution. To comprehend how dilutions are prepared,
you must grasp the following three concepts: Concentration, dilution factor, and the dilution.
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Microbiology Lab-2014
The dilution factor represents the multiple by which an initial concentration must be divided by
in order to obtain the desired final concentration. For example, if a solution contains 30g of
caffeine per litre of solution and you wish to reduce the caffeine concentration to 0.3 g/L, then
you will have to divide the initial concentration by 100, which represents the dilution factor. You
can use the following formula in order to determine a dilution factor.
Dilution Factor =
Initial Concentration
Final Concentration
The dilution represents the fraction of the component being investigated. For example, in the
previous problem a dilution of 1/100 was prepared. The dilution is expressed as a fraction of 1
over the dilution factor.
In order to properly setup dilutions you must learn to properly use pipettes. Here are some
general guidelines:
Choose the pipette whose capacity is closest to the volume you wish to measure. For
instance, to measure 0.1mL it is best to use a 1.0 mL pipette rather than a 10 mL pipette.
Minimize the number of pipetting’s done to minimize the chances of error. For instance if
you wish to dispense 1 mL in ten tubes, it is best to pipette 10 mL once and dispense 1 mL ten
times rather than pipetting 1 mL ten times and dispensing ten times. Change pipettes for each
different solution.
Performing serial transfers: When performing serial transfers as in serial dilutions multiple
pipettes are not required. Use the following guidelines to minimize the number of pipettes used:
Source
Going from a more concentrated to a less concentrated solution: Pipette the desired volume
from the source and then dispense into the new tube (“A” in the picture below). Rinse by
pipetting up and down several times (In “A” in the picture below). Pipette the desired volume
from this tube (“A”) and dispense into the next tube (“B”). Repeat the process from “A” to “B”
with the same pipette.
A
B
C
10
Dispense and rinse pipette in this tube before uptake of the
desired volume from this new solution.
Microbiology Lab-2014
Going from a less concentrated to a more concentrated solution: In this instance, pipetting is
even easier. Just use the same pipette to transfer the desired volume from a lower concentration
solution to one of higher concentration. No equilibration or rinsing is required in this case.
Calculation of serial dilutions
Dilutions essentially represent fractions and thus follow the same mathematical principals. That
being said, the dilution (or the fraction) indicates what fraction of the total is represented by the
compound being diluted.
Ex. You wish to dilute a solution by a factor of 4. To do so the fraction desired is therefore 1/4;
i.e. a quarter of the total volume must be represented by whatever is being diluted. Therefore,
two fractions which are equal, for example 2/4 and 4/8 represent the same dilutions or dilution
factors.
Preparing dilutions: The things you must determine before preparing dilutions are what final
total volume you want, what is the dilution factor desired, and what is the final concentration you
want (if this is known).
For example, I want a final volume of 50 mL and a dilution factor of 4X. The fraction desired is
thus 1/4 where the denominator represents the total. Since I want a final volume of 50 mL, 1/4 of
the 50 must represent the compound being diluted; thus 12.5. What this means is that 1/4 =
12.5/50. Therefore to prepare this dilution you would add 12.5 mL of the solution to be diluted in
(50 mL -12.5 mL = 37.5 mL) of solvent.
You can use the following formula to determine the volume of the stock solution to dilute if you
know the final concentration that you wish to obtain :
Concentration you want
Concentration you have
X
Final volume wanted
=
Volume of stock solution
to be added to the mixture
Serial dilutions are simply sequential dilutions where the stock solution used for each dilution
represents the previous dilution. The final dilution for the series is the product of each individual
dilution.
Final Dil. = Dil.1 X Dil. 2 X Dil. 3 etc.
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Microbiology Lab-2014
ASEPTIC TECHNIQUE
In the field of microbiology, the aseptic technique is one of the procedures used by
microbiologists to prevent the contamination of one’s self, which could lead to an infection, the
contamination of their workplace, as well as contamination of the materials and the specimens
with which they work. It is used in all cases involving the transfer of cultures or microbiological
media. The use of the flame from a Bunsen burner is central to the proper execution of this
technique. You will be using this technique throughout the semester and it is therefore
imperative that you master it.
This week, you will be demonstrated aseptic technique as it is used with serological pipettes.
Exercise 1.0
PREPARATION OF GROWTH MEDIA AND MICROBIAL GROWTH:
To familiarize yourselves with the ingredients of a growth medium, concentrations, and
dilutions, you will perform the following exercise which will allow you to examine the growth
yield of Saccharomyces cerevisiae in yeast extract-peptone growth medium (YEP medium) as a
function of different carbon sources. The composition of this medium is indicated below.
Reagent
Bacto peptone
Yeast extract
Final concentration (m/v)
2%
1%
Sterilize by autoclaving.
Materials (Per groups of 2)
7 sterile test tubes
One bottle or flask
10 mL of sterile water
5 mL of the assigned 10% (m/v) sugar solution (glucose, galactose, fructose or sucrose)
1 mL of a yeast culture grown in YEP + glucose, then centrifuged and suspended in sugar free
YEP medium.
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Microbiology Lab-2014
Method (Per groups of 2)
1. Prepare 75 mL YEP medium in the bottle or flask provided.
2. Bring your medium to the designated area so that it can be autoclaved. Make sure to
properly label the container with your group number.
3. Label 7 sterile test tubes from 1 to 7 and with the following information: Your group number
and the sugar
4. Using aseptic technique, add to test tubes 1 to 7 the following volume, 0.0, 0.1, 0.2, 0.5, 0.8,
1.0 and 2.0 mL of the sugar solution assigned to your group; either glucose, galactose,
fructose or sucrose
5. Using aseptic technique, complete the volumes to 2.0 mL with sterile water.
6. Once the YEP medium has been sterilized and cooled down, add 3 mL of medium to each
tube.
7. Inoculate each of the test tubes with 0.1 mL of the yeast suspension provided.
8. Incubate at 30oC until the next lab period.
9. In the next lab period obtain optical density readings of each culture at a wavelength of
600nm of each culture. Make sure to thoroughly mix the cultures before obtaining your
readings.
10. For your assignment, also obtain the readings of the groups which used different carbon
sources.
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Microbiology Lab-2014
Day 2
INTRODUCTION TO MICROSCOPY
Microbiology is the study of very small organisms, microorganisms, which can only be viewed
with the aid of a microscope. There are several groups of organisms that fit into this category
including bacteria, algae, fungi, and protists. Within this group there are several species
interesting to humans because of their ability to cause disease or their use in the food industry.
Our focus during the semester will be on bacteria. Given their very small size, microorganisms
cannot be observed with the unaided eye unless a very large number of cells are present in a very
small area. It is therefore necessary in order to see microbial cells to use an instrument that
allows magnification; the microscope.
The light microscopes used in this lab are binocular and have ocular lenses with a magnification
of 10X. In addition to this magnification there are also four different objective lenses to choose
from – 4X, 10X, 40X, and 100X. Magnification of the object being viewed is the product of the
ocular objective multiplied by the lens objective currently in use. For instance when viewing an
object on the 4X objective lens, the object is magnified a total of 40X. The highest objective for
our microscopes is 100X, which has a magnification of 1000X. With this objective, it is
necessary to place a drop of oil on top of the slide and immerse the lens. The oil immersion lens
(100X) is specially sealed and IS THE ONLY LENS THAT SHOULD BE PLACED IN OIL!
The importance of proper handling and use of the microscope is vital. It is critical that you clean
the microscopes before and after use. Use a new KimWipe to gently wipe the ocular lenses and
then wipe the 10x, 40x, and 100x objective lenses. If there is any excess oil on the microscope be
sure and remove that. If you have trouble removing oil, use the microscope cleaner provided.
Viewing a specimen
1. Use the coarse adjustment knob (1) to move the stage to its lowest level.
2. Clean all objective lenses.
3. Before use, clean all slides, top and bottom, with KimWipes.
4. Adjust the condenser (2) to its highest level. Turn on the lamp.
5. Rotate the objectives until the 10X objective clicks into place.
6. Place the slide on the stage so that it is held within the slide holder clamping device. The
slide must lie flat on the stage. Using the mechanical stage knobs (3), position the slide so
that the specimen is in the exact center of the light coming through the condenser.
7. While looking through the eyepieces (NOT AT THE COMPUTER SCREEN), adjust the
width between the eyepieces until a single, circular field is seen simultaneously with both
eyes.
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Microbiology Lab-2014
8. Light intensity is an essential aspect of microscopy. For optimal viewing, the light must be
adjusted at each magnification. Always adjust the light while looking through the eyepieces.
a. Initially adjust the light intensity (4) at a low/medium setting. The entire viewing
area (field) must be filled with light. The lighted area may become smaller while
focusing or changing magnifications.
b. Locate the thin, black iris diaphragm (5) lever under the stage. Adjust this lever
to a medium/low light level. The iris diaphragm will need to be adjusted as
magnifications increase.
9. Under low power (10X), SLOWLY focus with the coarse adjustment knob until the
specimen comes into view. Adjust the light as required.
10. Focus the image with the fine adjustment knob and by adjusting the light.
11. Before switching to the next objective, move the slide so that the desired specimen is located
in the center of the field.
100X Oil Immersion Procedure
Immersion oil is used with the 100X objective because it increases the resolution. The oil should
come in contact with both the lens of the 100X objective and the slide.
12. Be sure that the specimen is in the EXACT CENTER of the viewing field under the 40X
objective.
13. Rotate the 40X objective away from the slide but do not yet click the 100X objective into
place.
14. Put a small drop of immersion oil on the slide directly over the light.
15. Rotate the nosepiece until the 100X oil immersion objective is clicked into place.
16. DO NOT USE THE COARSE ADJUSTMENT KNOB WHEN FOCUSING UNDER
THE 100X OBJECTIVE! ONLY USE THE FINE ADJUSTMENT KNOB!
17. Adjust the light for optimal viewing.
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5
4
2
1
3
Taken from: http://coolessay.org/docs/index-32428.htmL
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Common Problems
The field is dark.
Is the light on?
Is the objective securely clicked into place?
Is the diaphragm open?
Is the slide lying flat on the stage?
You are not sure if you are looking at dirt on the objective lens or the specimen.
Use the mechanical stage knobs to move the slide slightly while looking through the eyepieces.
If what you are looking at does not move, it is probably dust or dirt on the objective. If it does
move, it is on the slide.
Rotate the ocular gently between your fingers. If what you are looking at rotates, it is probably
dirt on the ocular.
You cannot find the specimen.
Is the specimen directly over the light?
Is the slide secure and flat in the mechanical stage?
Did you start with a low power objective and focus on the lower objectives first?
Have you adjusted the light?
Are you moving the adjustment knobs too quickly? Work slowly so you do not miss the
specimen. Remember, bacteria look like specks at low magnifications.
You are having trouble focusing.
Always start on a low power objective, and focus here first.
Focus SLOWLY! It is very easy to move past the specimen if the adjustment knobs are moved
too quickly.
Be sure to look in the ocular (NOT THE COMPUTER SCREEN) while you are focusing with
the adjustment knobs or changing the light intensity.
Adjust the light.
You lose the specimen when switching from the 40X objective to the oil immersion objective.
Was the specimen in the exact center of the field before switching to the 100X objective?
Is the 100X objective lens clean?
Have you adjusted the light?
Have you refined the image with the fine adjustment?
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Microbiology Lab-2014
Exercise 1.1
Materials (Per groups of 2)
Slide of the letter "e"
Slides
Coverslips
Tooth picks
1 mL yeast suspension
Method (individually)
1. Initially you will start by examining microscopically a prepared slide of the letter "e".
2. Follow the directives previously presented to obtain pictures of the letter "e" at the following
objective magnifications: 10X, 40X and 100X (under oil immersion).
3. While observing the letter "e" under the 10X objective carry out the necessary steps to
answer the following questions:
a. What is the orientation of the letter "e" relative to your view with the naked eye?
b. When you move the slide away from you, in what direction does the letter "e" move?
c. When you move the slide towards your left, in what direction does the letter "e" move?
4. In this part of the exercise you will be preparing a wet mount of your cheek cells. This
is the only lab where wet mounts will be used.
5. Put one drop of water in the center of the slide.
6. Take a flat-sided toothpick and carefully (and repeatedly) scrape the inside of your cheek to
obtain some cheek cells.
7. Gently stir the toothpick in the drop of water.
8. Gently lower the cover slip over the cheek cells.
9. Follow the directives previously presented to obtain pictures of the cells at the following
objective magnifications: 10X, 40X and 100X (under oil immersion). Locate and identify
the cytoplasm, cell membrane and the nucleus.
10. In this part of the exercise you will be preparing a wet mount of a yeast suspension.
11. Put one drop of the yeast suspension in the center of the slide.
12. Gently lower the cover slip over the yeast cells.
13. Follow the directives previously presented to obtain pictures of the cells at the following
objective magnifications: 10X, 40X and 100X (under oil immersion).
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Microbiology Lab-2014
LAB #2
MICROBIAL GROWTH IN THE LAB
Day 1
In their natural setting, not only are the number of microorganisms relatively low, but in addition
several different species live together. For example, the tips of your fingers are probably covered
by at least ten different species of bacteria in relatively low numbers (between a few hundred to
several thousands). In addition, optimal conditions required for the growth of a given species (for
example its nutritional requirements, temperature, pH, etc.) are very diverse. By keeping in mind
these characteristics; microbiologists have developed several different strategies to enable the
study of microorganisms.
MICROBIOLOGICAL MEDIA
It is often impractical to study a microorganism in its natural environment. For instance, if one
wanted to investigate the effects of newly developed antibiotics on a given bacterial pathogen it
would be unethical to do this on humans themselves. Furthermore, since bacterial populations
are heterogeneous, the effect on a single isolated species would be difficult to interpret.
Consequently, methods are necessary to grow bacteria in culture in a laboratory environment.
This is achieved by using a variety of different media, which can be synthetic or non-synthetic in
nature.
Growth media are available in liquid, semi-solid, or solid form. Semi-solid and solid media are
obtained by the addition of different concentrations of a solidifying agent. The most common
solidifying agent used in microbiology is agar; a polysaccharide derived from seaweed. Agar
possesses several advantageous characteristics. 1) it can be easily liquefied by boiling, and can
be maintained in its molten form at temperatures as low as 45oC. 2) agar solidifies at
temperatures below 45 degrees, and finally 3) most bacteria do not digest agar. Another
solidifying agent that is less commonly used is gelatine. Its use is less common since many
bacteria can digest it.
Liquid media are usually referred to as broths. Solid media can be in the form of plates or slants.
All media must include the necessary nutrients required for microbial growth. Other conditions
such as temperature and the presence or absence of oxygen are controlled by other pieces of
equipment such as an incubator. These conditions allow the microbiologist to grow and maintain
large numbers of bacteria, which are necessary for experimental studies.
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Microbiology Lab-2014
INOCULATING SOLID MEDIA: STREAKING
Streaking can be done from a solid or a liquid medium, whereas spreading is always performed
from a liquid medium. The instrument used in this case is an inoculation loop, which is basically
a metal wire used to pick up and deliver the bacteria. Do not confuse this instrument with the
inoculation needle, which has a straight end rather than a loop.
Exercise 2.0
Materials (Per groups of 2)
Agar plate with E. coli
Two TSA plates
Two TSA slants
Method (individually)
1. Before using the loop, it must be sterilized. To do this, place the loop in the flame of the
Bunsen burner until it turns red. Allow the loop to cool for a minute or so before using it.
DO NOT PUT IT ON YOUR BENCH.
2. Once it has cooled down, grab some bacteria from the plate culture supplied. Be careful not
to pick up too much.
3. Now streak the surface of the new plate as shown.
4. Repeat to inoculate a slant as shown.
5. Place samples, plate and slant, in the designated area so that they can be incubated at 37oC
until the next lab period.
Plate
Slant
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Microbiology Lab-2014
INOCULATING SOLID MEDIA: STREAKING FOR SINGLE COLONIES
Streaking for single colonies is simply a more elaborate method of streaking which is used to
generate pure cultures in which only one organism can be found. Several different methods exist
to generate pure cultures. In general, all of these methods rely on the principal of dilutions to
isolate the desired organism from all others. Let us take for example a population of millions of
individuals from which you want to identify and pick out a specific individual. If one could
dilute the population such that in any given area there were only a couple of individuals, it would
then be easy to pick out the individual you are interested in. As mentioned above, diluting a
heterogeneous starting culture to such a point usually generates pure cultures. Streaking for
single colonies achieves this.
The procedure is essentially as follows. Initially bacteria are picked up from a broth or solid
culture with a sterile loop. The bacteria are then streaked on an area of the plate, essentially
diluting it. The loop is then sterilized once again and used to streak a new area of the plate by
picking up bacteria from the initial streak, thus diluting it even more. (See figure below) Note:
You must flame the loop between each streaking and you must not go back to the source.
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Microbiology Lab-2014
Exercise 2.1
Materials (Per groups of 2)
Mixed broth culture
Agar plate with E. coli
2 TSA plates
Method (individually)
1. As previously, with a sterile inoculation loop grab some bacteria from the E.coli plate culture
supplied and streak the TSA plate as shown in the first panel of the figure on the previous
page. STERILIZE YOUR LOOP after this initial streaking.
2. Make a second set of streaks with the sterile loop as shown.
3. Continue as many times as possible, sterilizing the loop each time between streaks to isolate
single colonies.
4. On a new plate, repeat the procedure for single colony isolation from the mixed broth
culture.
5. Place both single colony isolations in the designated area so that they can be incubated at
37oC until the next lab period.
PLATING BACTERIA: SPREADING
In contrast to streaking, spreading is only used with liquids. It allows spreading the bacteria
evenly over the surface of the plate. However, in contrast to streaking it does not allow any
dilutions to be performed on the plate itself. Consequently, if dilutions are required, these must
be prepared independently before. The instrument used is a glass spreader commonly referred to
as a “hockey stick”. As with the loop, the spreader must be sterilized before each use.
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Microbiology Lab-2014
o STERILIZING THE SPREADER
In order to sterilize the spreader, it is dipped into ethanol, ignited, and the ethanol is
allowed to burn off. Do not hold the spreader in the flame as it will get to hot! The
spreader is then allowed to cool and used to spread the sample of bacteria onto the surface
of the plate.
Ethanol
Dip spreader in
ethanol
Allow ethanol to
burn off
Ignite ethanol
23
Spread bacteria
Microbiology Lab-2014
VIABLE COUNTS
There are several ways one may obtain an estimate of the number of microbes in a given
environment. Amongst the most common ways, are the viable counts. Viable counts determine
the number of live microbes within a given sample. This method requires that the microbes be
grown on suitable media until single colonies are observed. Since a single colony is assumed to
have arisen from a single cell, it is concluded that the number of single colonies obtained
represents the number of viable microbes present within the sample tested. In addition, following
their growth, a researcher can estimate the number of different species present according to the
different morphologies observed. There exist several variations of the viable count. One of the
criterions which dictate the choice of the method used is the sampling source. For example, do
you wish to sample a surface, a solid sample or a liquid sample?
Exercise 2.2
Materials (Per groups of 2)
E. coli broth of known concentration
3 TSA plates
100 mL of TSB medium
8 sterile test tubes
Method (individually)
1. Prepare in a final volume of 10 mL of TSB broth 10-5, 10-6 and 10-7 dilutions of the E. coli
broth.
2. Appropriately label 3 TSA plates.
3. Transfer and spread 0.1 mL of each of your dilutions on the corresponding plates.
4. Incubate your plates in the inverted position at 37oC until the next lab period.
Exercise 2.3: ENVIRONMENTAL (SOIL) SAMPLING
There are a number of different methods for determining the number of microbial colonyforming units (CFUs) in soil. The results depend on diluting the CFUs to a concentration where
the growth of one colony does not inhibit neighboring colonies. Bacteria are the most numerous
culturable organisms in soils (viruses are more numerous, but difficult to culture—you need a
suitable host). The predominant genera are Arthrobacter, Bacillus, Pseudomonas, and
Streptomyces. Arthrobacter, and Streptomyces are actinomycetes which produce cells that
resemble fungi. The results you will get depend on the media used. Media for isolating bacteria
is usually not ideal for the growth of fungi, while that used for fungi often has antibiotics to
inhibit bacterial growth.
Materials (Per groups of 2)
10g of soil
Flask with 90 mL of sterile water
100 mL sterile water
3 sterile test tubes
3 TSA plates
3 plates of Sabouraud Dextrose Agar containing 100µg/mL chloramphenicol
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Microbiology Lab-2014
Method (Per groups of 2)
1. Transfer 10g of soil to the flask containing 90 mL of sterile water. Shake on shaking
platform for 10 minutes.
2. Prepare 10-1, 10-2, and 10-3 dilutions of the soil suspension.
3. Plate 0.1 mL from each dilution on 3 appropriately labelled TSA plates and 3 appropriately
labelled Sabouraud Dextrose Agar plates using the spread plate technique.
4. Incubate the inverted plates at 28oC. The bacterial plates will be incubated for 48 hours
whereas the fungal plates will be incubated until next week.
AN EPIDEMIC OF POTATO CROPS
Plants, just as with animal species, are susceptible to several microbial pathogens. These
invaders can have an important impact on the crops, the preservation of these, human health and
obviously the economy. One of the most prominent examples was the great famine in Ireland
(1845-1848). We attribute to it between 500 000 and 1 million deaths, following the devastation
of Irish crops. In 1845 a microbial pathogen propagates itself on the Irish potato crops. The
tuber of the potato thus becomes inedible: it rapidly rots, preventing any form of partial recovery
of this vegetable. As a consequence of this natural disaster, Ireland is plunged in a large scale
food shortage.
Imagine that such a scenario occurred today. You are recruited as a microbiologist to identify the
pathogen and then elaborate prevention and control methods. Today you will begin a long-term
project to accomplish these goals. You should keep a journal of your observations and your
results which you will use for a final report.
Exercise 2.4
Materials (Per groups of 2)
1 TSA plate
One infected slice of potato
One uninfected slice of potato
Method (Per groups of 2)
1. Examine the two potato slices which were supplied by the farmer. Record precise
observations on the appearance and any other characteristics that you may notice.
2. Take pictures of the potato slices.
3. The first step in order to identify the microbe responsible is to isolate it and to generate pure
cultures. To this end, use the technique of streaking for single colonies which you saw
earlier to isolate the potential pathogen.
4. Incubate the inverted plates at 28oC until the next lab period.
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Microbiology Lab-2014
Day 2
Exercise 2.1
STREAKING FOR SINGLE COLONIES
1. Obtain and examine the plates on which you performed your streaking for single colonies.
Did you obtain single isolated colonies?
2. Ask one of your teaching assistants to evaluate your streaking technique for single colonies.
3. Compare your single colony streaks to your regular streaks and slants.
Exercise 2.2
VIABLE COUNTS
1. Obtain the plates on which you spread your dilutions of the E. coli culture.
2. Count the number of colonies on plates which have between 30-300 colonies.
3. Determine the viable count (CFU/mL) in the original broth.
Exercise 2.3 ENVIRONMENTAL (SOIL) SAMPLING: MACROSCOPIC
VISUALIZATION – COLONY MORPHOLOGIES
Preliminary identification of microbes can be based on colony morphology. Each colony
represents a population of cells originating from a single cell which after multiple cycles of
cellular division has generated a colony of cells that are stacked one on top of the other and give
rise to characteristic colonies. For example, a stack of bananas would look differently from a
stack of potatoes. The morphology of a colony is a function of many factors including cell shape,
cell size, and physiology. Colonies also often have distinctive colors, textures and smells.
BACTERIAL COUNTS
Following an appropriate incubation period, you should now see colonies on the TSA plates
typical of bacteria isolated from your soil sample. You may notice the large variety of colors,
shapes, and textures of colonies from different species. Use the following schematic to determine
the colony morphologies observed for the bacteria from your soil. This will allow you to obtain a
preliminary estimate of the number of different species found within your samples.
BACTERIAL COLONY MORPHOLOGIES
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Microbiology Lab-2014
Once you’ve obtained an estimate of the number of different bacterial species, your next step
will be to determine the number of bacterial CFUs/g of soil. To accomplish this, choose a
dilution which has between 30-300 colonies. Determine the number of CFUs for this dilution.
Record this number and the corresponding dilution in your note book.
Exercise 2.4: AN EPIDEMIC OF POTATO CROPS
Materials (Per groups of 2)
1 TSA plate
Method (Per groups of 2)
1. Examine your streaking for single colonies. Did you obtain pure cultures? Are these all the
same? Did different people obtain similar colonies?
2. Take a picture of single colonies of the isolate.
3. Record the colony morphology in your journal.
4. Perform a streaking for single colonies from the predominant colony type. Incubate the
inverted plates at 28oC until the next lab period.
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Microbiology Lab-2014
MICROSCOPIC EXAMINATION – SIMPLE STAINS
Microbial cells are colorless and thus very difficult to visualize even with a microscope.
Consequently, various staining procedures have been developed to both visualize and classify
microbes. Staining procedures are generally classified into two categories; either simple staining
or differential staining. Simple staining involves the use of a single stain such as methylene blue
which stains all cells the same color. This type of stain is useful to look at the different shapes
and aggregations of bacterial cells. (See figure on page 30)
Exercise 2.5
Materials (Per groups of 2)
Microscope slides
Bacterial plates from environmental samples
Stains
Method (Bacterial staining) (Per groups of 2)
1. Prepare smears from three different bacterial colonies from your environmental sampling.
Use a sterile loop to transfer a drop of water onto a microscope slide. Then use the sterile
loop once again to transfer some bacteria from a chosen colony (VERY LITTLE) to the drop
of water on the microscope slide.
2. Suspend the bacteria in the drop of water and spread over a small area of the slide.
3. Allow your smears to completely air dry on your work bench.
Too little
OK
28
Too thick
Microbiology Lab-2014
4. Heat fix your bacterial smears by rapidly passing the slides through the flame of the Bunsen
burner. Do not over heat!! Burnt bacteria do not stain well.
5. Setup your staining station as follows:
Absorbent paper mat (Plastic coating under)
Staining Container (Gladware)
6. Deposit the slides over the slits of the Gladware. Stain each of the slides with one of the
stains indicated below as follows: Add one drop of one of the following stains to each of
your smears.



Methylene blue
Crystal violet
Safranin
7. Leave the stain on the smears for approximately 30 seconds.
8. Wash off the excess dye with distilled water.
9. Blot the slides dry by pressing them between the layers of absorbent paper.
10. Examine under the microscope and obtain digital pictures. Save these.
Dispose of the staining solutions in the drums provided to that effect.
DO NOT THROW STAINS DOWN THE DRAINS
PowerPoint Presentation
Each group must prepare a PowerPoint presentation of their images. The first slide of the
presentation must include a title, the names of all group members, the group number and the
date. The presentation must include the following images (One Per slide):
Pictures of simple stains of bacteria with each of the different stains including the following
information (3 images).
 Type of staining and stain used
 Cellular morphology
 Cellular aggregation
 Magnification
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Microbiology Lab-2014
BACTERIAL CELL MORPHOLOGIES
Neisseria
Micrococci
Micrococci
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Microbiology Lab-2014
LAB#3
COUNTING MICROORGANISMS
Day 1
Exercise 2.3 ENVIRONMENTAL (SOIL) SAMPLING: MACROSCOPIC
VISUALIZATION– COLONY MORPHOLOGIES
FUNGAL COUNTS
Fungi are eukaryotic organism and they are classified into two main groups; yeasts and molds.
These groups can easily be discriminated based on the macroscopic appearance of the colonies
formed. The yeasts produce moist, creamy, opaque or pasty colonies, while molds produce
fluffy, cottony, woolly or powdery colonies. These microorganisms are useful as well as harmful
to human beings. Useful because they produce many antibiotics, natural products and are used in
industrial fermentation processes. They may be harmful since they may cause human diseases,
produce toxic substances as well as harm important crops. It is therefore very important to study
fungal species. The branch of science that deals with study of fungal species is called Mycology.
The colonial texture describes the height of the aerial hyphae above the agar surface. Colonial
topography describes the various designs of hills and valleys seen on fungal cultures. The
topography is often masked by the aerial hyphae; therefore, this characteristic is better observed
on the reverse side of the colony. A colony may possess no topography, that is, it is flat.
The last characteristic of macroscopic morphology is the color of the colony. Be as specific
about the colony colors as possible. For example, instead of describing a culture as brown, use
words such as beige, tan, khaki, or mahogany. If there are concentric rings of different colors
describe each one. Be sure to characterize both the front and reverse sides of the culture.
Once you’ve obtained an estimate of the number of different fungal species, your next step will
be to determine the number of fungal CFUs/g of soil. Choose a dilution which has between 1050 colonies. Determine the number of CFUs for this dilution. Record this number and the
corresponding dilution in your note book.
MICROSCOPIC EXAMINATION – SIMPLE STAINS
As with bacteria, staining of fungal cells is a very important step towards their identification.
Here we are going to stain fungal cells using Lactophenol cotton blue. This staining technique
will allow you to identify the most common fungal genera found in soil; namely Rhizopus,
Mucor, Aspergillus and Penicillium.
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Microbiology Lab-2014
Materials (Per groups of 2)
Microscope slides
Fungi plates from environmental samples
Lactophenol blue
Method (Fungal staining) (Per groups of 2)
1. Prepare wet mounts of three of your fungal colonies as follows: Using a sterile loop, transfer
a small amount of one of your fungal colonies to a drop of lactophenol blue deposited on a
microscope slide. It is often useful to take a little of the agar medium together with the
fungus.
2. Very briefly pass through the flame to melt any agar. Then overlay with a coverslip.
3. Examine under the microscope at 40X and obtain digital pictures. Save these.
For your assignment you will need to submit the following:
Pictures of simple fungal stains (3 images).
 Type of staining and stain used
 Fungal genera
 Magnification
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Microbiology Lab-2014
FUNGAL COLONY AND MICROSCOPIC MORPHOLOGIES
Colonial morphology
Microscopic morphology
White colored fungus. Cottony
Colorless or brown spores.
and fuzzy
Nonseptae hyphae with root like
hyphae (rhizoids)
Colonies similar to Rhizopus
Colorless or brown spores.
Nonseptae hyphae with no rhizoids
White colonies become greenish
blue, black, or brown as culture
matures
Spores developing in chains
(Conidia) arising from terminal
bulb of conidiophore which arise
from septae hyphae
Mature cultures usually greenish
or blue green
Spores developing in chains
(Conidia) arising from branching
conidiophore which arise from
septae hyphae
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Microbiology Lab-2014
Microscopic Fungal Structures
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Microbiology Lab-2014
MOST PROBABLE COUNTS
A variation of viable counts is based on probabilities to determine the number of bacteria in a
sample. As with viable counts, this method requires the growth in an appropriate medium.
However, in contrast to viable counts, detection is based on the presence or absence of growth or
on the production of a by-product.
In the following exercise we will use the method of the most probable number to estimate and
compare the number of bacteria of the genus Lactobacillus in different brands of yogurt. To this
end, the lactobacilli growth medium MRS will be used. This medium contains peptone as a
source of nitrogen and glucose as a carbon source. Polysorbate 80, magnesium acetate and
manganese provide growth factors and inhibit the growth of microorganisms other than
lactobacilli.
Exercise 3.0
Materials (Per groups of 2)
Test tube with 1g of yogurt (Brand indicated)
Physiological saline
18 X 5 mL MRS broth
3 sterile test tubes
Method (Per groups of 2)
1. Add 10 mL of saline to the yogurt sample. Mix well.
2. Prepare the following dilutions in saline of the yogurt suspension in a final volume of 10
mL: 10-4, 10-6 and 10-8.
3. Label 6 sets of 3 tubes each containing 5 mL of MRS broth 10-4, 10-5, 10-6, 10-7, 10-8, 10-9.
4. Inoculate 3 MRS broths labelled 10-4 with 1.0 mL of the (10-4) dilution of yogurt.
5. Inoculate 3 MRS broths labelled 10-5 with 0.1 mL of the (10-4) dilution of yogurt.
6. Inoculate 3 MRS broths labelled 10-6 with 1.0 mL of the (10-6) dilution of yogurt.
7. Inoculate 3 MRS broths labelled 10-7 with 0.1 mL of the (10-6) dilution of yogurt.
8. Inoculate 3 MRS broths labelled 10-8 with 1.0 mL of the (10-8) dilution of yogurt.
9. Inoculate 3 MRS broths labelled 10-9 with 0.1 mL of the (10-8) dilution of yogurt.
10. Incubate your tubes at 37oC until the next la period.
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Microbiology Lab-2014
VIABLE COUNTS OF LACTOBACILLI
Exercise 3.1
Materials (Per groups of 2)
Yogurt dilutions prepared in the previous exercise
3 Petri dishes
3 test tubes with 15 mL molten MRS agar
1L beaker
Method (Per groups of 2)
1. Allow the hot tap water to run until it reaches its maximum temperature.
2. Fill about half way a beaker.
3. Obtain 3 test tubes of molten MRS media and place them in the hot water beaker.
4. Label each of the three Petri dishes 10-4, 10-6 and 10-8 respectively.
5. Inoculate one of the test tubes of molten MRS agar with 1.0 mL of the 10-4 dilution.
6. Mix well for a few seconds and then pour the contents into the Petri dish labelled 10-4.
7. Repeat steps 5 and 6 for the 10-6 and 10-8 dilutions of yogurt.
8. Allow the poured Petri plates to cool down until the agar solidifies. Approximately 10
minutes.
9. Incubate the plates at 37oC until the next lab period.
DIRECT COUNTS (hemocytometer slide)
As the name implies, direct counts involves taking a direct measurement of the actual number of
microorganisms present within a sample without a priori growing them. This can be achieved by
different visualization techniques two of which you will examine in the following exercises.
Note that a direct count does not distinguish between whether a microorganism is alive or dead.
One method involves using a special slide, a haemocytometer slide, which possesses a counting
chamber of a fixed and known volume. A standard haemocytometer slide has two identical
counting areas consisting of nine 1mm2 etched squares (1mm X 1mm; see figure). When the
counting chamber is overlaid with a coverslip, the free space available is 0.1mm deep. The
volume of each square is thus 0.1mm3 or 10-4 cm3. After applying an aliquot of the sample to be
counted, the cells are visualized and enumerated. The cells within each of three squares are
counted. The average is then calculated and the total number of cells within the original sample
is interpolated.
Example of calculation:
If 10, 14, and 6 bacterial cells were counted in each of three independent squares; the average
number of bacteria Per square is 10. This number is equivalent to having 10 bacteria/ 0.1mm3, or
10 bacteria/ 0.0001cm3 or 10 bacteria/0.0001mL.
Therefore, the concentration of bacteria in the sample being examined is 1 X 105/mL.
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Microbiology Lab-2014
Hemocytometer
Y
Y
Exercise 3.2
Materials (Per groups of 2)
Yeast suspension
Hemocytometer slide
Sterile water
Tubes
Pasteur pipettes/micropipettor
Method (Per groups of 2)
1. Prepare the following dilutions of the yeast suspension: 10-1, 10-2, and 10-3. Make sure to
mix the yeast suspension well before sampling.
2. Fill the hemocytometer counting chamber with a sample from the highest dilution (see image
below or ask a teaching assistant to show you). Make sure to mix the yeast suspension well
before sampling.
3. Count the number of cells observed in each of three squares of the size indicated by a “Y” in
the above image.
4. If the number of cells is too low, start over with the previous dilution. If the number of cells
is too high, prepare a 10 fold higher dilution and start over.
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Microbiology Lab-2014
DIRECT COUNTS (Calibration beads)
Another method commonly used to perform a direct count involves the use of microscopic beads
at a known concentration. A given volume of these beads are added to the sample to be examined
and then observed under the microscope. The number of beads observed in a field of view is then
counted. Given that the concentration of beads is known, one may then derive the volume of the
field under observation.
Ex.
Beads added to a
final concentration
of 1 X 103 beads/mL
Field of view under microscope
Number of beads observed in full field of view: 8 beads/field
Bead concentration (given) 1 X 103beads/mL
Therefore:
1 X 103 beads → 1mL
8 beads
→ χ mL
Volume of field of view = 8/1 X 103 = 0.008 mL or 8µL
Exercise 3.3
Materials (Per groups of 2)
0.5mL Calibration beads (1 X 104/mL)
Slides
Coverslip
Method (Per groups of 2)
1. Mix the suspension of beads provided on the vortex.
2. Using a micropipettor, apply 20µL of the suspension to a slide and overlay with a coverslip.
3. Observe under the microscope at 40X and 100X magnifications.
4. Count the total number of beads in the field of view under the 40X and 100X magnifications.
5. For your assignments, determine the volume observed under the 40X and 100X
magnifications.
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Microbiology Lab-2014
AN EPIDEMIC OF POTATO CROPS (Cont'd)
Exercise 3.3
Materials (Per groups of 2)
1 TSA plate
Method (Per groups of 2)
1. Obtain your streaking for single colonies which you performed last week.
2. From a single colony, do another streaking for single colonies.
3. Incubate the inverted plates at 28oC until the next lab period.
MICROSCOPIC VISUALISATION - GRAM STAINING
Previously, you performed a simple staining procedure, which allowed you to compare microbial
cell sizes and shape. Several other staining procedures have been developed which are said to be
differential. These procedures stain microbes differently as a function of different cellular
properties.
Dr. Christian Gram developed a differential staining technique, the Gram stain. Gram staining
allows not only to observe cell shape and size, but also to classify bacteria in one of two groups:
Gram positive or Gram negative.
The Gram stain technique makes use of four chemical components: Crystal violet, a primary
dye that stains all cells blue indiscriminately, Gram’s iodine, which acts as a mordant that
interacts with the primary stain, Ethanol, whose which dehydrates the cell wall and Safranin, a
counter stain that stains all cells indiscriminately red. The sequential addition of these 4
components will cause some bacterial species to be stained red (Gram negative), whereas others
will be stained blue (Gram positive), according to their species.
The differential property of Gram staining is a function of their different cell wall composition.
Typically, Gram-positive bacteria possess a very thick cell wall composed of 10-20
peptidoglycan layers. In Contrast, Gram-negative organisms have relatively thin cell walls
consisting of one-three peptidoglycan layers covered by a lipid layer. The critical step in the
Gram staining technique, which confers its discriminatory properties, is the ethanol wash. In the
case of Gram-positive organisms, the cell walls are very rapidly dehydrated due to the lack of
lipids. Consequently, these cells tend to retain the crystal violet-Gram’s iodine complex. In
contrast, the cell wall of Gram-negative organisms is not easily dehydrated by the ethanol wash
due to its high lipid content and thus retains its high permeability. Consequently, the ethanol
wash effectively leaches any of the crystal violet-Gram’s iodine complexes, and the cells remain
susceptible to safranin staining. These cells will thus appear red.
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Microbiology Lab-2014
Exercise 3.4
Materials (Per groups of 2)
Microscope slides
Broth cultures of S. aureus and E. coli
Stains
Method (Per groups of 2)
1. Prepare heat fixed bacterial smears of the broth cultures supplied.
2. Stain with crystal violet for one minute.
3. Carefully wash of the stain with distilled water.
4. Apply Gram’s iodine for one minute
5. Carefully wash of the Gram’s iodine with distilled water.
6. Carefully wash with 95% ethanol by adding it one drop at a time until the alcohol runs clear.
7. Wash the excess ethanol with distilled water
8. Counterstain with safranin for 45 seconds.
9. Wash off with distilled water
10. Blot dry with bilbous paper.
11. Examine under the microscope and take digital pictures. Save these.
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Microbiology Lab-2014
DAY 2
MOST PROBABLE COUNTS
To understand the theory behind the most probable counts (MPN), think about 10 fold serial
dilutions with 1mL samples from each dilution inoculated in different tubes containing a given
growth medium.
Following the incubation, the broths are examined for the presence or the absence of growth. In
theory, if a least one organism was present in any of the inoculums visible growth should be
observed for that tube. If the broth inoculated from the 10-3 dilution shows growth, but the broth
inoculated from the 10-4 dilution does not, it is thus possible to affirm that there were more than
1X103 organisms per mL of sample, but less than 1 X 104 per mL.
Bacteria are only rarely, if ever, evenly distributed within a sample. For example, if a 10 mL
sample contains a total of 300 organisms, not all 1 mL aliquots will contain 30 organisms; some
will have more or less than 30 organisms, but on average all ten aliquots in the whole 10 mL
sample will be 30. This holds true for any of the dilutions from which an inoculum is taken.
To increase the statistical accuracy of this type of test, more than one broth is inoculated for each
dilution. The standard MPN makes use of a minimum of three dilutions and 3, 5 or 10 tubes per
dilution. Following the incubation, the pattern of positive and negative tubes is recorded after
which a table of standard MPN is consulted in order to determine the most probable number of
organisms (which cause the positive results) Per unit volume of the original sample.
Suspension of
1g/100 mL
In the following example, sets of three tubes of broth are inoculated with 1 mL from each of the
10 fold dilutions of a soil suspension at 1g/100 mL.
Inoculum:
Results:
1mL of 100 0.1mL of 100 1mL of 10-2
+++
+++
+-+
0.1mL of 10-2
+--
1mL of 10-4
---
Following the incubation, the number of tubes which show growth is recorded and expressed as
the number of positive tubes over the total number of tubes for that dilution. For example, for the
10-3 dilution the result would be expressed as 2/3. At a certain point the dilution will be so high
that no organism is found within the inoculums in any of the tubes for that dilution. In this case
the result would be expressed as 0/3.
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Microbiology Lab-2014
Exercise 3.0
MPN of lactic bacteria
Obtain your assays for the most probable number and record the positive and negative results. A
positive test is represented by an abundant growth. Determine the MPN of lactic bacteria as
follows:
MPN determination
When more than three dilutions are used in a decimal series of dilutions, use the results from
only three of these to determine the MPN. To select the three dilutions to be used in determining
the MPN index, determine the highest dilution (most dilute sample) that gives positive results in
all three samples tested (so 3/3) and for which there are no lower dilution (less dilute sample)
giving any negative results. Use the results for this dilution set and the two next succeeding
higher dilutions to determine the MPN index from the MPN table. (See examples “a” and “b”
below) If none of the dilutions yield all positive tubes, then select the three lowest dilutions for
which the middle dilution contains the positive result, as shown in example “c”.
Example 100 10-1 10-2 10-3 Combination of positives MPN index/ mL
a
3/3 3/3 2/3 0/3
3-2-0
9.3
b
3/3 2/3 1/3 0/3
3-2-1
15
c
0/3 1/3 0/3 0/3
0-1-0
0.3
Having chosen the appropriate combination of positives, consult the table on the next page to
determine the MPN index. Once you’ve obtained the MPN index, multiply it by the dilution
factor of the middle set of dilutions. For instance in example “a” you would get 9.3 X 101.
C. Gram Staining
1. Perform a Gram stain on one of your positive broths.
2. Visualize with the microscope and take digital pictures. Save.
42
Microbiology Lab-2014
Pos. tubes
0.10 0.01 0.001
Pos. tubes
MPN/g
(mL)
0.10 0.01 0.001
MPN/g (mL)
0
0
0
<3.0
2
2
0
21
0
0
1
3.0
2
2
1
28
0
1
0
3.0
2
2
2
35
0
1
1
6.1
2
3
0
29
0
2
0
6.2
2
3
1
36
0
3
0
9.4
3
0
0
23
1
0
0
3.6
3
0
1
38
1
0
1
7.2
3
0
2
64
1
0
2
11
3
1
0
43
1
1
0
7.4
3
1
1
75
1
1
1
11
3
1
2
120
1
2
0
11
3
1
3
160
1
2
1
15
3
2
0
93
1
3
0
16
3
2
1
150
2
0
0
9.2
3
2
2
210
2
0
1
14
3
2
3
290
2
0
2
20
3
3
0
240
2
1
0
15
3
3
1
460
2
1
1
20
3
3
2
1100
2
1
2
27
3
3
3
>1100
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Microbiology Lab-2014
AN EPIDEMIC OF POTATO CROPS (Cont'd)
Exercise 3.3
Materials (Per groups of 2)
1 TSB broth
Method (Per groups of 2)
1. Obtain your streaking for single colonies that you did in the previous lab period.
2. Perform a Gram stain on the predominant colony type.
3. Observe under the microscope and take a digital picture. Save.
4. Inoculate the TSB broth with an isolated colony and place in the rack provided to that effect.
MICROSCOPIC VISUALISATION - ACID-FAST STAINING
Another specialized staining procedure is the Acid-Fast stain. This staining technique is
particularly useful to colorize bacteria, which possess wax-like constituents in their cell wall.
These include many human pathogens such as the Mycobacteriacae, some members of which
cause tuberculosis and leprosy. The high content of mycoic acid, a wax like compound, of the
cell wall of bacteria within this family makes them literally impermeable to stains. However,
once a stain has penetrated, it cannot be easily removed by decolorizing agents such as ethanol.
The principal of the acid-fast technique is thus to render the bacteria permeable to the stain and
to then verify resistance to harsh decolorizing conditions. These objectives are reached by using
carbol fuchsin as the primary stain. Carbol fuchsin is a highly lipid soluble compound and thus
easily permeates the waxy layer of Mycobacteriacae. These bacteria then resist decolorization by
the harsh acid wash treatment done with acid alcohol.
Exercise 3.5
Materials (Per groups of 2)
Microscope slides
Slant cultures of B. subtilis and M. smegmantis
Carbol Fuschine of Kinyoun
Acid alcohol
Method (Per groups of 2)
1. Prepare heat fixed bacterial smears of each of the cultures.
2. Flood each of the smears with carbol fuchsine for 5 minutes.
3. Rinse with distilled water.
4. Destain with acid alcohol until no more stain runs off.
5. Rinse with distilled water.
6. Counter stain with methylene blue for 30 seconds.
7. Destain with distilled water.
8. Blot dry the slide with blotting paper.
9. Examine under the microscope and take digital pictures. Save these.
44
Microbiology Lab-2014
MICROSCOPIC VISUALISATION – SPORE STAINING
Bacterial cells belonging to both the Bacillus and the Clostridium genera can assume one of two
states: A vegetative cell, which is metabolically active, or that of a spore, which is metabolically
inactive. The spore represents a dormant state of the cell that is highly resistant to several
different harsh conditions such as heat and dehydration. When the environmental conditions
become unfavourable for continued growth, vegetative cells initiate sporogenesis, which gives
rise to a novel intracellular structure, the endospore. Just like the seeds of plants, the endospore is
composed of several layers, which confer to it a high level of resistance. Eventually, the
endospore is released as a spore that is independent of the vegetative cell. When the
environmental conditions become favourable for growth, the spores germinate and return to their
vegetative state.
The resilient nature of both the spore and the endospore make them resistant to standard staining
techniques and thus require specialized staining procedures to stain them. Briefly, spore staining
is accomplished as follows. A bacterial smear is initially exposed to a primary stain, malachite
green. Due the high level of impermeability of the spores, the stain is simultaneously exposed to
heat, to increase the permeability to the stain. At this stage, both the spores and the vegetative
cells are stained green. The smear is subsequently thoroughly washed with water to remove the
excess of the stain. Since malachite green has relatively little affinity for structures of the cell,
the vegetative cell is decolourized by this treatment whereas the spores retain the stain. Finally, a
counterstain is applied, safranin, which colors the cells red whereas it does not stain the spore.
See the demonstration slide
1. Prepare heat fixed smears.
2. Flood each smear with malachite green stain.
3. Boil over the flame of the Bunsen burner for five minutes. Do not let the stain dry out. Add
more stain as needed.
4. Rinse the smear with distilled water.
5. Counter stain with safranin
6. Rinse with distilled water and blot dry.
7. Examine under the microscope.
45
Microbiology Lab-2014
PowerPoint Presentation
 Each group must prepare a PowerPoint presentation of their images. The first slide must
include a title, the names of the group members, the group number and the date. The
presentation must include the following images (one per slide) :
 A Gram stain of each broth culture supplied. (2 images)
 A Gram stain obtained from the potatoes (1 image)
 A Gram stain obtained from the MPN (1 image)
 An acid fast stain of Mycobacterium smegmantis and B. subtilis (2 images)
 Lactophenol blue stains of Fungi (3 images)
 Each image must include an appropriate legend which includes the following information :
o Bacterial (Fungal) genus and species (if known) or source
o Type of staining used
o Cell morphology
o Cell aggregation
o Magnification
46
Microbiology Lab-2014
LAB # 4 `
GROWTH MEDIA AND SOIL BACTERIA
Day 1
There are several types of microbiological media which are classified as either defined,
differential or selective. Defined media are quite varied and have very different compositions.
They may contain very few or a great number of ingredients. There particularity is that the exact
composition and thus all the ingredients that compose it are known. In contrast, complex media
are made from ingredients of which the exact composition is unknown; for example a medium
made from beef extract. In both cases these media can be made differential or selective.
Differential media contain compounds which allow visual discrimination, usually by color
changes, different bacterial metabolisms. However, media which are said to be selective contain
compounds that prevent the growth of certain microorganisms.
Different differential growth media have been elaborated in order to assess the type of
metabolism used. These usually include a pH indicator that changes color as a result of the byproducts generated from the carbon source and the metabolism used. In general, the presence of
acid is indicative of the metabolism of a sugar by either an oxidative or fermentative metabolism.
In contrast, the accumulation of alkaline by-products is indicative of protein catabolism by an
oxidative mode.
SOIL BACTERIA
Bacteria represent the most abundant microorganisms in soils. Their numbers can be as high as 1
X 108 per gram of soil. They can be generally classified in the following categories:
Decomposers: These bacteria have an important role in the decomposition of organic matter.
Bacteria of the Bacillus and Pseudomonas genera are prime examples of decomposers. Since the
bulk of the organic matter present in soils is initially in the form of complex polymers such as
cellulose or starches, several of these bacteria possess the ability to secrete exocellular enzymes
which can degrade these polymers into units which are more easily digested.
Bacteria which fix nitrogen: Amongst this type of bacteria are the Rhizobiums which can
establish symbiotic relationships with the roots of plants. These bacteria extract atmospheric
nitrogen gas which they convert in a usable form for plants. Other bacterial genera, such as
Azotobacter, Azospirillum, Agrobacterium, Gluconobacter, Flavobacterium and Herbaspirillum
can also fix nitrogen, but they are not symbiotic.
Nitrifying bacteria: These bacteria convert ammonium (NH4+) to nitrite (NO2-) and to nitrate
(NO3-) – a nitrogen source which is preferred by most plants.
Denitrifying bacteria: These bacteria convert nitrate to nitrogen gas. These bacteria are
typically anaerobic and live in the absence of oxygen.
47
Microbiology Lab-2014
Aerobic and anaerobic: The availability of oxygen being highly variable in soils, the oxygen
requirements amongst bacteria are very diverse. Oxygen may be used by some organisms,
including humans, and some bacteria, as a final electron acceptor in aerobic respiration. With
respect to their needs, bacteria can be placed into different classes based on their ability to use
oxygen as a final electron acceptor and their ability to grow in the presence of oxygen.
Strict aerobes:
Facultative anaerobes:
Anaerobes:
Obligate anaerobes:
Have an absolute oxygen requirement for survival.
Oxygen requirement is optional.
Do not make use of oxygen, but can grow in its presence.
Do not use oxygen and cannot grow in its presence.
Strict
Facultative
Facultative
Anaerobes
Obligate
Aerobes
Aerobes
Anaerobes
Anaerobes
Growth in O2
+
+
+
+
Growth w/o O2
+
+
+
+
Final e -acceptor O2
+
+
+
The presence or absence of molecular oxygen can be a critical factor in determining if a
particular species of bacteria will grow in a given environment.
48
Microbiology Lab-2014
Exercise 4.0
BACTERIA IN COMPOST
Sample preparation
Materials (Per groups of 2)
Compost (beside the balance)
250 mL bottle containing 90 mL sterile water
50 mL sterile Falcon tube
6 sterile tubes
Sterile water
Method (Per groups of 2)
1. Weigh one gram of compost.
2. Add the compost to the bottle of sterile water.
3. Mix vigorously for 1-2 minutes.
4. Allow to settle on your table for 5 minutes, and then repeat step 3 one more time.
5. Allow to settle on your table for 10 minutes.
6. Carefully pour approx. 20-30 mL of the supernatant into a sterile 50 mL Falcon tube. Avoid
as much as possible the transfer of any sediment.
7. Prepare a series dilutions in a final volume of 10 mL of sterile water representing the
following dilution factors: 102X, 103X, 104X, 105X and 106X.
Exercise 4.1
AEROBIC HETEROTROPHY COUNT (even numbered groups)
Materials (Per each even numbered group)
Dilutions of compost prepared in the previous exercise
5 TSA + 0.1% glycerol plates
Method (even numbered groups)
1. Spread 0.1 mL from each of the compost dilutions (10-2 to 10-6) on appropriately labelled
TSA + 0.1% glycerol plates.
2. Incubate at 28oC.
ANAEROBIC HETEROTROPHY COUNTS (odd numbered groups)
Materials (Per each odd numbered group)
Dilutions of compost prepared in the previous exercise
5 TSA + 0.1% glycerol plates
Method (odd numbered groups)
1. Spread 0.1 mL from each of the compost dilutions (10-2 to 10-6) on appropriately labelled
TSA + 0.1% glycerol plates.
2. Incubate in the anaerobic chamber at 28oC.
49
Microbiology Lab-2014
COUNTS OF GRAM NEGATIVE AND POSITIVE BACTERIA
To obtain an estimate of Gram negative and Gram positive bacteria in soil, we will make use of
selective and differential media; MacConkey agar and CNA of Columbia agar
MacConkey agar is a selective and differential medium which contains lactose and proteins as
potential carbon sources. The selectivity of this medium is due to the inclusion of crystal violet
which inhibits the growth of Gram positive organisms and yeasts. The differential property of
this medium allows one to discriminate lactose fermentation by the addition of a pH indicator.
Bacteria which ferment lactose are pink, whereas non-fermenters are colorless.
CNA of Columbia agar is a rich medium which contains a combination of peptones (protein
peptides) obtained from animal tissues as well as beef extract. Yeast extract is included as
sources of B vitamins. The addition of nalidixic acid and colistin make this medium selective for
Gram positive bacteria. Nalidixic acid blocks DNA replication whereas colistin perturbs the
plasma membrane of Gram negative organisms.
Exercise 4.2
Materials (Per groups of 4)
Dilutions of compost prepared in the previous exercise
5 MacConkey plates (Even numbered groups)
5 CNA of Columbia plates (Odd numbered groups)
Method (Even numbered groups)
1. Spread 0.1 mL from each of the compost dilutions (10-2 to 10-6) on appropriately labelled
MacConkey plates.
2. Incubate at 28oC.
Method (Odd numbered groups)
1. Spread 0.1 mL from each of the compost dilutions (10-2 to 10-6) on appropriately labelled
CNA of Columbia plates.
2. Incubate at 28oC.
50
Microbiology Lab-2014
NITROGEN CYCLE
The nitrogen cycle involves 5 processes -- fixation, absorption, mineralization, nitrification, and
denitrification – which are all carried out by microorganisms.
Fixation (N2 → NH4+) is a process by which N2 is converted to ammonium; an essential
compound since it is the only way that organisms can obtain nitrogen directly from the
atmosphere.
Nitrogen absorption (NH4+ → organic N) generated by bacteria is rapidly incorporated by
plants and other bacteria into proteins as well as other organic nitrogenous compounds.
Mineralization (Organic N → NH4+) arises as a result of decomposition. When organisms die,
decomposers such as bacteria consume the organic matter and convert significant amounts of
nitrogen in these dead organisms to ammonium. In the ammonium state, nitrogen is available for
use by plants or to be transformed into nitrate (NO3-) by a process called nitrification.
Nitrification (NH4+ → NO3-) several bacteria obtain energy by converting part of the
ammonium, which they use as an electron source, produced by the decomposition to nitrate.
Denitrification (NO3- → NO2-) is performed by denitrifying organisms which convert nitrate
(NO3-) to nitrite (NO2-) and in some instances to nitrogen gas:
NO3- → NO2- → NO → N2O → N2.
Exercise 4.3
MINERALISATION (Even numbered groups)
Materials (For each even numbered group)
Dilutions of compost prepared in the previous exercise
20 phenol red broths
Method (Even numbered groups)
1. Perform an MPN count as indicated on the next page in 4 sets of 5 tubes of phenol red broths
for the dilutions 100 to 10-3.
2. Incubate at 28oC.
DENITRIFYING BACTERIA (Odd numbered groups)
Materials (For each odd numbered group)
Dilutions of compost prepared in the previous exercise
20 nitrate broths
Method (Odd numbered groups)
1. Perform an MPN count as indicated below in 4 sets of 5 tubes of nitrate broths for the
dilutions 100 to 10-3.
2. Incubate at 28oC.
51
Microbiology Lab-2014
Compost sample
Inoculum volume
100
100
10-2
10-2
1.0 mL
0.1 mL
1.0 mL
0.1 mL
Vol. of medium
5 tubes
5mL
5mL
5mL
5mL
Final dilution
100
10-1
10-2
10-3
CARBON ASSIMILATION
The bulk of the available carbon is available in soils is from plants and represents mostly sugar
polymers, such as cellulose, an important component of plant cell walls, and starches, main sugar
storage in plants. Utilization of these compounds requires the synthesis and secretion of
exocellular enzymes whose goal are to degrade these polymers in simpler compounds which can
be transported inside the cell and metabolized. These enzymes are thus quite common in
decomposing microorganisms.
Amylase
or
Cellulase
Starch
or
Cellulose
Exercise 4.4
BACTERIA THAT DEGRADE STARCH (Even numbered groups)
Materials
Dilutions of compost prepared in the previous exercise
5 Starch agar plates
Method
1. Spread 0.1 mL from each of the compost dilutions (10-2 to 10-6) on appropriately labelled
Starch agar plates
2. Incubate at 28oC.
52
Microbiology Lab-2014
BACTERIA THAT DEGRADE GELATIN (Odd numbered groups)
Materials
Dilutions of compost prepared in the previous exercise
Molten NA + 0.8% gelatine
5 Petri dishes
1L beaker
Method
1. Run the hot water from the tap until it has reached the maximal temperature.
2. Fill the beaker provided about half way.
3. Obtain five tubes of molten NA + gelatine agar and put them in the beaker of hot water.
4. Transfer 0.1mL of your 10-2 dilution of compost to a first tube of molten agar.
5. Mix well for a few seconds, and then pour in a first Petri dish labelled appropriately.
6. Repeat step 4-5 for each of the following dilutions (10-3-10-6).
7. Allow the plates to cool until the agar has solidified.
8. Incubate your plates at 28oC.
AN EPIDEMIC OF POTATO CROPS (Cont'd)
HOST RANGE
A pathogen’s host represents an environment in which its growth is adapted and thus defines the
host range that it can invade. In the following exercise you will attempt to reproduce the
infection observed on potatoes in order to confirm that the isolated microbe is indeed responsible
for the disease. In addition, you will determine the range of hosts that the pathogen can infect.
Exercise 4.5
Materials (Per groups of 2)
Broth of the bacteria
2 potatoes slices (all groups)
2 slices each from two fruits or vegetables amongst the following (assigned groups)
 Radish
 Apple
 Carrot
 Onion
 Cucumber
 Turnip
6 Petri dishes
5mL TSB
53
Microbiology Lab-2014
Method (Per groups of 2)
1. Label each of the 6 Petri dishes with your names.
2. Label two Petri dishes control potato and infected potato respectively.
3. Label two Petri dishes with the name of the assigned fruit or vegetable control and infected
respectively.
4. Label two Petri dishes with the name of the assigned fruit or vegetable control and infected
respectively.
5. Immerse for 2-3 minutes the slices of potatoes and the two other fruits or vegetables in
approximately 100 mL of the hypochlorite solution.
6. Transfer and immerse for 2-3 minutes the slices of potatoes and the two other fruits or
vegetables in approximately 100 mL of sterile water.
7. Place each of the slices in the appropriate Petri dishes.
8. Inoculate the surface of each of the control slices with 0.1mL TSB.
9. Inoculate each of the slices to be infected with 0.1 mL of the bacterial culture.
10. Place all Petri dishes in the designated area so that they are incubated at 28oC until the next
lab period.
54
Microbiology Lab-2014
Day 2
SURVEY OF BACTERIA IN COMPOST
Each group must obtain the following counts and determine the bacterial count/g of original
compost:




Aerobic heterotrophic count
Anaerobic heterotrophic count
Count of Gram positive bacteria
Count of Gram negative bacteria

MPN of bacteria which perform mineralization
Phenol red broth contains proteins as the carbon source and organic nitrogen source. In
addition, a pH indicator, phenol red is included. The phenol red turns yellow in an acid
medium and becomes deep red in an alkaline medium. The degradation of organic
sources of nitrogen generates large amounts of ammonium ions which are released into
the medium. Its presence can be detected by a change to an alkaline pH.

MPN of denitrifying bacteria
To determine whether nitrate has been reduced, add 3 drops of sulfanilic acid and 3 drops
of alpha-naphtalamine to the tubes in which growth has been observed. These reagents
react with nitrite generating a red color. Observe whether there is a change of color
within one minute. If no change is observed, add a pinch of zinc powder and observe
once again whether there is a change in color. A color change to red after the addition of
the first reagents or no change in color after the addition of zinc indicates a positive
result.

Count of bacteria that degrade starch
To determine which bacteria degrade starch, flood the plates with Gram’s iodine. Gram’s
iodine reacts with starch generating a dark blue to black color. In the absence of starch,
the iodine remains brownish. Count the number of colonies which show a halo of iodine
that does not react with starch.

Count of bacteria that degrade gelatin
To determine which bacteria degrade gelatin, look for zones of turbidity around the
colonies. Count those colonies which show a cloudy halo around them.
55
Microbiology Lab-2014
Pos. Tubes
0.1
0.01
0.001
0
0
0
0
0
0
Pos. tubes
MPN/g
0.1
0.01
0.001
<1.8
3
2
2
1
1.8
3
3
1
0
1.8
3
0
1
1
3.6
0
2
0
0
2
0
MPN/g
Pos. tubes
MPN/g
0.1
0.01
0.001
20
5
4
1
400
0
17
5
4
2
440
3
1
21
5
4
3
710
3
3
2
24
5
4
4
710
3.7
3
4
0
21
5
4
5
1,100
1
5.5
3
4
1
24
5
5
0
710
3
0
5.6
3
5
0
25
5
5
1
1100
1
0
0
2
4
0
0
13
5
5
2
1700
1
0
1
4
4
0
1
17
5
5
3
2600
1
0
2
6
4
2
3
38
5
5
4
4600
1
1
0
4
4
3
0
27
5
5
5
>1600
1
1
1
6.1
4
3
1
33
1
1
2
8.1
4
3
2
39
1
2
0
6.1
4
4
0
34
1
2
1
8.2
4
4
1
40
1
3
0
8.3
4
4
2
47
1
3
1
10
4
5
0
41
1
4
0
11
4
5
1
48
2
0
0
4.5
5
0
0
23
2
0
1
6.8
5
0
1
31
2
0
2
9.1
5
0
2
43
2
1
0
6.8
5
0
3
58
2
1
1
9.2
5
1
0
33
2
1
2
12
5
1
1
46
2
2
0
9.3
5
1
2
63
2
2
1
12
5
1
3
84
2
2
2
14
5
2
0
49
2
3
0
12
5
2
1
70
2
3
1
14
5
2
2
94
2
4
0
15
5
2
3
120
3
0
0
7.8
5
2
4
400
3
0
1
11
5
3
0
220
3
0
2
13
5
3
1
250
3
1
0
11
5
3
2
400
3
1
1
14
5
3
3
400
3
1
2
17
5
3
4
400
3
2
1
17
5
4
0
400
56
Microbiology Lab-2014
Exercise 4.4 ISOLATION OF UNKNOWNS THAT ARE GRAM POSITIVE RODS
Materials (Per groups of 2)
2 TSA plate
Method (Per groups of 2)
1. From your CNA, gelatine or starch plates, find two different colonies that represent Gram
positive rods. To do so, perform Gram stains on a few colonies. Note: do not use the entire
colony since you will have to streak the chosen colonies for single colonies.
2. Take pictures of the Gram stains of the chosen colonies.
3. Streak each of the chosen colonies for single colonies on a TSA plate.
4. Incubate at 28oC.
AN EPIDEMIC OF POTATO CROPS (Cont'd)
HOST RANGE
Exercise 4.5
1. Examine each of your slices of fruits or vegetables. Determine whether there are any
evidences of infection and deterioration.
2. Take pictures.
3. Record your observations in your journal.
57
Microbiology Lab-2014
LAB #5
BACTERIAL DIAGNOSTICS – GRAM POSITIVE BACTERIA
Day 1
IDENTIFICATION OF GRAM POSITIVE RODS
The two main shapes of Gram positive bacteria include rods and cocci. Those belonging to the
cocci group will be discussed further on. The predominant families including Gram positive rods
include the Bacillaceae, Clostridiaceae and the Lactobacillaceae. These families are divided into
two general groups; sporulating bacteria which includes the Bacillaceae and the Clostridiaceae,
and the non-sporulating bacteria, the Lactobacillaceae.
The Bacillaceae encompass the genus Bacillus which includes aerobic and facultative anaerobic
bacteria. These bacteria possess a vast diversity of growth requirement. A great variety of
biochemical activities are noted in this family, including activities of fermentation, proteolysis as
well as the capacity to degrade complex carbon compounds.
The Clostridiaceae family, which includes the genus Clostridium, has characteristics which are
very similar to the Bacillaceae, but these distinguish themselves from the latter by the fact that
they are strict anaerobes.
Organisms within the Lactobacillaceae family can be either anaerobes or facultative anaerobes
with very complex nutritional requirements. Some of them produce lactic acid from the
fermentation of carbohydrates. Members of this family are rarely pathogenic, with the exception
of bacteria of the genus Listeria, an aerobe or microaerophile.
Exercise 5.0
Materials (Per groups of 2)
Plates on which you isolated and streaked unknown Gram positive rods
Methods (Per groups of 2)
COLONY MORPHOLOGY
1. Look at the plates on which you streaked the unknowns for single colonies.
2. Using the dissecting microscope, obtain pictures of typical colonies from each unknown.
3. Record the colony morphology as well as the colors of each unknown.
GRAM STAIN
1. Perform a Gram stain of each of your unknowns.
2. Take a digital picture for your report.
3. Record the Gram reaction, cell shape and aggregation.
58
Microbiology Lab-2014
SIMPLE CARBON METABOLISM
The carbon source and the metabolism used are quite varied amongst different bacteria. Growth
in phenol red broths is commonly used to assess the metabolism of simple carbon sources. These
broths contain two carbon sources; one being a sugar of your choice and the other protein. In
addition a pH indicator, phenol red is included. Phenol red turns yellow in an acid environment
and turns red in an alkaline environment. These broths also contain an inverted vial that permits
to detect the accumulation of gases. Generally, the presence of acid and gas indicates a
fermentative metabolism, whereas any growth in the absence of gas production indicates an
oxidative metabolism. Note that the production of neutral or acidic by-products indicates the
catabolism of a sugar. In contrast, generation of an alkaline reaction indicates the catabolism of
proteins.
Exercise 5.1
Materials (Per groups of 2)
Single colony streaking of unknown Gram positive rods isolated last week
2 pairs of phenol red broths with glucose, arabinose, xylose or mannitol
Physiological saline (0.9% m/v)
Sterile mineral oil
2 sterile tubes
Bact. 1
Bact. 1
PRG:
PRG:
Method (Per groups of 2)
1. Prepare 1 mL suspensions in physiological saline of each of your unknowns. You wish to
obtain a detectable turbidity.
2. Inoculate one pair of phenol red glucose broths with 0.1 mL of one of your unknown rods.
3. Overlay ONE tube of the inoculated pair of phenol red glucose broths with 1 mL of sterile
mineral oil.
4. Inoculate one pair of phenol red arabinose broths with 0.1 mL of the same unknown rods.
5. Overlay ONE tube of the inoculated pair of phenol red arabinose broths with 1 mL of sterile
mineral oil.
6. Inoculate one pair of phenol red xylose broths with 0.1 mL of the same unknown rods.
7. Overlay ONE tube of the inoculated pair of phenol red xylose broths with 1 mL of sterile
mineral oil.
8. Inoculate one pair of phenol red mannitol broths with 0.1 mL of the same unknown rods.
9. Overlay ONE tube of the inoculated pair of phenol red mannitol broths with 1 mL of sterile
mineral oil.
10. Repeat steps 2-9 with the second unknown. Thus 8 tubes/unknown for a total of 16 tubes.
11. Incubate at 28oC.
Mineral oil overlay
Pair of Phenol red glucose (PRG) broths inoculated with same
bacteria.
Tube 1 represents aerobic conditions
Tube 2 is overlaid with mineral oil to create anaerobic conditions
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Microbiology Lab-2014
GLUCOSE FERMENTATION; PRODUCTION OF MIXED ACIDS OR ACETOIN
Some bacteria that ferment glucose generate large quantities of acids that can be detected with
the reagent methyl red. In contrast, other bacteria generate only low levels of acids. However,
these bacteria generate large quantities of neutral by-products such as ethanol and butanediol.
One of the intermediates in the production of these by-products is acetoin whose presence can be
detected with a chemical reagent. The Methyl Red Vogues-Proskauer test is used to
differentiate between these two types of glucose fermentations.
MR/VP : Bact. 1
Materials (Per groups of 2)
Suspensions prepared in the previous exercise of unknown rods
2 MR-VP broths
MR/VP : Bact. 2
Exercise 5.2
Method (Per groups of 2)
1. Inoculate with 0.1 mL of each of your unknown rods two MR-VP broths.
2. Incubate at 28oC.
UREASE
Some bacteria have the ability to use urea, a by-product generated from the metabolism of
proteins by vertebrates, as a nitrogen source. The catabolism of urea requires the enzyme urease,
which hydrolyses urea and consequently generates ammonia, carbon dioxide, and water. The
ammonia that is released in the growth medium produces an alkaline reaction, which can be
detected by a pH indicator within the medium, phenol red, which turns pink in the presence of
alkali products.
Exercise 5.3
60
Bact. 1
Bact. 2
Urea:
Method (Per groups of 2)
1. Streak your unknown rods on urea agar slants.
2. Incubate at 28oC.
Urea:
Materials (Per groups of 2)
Single colony streaking of an unknown Gram positive rod isolated last week
2 urea agar slants
Microbiology Lab-2014
USE OF CITRATE AS A CARBON SOURCE
This test was conceived to verify whether a microorganism can use citrate as its sole carbon
source and inorganic ammonium salts as sole nitrogen source. The use of citrate generates
alkaline by-products which can be detected by the inclusion of a pH indicator, bromothymol
bleu, which is green at a pH of 6.8 and blue at a pH of 7.6 or above.
Bact. 2
Citrate
Method (Per groups of 2)
1. Streak each of your unknowns on a Simmons citrate agar slant
2. Incubate at 28oC.
Bact. 1
Materials (Per groups of 2)
Single colony streaking of unknown Gram positive rods isolated last week
2 Simmons citrate agar slants
Citrate
Exercise 5.4
UTILIZATION OF COMPLEX CARBON SOURCES: EXOCELLULAR ENZYMES
Simple carbon sources such as monosaccharides, disaccharides, and amino acids enter the cell
either by simple diffusion or by making use of specific transporter systems. In contrast, more
complex carbon sources such as polysaccharides and proteins are too large to use either of these
transport mechanisms. Consequently, they must first be broken down into smaller manageable
units before they can be utilized. This is achieved by the secretion of specialized exocellular
enzymes, which operate outside of the cell to break down polymers into smaller monomeric
compounds. Examples of complex polymeric carbon sources utilized by some bacteria are
starch, a polysaccharide, casein, a polypeptide or protein, tributyrin, a lipid or fatty acid polymer
and DNA, a nucleotide polymer. Each of these complex carbon sources require the secretion of a
specific enzyme, which degrades them into monomers, which can be easily transported into the
cell to be metabolized. -amylase, is an enzyme that can cleave the -1,4 linkage joining the
glucose monomers in starch. Caseinase is a protease, which can cleave the peptide linkages
joining the amino acid monomers in the protein casein. Lipase can degrade into individual fatty
acids. Finally, DNase cleaves the phosphodiester linkages between the nucleotides within a
polynucleotide chain.
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Microbiology Lab-2014
Exercise 5.5
Materials (Per groups of 2)
Single colony streaking of unknown Gram positive rods isolated last week
2 starch plates, 2 milk plates, 2 “Spirit Blue” plates and 2 DNA plates
Method (Per groups of 2)
1. Streak for single colonies one of your unknowns on each of the starch, milk, “Spirit Blue”
and DNA plates.
2. Repeat step 1 with your second unknown rod.
3. Incubate at 28oC.
NITRATE AND NITRITE REDUCTION
Some bacterial species can reduce nitrates to nitrite and subsequently the nitrite to ammonia,
which can be used for the synthesis of amino acids. Enzymes called nitrate reductases, which are
necessary for the assimilation of nitrates, catalyze these reactions. Other bacterial species can use
nitrates instead of oxygen as a final electron acceptor for the generation of energy. This nitrate
reduction pathway is said to be dissimilatory. The use of nitrates as a final electron acceptor is an
example of anaerobic respiration.
NO3NO2NH4+
Nitrate reductase
Nitrite reductase
other enzymes
N2
Exercise 5.6
Materials (Per groups of 2)
Suspensions prepared in the previous exercise of unknown rods
2 nitrate broths
Method (Per groups of 2)
1. Inoculate with 0.1 mL of each of your unknown rods nitrate broths.
2. Incubate at 28oC.
TOLERANCE TO HIGH SALT CONCENTRATIONS
At high concentrations, salt acts as a selective agent that interferes with membrane permeability
and osmotic equilibrium in most bacteria. Organisms which tolerate salt will have no problem
growing in such an environment.
Exercise 5.7
Materials (Per groups of 2)
Single colony streaking of unknown Gram positive rods isolated last week
2 TSB + 6.5% NaCl broths
Method (Per groups of 2)
1. Inoculate each of your unknowns in TSB + NaCl broths.
2. Incubate at 28oC.
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Microbiology Lab-2014
CYTOCHROME OXIDASE
Many aerobic and some facultative anaerobic bacteria possess cytochrome oxidase in their
electron transport chain, which transfers electrons to oxygen generating water. The presence of
cytochrome oxidase is easily determined by the addition of the test reagent paminodimethylaniline oxalate, which can donate electrons (becomes oxidized) to cytochrome
oxidase. p-aminomethylaniline oxalate turns pink and eventually black when it is oxidized.
Exercise 5.8
Materials (Per groups of 2)
Single colony streaking of unknown Gram positive rods
isolated last week
p-aminodimethylaniline oxalate reagent
Method (Per groups of 2)
1. Ask a teaching assistant to add a drop of the reagent
allowing the detection of the cytochrome oxidase on one
of the colonies of your bacterial unknowns.
2. Examine whether a change in color occurred, of the
colony not the medium, after 1-2 minutes.
BACTERIAL MOTILITY
Several bacteria can swim by using flagella. The flagellum is similar to those of eukaryotes but
of a very different structure. Not all bacteria have flagella, the latter are much more common
amongst rod shaped bacteria, but there are a few rare cocci that also have them. Whether a
bacterium is motile or not can be used towards their identification. One of the ways to detect
motility is to grow the bacteria in a medium with a low agar concentration and then observe
whether the bacteria moved away from the initial inoculation site. Triphenyl tetrazolium chloride
is often included in these media to help in the visualization. This indicator is reduced by most
bacteria, making it red.
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Microbiology Lab-2014
Exercise 5.9
Materials (Per groups of 2)
Single colony streaking of an unknown Gram positive rod isolated last week
2 TSA stabs containing 0.001% triphenyl tetrazolium chloride and 0.5% agar
Method (Per groups of 2)
1. Use a sterilized inoculation needle (NOT the loop) to pick some of the growth from one of
your unknowns and then stab the motility assay medium.
2. Repeat with your second unknown in a second stab tube.
3. Incubate at 28oC.
CATALASE
Catalase is an enzyme, which is found in most organisms that live in the presence of oxygen,
such as aerobic and facultative microorganisms. Oxygen
metabolism generates free radicals, such as hydrogen peroxide,
which damages the cell. Catalase reduces peroxide converting it to
water and oxygen.
2H2O2
2H2O + O2
The generation of oxygen can easily be detected as fine bubbles.
Exercise 5.10
Materials (Per groups of 2)
Single colony streaking of unknown Gram positive rods isolated
last week
3% (v/v) Peroxide
Method (Per groups of 2)
1. Add 1-2 drops of 3% hydrogen peroxide to the growth of your bacterial unknowns.
2. Observe if there is the production of air bubbles.
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Microbiology Lab-2014
IDENTIFICATION OF GRAM POSITIVE COCCI: MICROCOCCACEAE AND
STREPTOCOCCACEAE FAMILIES
The Micrococcaceae
The Micrococcaceae family includes pathogenic and non-pathogenic organisms often associated
to the natural human flora. This family includes two main genera, the Staphylococcus and the
Micrococcus. Both can make use of oxygen and possess a typically respiratory metabolism.
Specifically, members of the genus Micrococcus are strict aerobes whereas those from the genus
Staphylococcus are facultative aerobes. Indeed, the Micrococci produce acid from glucose only
under aerobic conditions whereas the Staphylococci do so under both aerobic and anaerobic
conditions.
Several species of the genus Micrococcus have pigmented colonies, such as M. luteus (yellow) or
M. roseus, (pink). Their cells often have a tetrad arrangement. Residents of the skin, this genus is
rarely pathogenic, being rather opportunistic. Contrary to the Micrococcus, the Staphylococci are
human parasites which very often under certain conditions are the cause of serious illness. The
three major species of the genus Staphylococcus are S. aureus, S. saprophyticus and S.
epidermidis. S. epidermidis is a non-pigmented non-pathogenic organism usually found on the
skin and mucus membranes. S. aureus, a yellow colored species, is commonly associated to
acne, pneumonias, meningitis, and toxic shock syndrome. S. saprophyticus, another organism
often found on skin is non-pigmented and often implicated in urinary infections.
The Streptococcaceae
This family includes bacteria of the genus Streptococcus, which includes pathogenic and nonpathogenic species. This genus is divided into three groups of related species; the Lactococcus,
streptococci of importance to the dairy industry, the Enterococcus, which includes streptococci
of fecal origins, and the Streptococcus, which includes most of the pathogenic species. The latter
are classified according to the Lancefield classification system, which divides these bacteria into
8 groups (A-H and K-U). This classification is based on the immunological reaction of
polysaccharides within their cell walls. Members of clinical importance of the Streptococcus
genus include those from the group A; S. pyogenes. S. pyogenes is the principal cause of strep
throats and in rare cases can cause the massive destruction of tissues (flesh eating bacteria). S.
agalactiae, only member of the B group, causes septicemias in newborns resulting in death in
75% of cases. The group D Enterococci are implicated in urinary infections, endocarditis, and
wound infections. Other Streptococci that are not classified according to the Lancefield
classification system include S. pneumoniae, the principal causing agent of Pneumonias as well
as S. mutans and S. mitis which are the main causes of cavities.
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Microbiology Lab-2014
Exercise 5.11
Materials (Per groups of 2)
Catalase positive broth culture (C+#)
Catalase negative broth culture (C-#)
2 chocolate agar plates
Methods (Per groups of 2)
Colony morphology
1. Streak each of your unknowns for single colonies on one of the chocolate agar plates.
2. Incubate the catalase negative culture at 37oC in the candle jar.
3. Incubate the catalase positive culture at 37oC.
Gram stain
1. Perform a Gram stain of each of your unknowns.
2. Take a digital picture for your report.
3. Record the Gram reaction, cell shape, and aggregation.
DIAGNOSTIC OF CATALASE NEGATIVE GRAM POSITIVE COCCI
BLOOD HEMOLYSIS
Blood agar (BA) contains general nutrients and 5% sheep blood. It is useful for cultivating
fastidious organisms and to determine the hemolytic activities of an organism. Some bacteria
produce exoenzymes that lyse red blood cells and degrade hemoglobin; called hemolysins.
Bacteria can produce different types of hemolysins. Beta-hemolysin breaks down the red blood
cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. This is
referred to as β-hemolysis (beta hemolysis). Many pathogenic species of Streptococcus and some
of Staphylococcus belong to this group. Alpha-hemolysin partially breaks down the red blood
cells and leaves a greenish color behind. This is referred to as α-hemolysis (alpha hemolysis).
The greenish color is caused by the presence of biliverdin, a by-product of the breakdown of
hemoglobin. Many non-pathogenic species of Streptococcus belong to this group. If the
organism does not produce hemolysins and does not break down the blood cells, no clearing will
occur. This is called γ-hemolysis (gamma hemolysis). Most Streptococcus within this group are
non-pathogenic.
Exercise 5.12
Materials (Per groups of 2)
Catalase negative broth culture (C-#)
Blood agar plate
Method (Per groups of 2)
1. Streak your catalase negative unknown for single colonies on a blood agar plate.
2. Incubate at 37oC in the candle jar.
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Microbiology Lab-2014
BILE-ESCULIN
This test is useful for the identification of group D streptococci; the Enterococcus. These bacteria
hydrolyze esculin to esculitin and glucose. Esculitin reacts with an iron salt, ferric citrate,
generating a dark brown or black complex. Bile is included to inhibit the growth of Gram
positive bacteria other than the Enterococci.
Exercise 5.13
Materials (Per groups of 2)
Catalase negative broth culture (C-#)
Bile esculin agar slant
Method (Per groups of 2)
1. Streak a slant of bile esculin with your catalase negative unknown.
2. Incubate at 37oC.
BACITRACIN AND OPTOCHIN SENSITIVITY
Sensitivity to these antibiotics allows the presumptive identification of different members of the
genus Streptococcus; such as S. pyogenes and S .pneumoniae.
Exercise 5.14
Materials (Per groups of 2)
Catalase negative broth culture (C-#)
Sterile cotton swab
1 chocolate agar plate
1 disc of bacitracin
1 disc of optochin
Method (Per groups of 2)
1. Dip a sterile cotton applicator in the broth culture of your catalase negative culture.
2. Spread the bacteria on the surface of a chocolate agar plate, covering as much of the surface
as possible.
3. Use sterile forceps to deposit a bacitracin disc and an optochin disc on the plate’s surface.
4. Incubate the plate at 37°C in the candle jar.
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Microbiology Lab-2014
DIAGNOSTIC OF THE MICROCOCCACEAE
MANNITOL + SALTS AGAR
This medium contains a high salt concentration (7.5%) which allows the enrichment of bacteria
of the genus Staphylococcus. As with phenol red broths, this medium provides two carbon
sources, either mannitol or proteins. The inclusion of a pH indicator, phenol red, allows the
discrimination of Staphylococcus fermenters, such as S. aureus, from the non-fermenters.
Exercise 5.15
Materials (Per groups of 2)
Catalase positive broth culture (C+#)
1 Mannitol + salts agar plate
Method (Per groups of 2)
1. Streak your catalase positive unknown for single colonies on a mannitol salts agar plate.
o
2. Incubate at 37 C.
TELLURITE AGAR OR BAIRD PARKER AGAR
This is a selective medium for the presumptive identification of coagulase-positive
staphylococci. The selectivity of the medium is due to Lithium Chloride and a 1% Potassium
Tellurite Solution, suppressing growth of organisms other than staphylococci. The differentiation
of coagulase-positive staphylococci is based on Potassium Tellurite and Egg Yolk Emulsion.
Staphylococci that contain lecithinase break down the Egg Yolk and cause clear zones around
the colonies. Reduction of Potassium Tellurite, a characteristic of coagulase-positive
staphylococci, causes blackening of colonies.
Exercise 5.16
Materials (Per groups of 2)
Catalase positive broth culture (C+#)
1 Tellurite agar plate
Method (Per groups of 2)
1. Streak your catalase positive unknown for single colonies on a Tellurite agar plate.
2. Incubate at 37oC.
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Microbiology Lab-2014
NOVOBIOCIN SENSITIVITY
Novobiocin sensitivity allows the discrimination of S. saprophyticus from other bacteria of the
Staphylococcus genus; S. saprophyticus being resistant.
Exercise 5.17
Materials (Per groups of 2)
Catalase positive broth culture (C+#)
One chocolate agar plate
Sterile cotton swab
Method (Per groups of 2)
1. Dip a sterile swab in the catalase positive unknown culture.
2. Spread the bacteria onto the surface of a chocolate agar plate such that you cover as much of
the surface area as possible.
3. Use sterile forceps to deposit a novobiocin on the surface of the plate.
4. Incubate at 37°C.
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Microbiology Lab-2014
Day 2
IDENTIFICATION OF GRAM POSITIVE RODS
- INTERPRETATION OF TESTS
Simple carbon metabolism
1. Obtain your pairs of phenol red broths and make the following observations :
 Was there any growth
 What is the color of the broth
 Is there gas accumulation
Acid
Alkaline
Acid + Gas
2. According to your observations, determine which sugars were metabolized and by what
metabolism.
GLUCOSE FERMENTATION; PRODUCTION OF MIXED
ACIDS OR ACETOIN
1. To complete the MR and VP tests, transfer 1 mL of the MRVP
broth culture you prepared in the previous lab to each of two new
test tubes.
2. For the MR test, add 2 drops of methyl red to one of the two
tubes. Observe the color at the surface of the broth. A red color
indicates the presence of a large quantity of acids whereas a
yellow color indicates the absence of any acids.
3. To complete the VP test, add 6 drops of alpha-naphtol to the
second tube.
4. Add 3 drops of KOH to the tube.
5. Mix well and allow the reaction to proceed for 10-15 minutes.
6. Observe whether there is a color change. If a red color develops
it indicates the presence of acetoin.
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Negative
Positive
Positive
Negative
Microbiology Lab-2014
UREASE
1. Examine your slants for a color change to pink. This color indicates
that alkaline by-products were generated by the action of urease.
USE OF CITRATE AS A CARBON SOURCE
1. Examine your slants for a color change to blue. This color indicates
that alkaline by-products were generated indicating citrate could be
used as a carbon source.
Negative Positive
Negative Positive
UTILIZATION OF COMPLEX CARBON SOURCES: EXOCELLULAR ENZYMES
1. Obtain the starch, milk, tributyrin and DNA plates you inoculated.
2. Examine your milk, spirit blue and DNA plates for any clearing surrounding the bacterial
growth. Such a clearing is indicative of the production of exocellular enzymes.
3. For the starch plate, flood the bacterial growth with Gram's iodine. Gram's iodine reacts with
starch to produce a dark blue color. Lack of color development indicates that starch was
degraded.
Amylase
Caseinase
Lipase
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DNAse
Microbiology Lab-2014
NITRATE TO NITRITE REDUCTION
1. Add 3 drops of sulfanilic acid and 3 drops of alpha-naphtylamine to your nitrate broth
culture. These reagents react with nitrite to give a red color.
2. Wait for 1 minute. If no color change occurs, add a pinch of zinc powder.
3. Observe if a color change occurs. Zinc reduces nitrates to nitrites which then react with
the sulfanilic acid and the alpha-naphtylamine to give a red color.
Results after the addition of sulfanilic
acid and alpha-naphtylamine
Results following the addition of
zinc
BACTERIAL MOTILITY
Examine your stab tubes to determine whether your bacteria
are motile
TOLERANCE TO HIGH SALT CONCENTRATIONS
Examine your cultures for the presence of an abundant growth.
IDENTIFICATION OF YOUR UNKNOWN GRAM POSITIVE RODS
Use the flow chart on the web page of this course to identify the bacterial genera and species of
your unknowns. (http://mysite.science.uottawa.ca/jbasso/microlab/IDFlowcharts.pdf)
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Microbiology Lab-2014
DIAGNOSTIC OF THE MICROCOCCACEAE AND STREPTOCOCCACEAE
DIAGNOSTIC OF CATALASE NEGATIVE GRAM POSITIVE COCCI
BLOOD HEMOLYSIS
1. Examine your blood agar plates and determine the type of hemolysis observed: alpha, beta
or gamma.
BILE-ESCULIN
1. Examine your Bile esculin slants. Darkening of the medium indicates
the presence of esculitin as a result esculin hydrolysis.
BACITRACIN AND OPTOCHIN SENSITIVITY
The bacitracin and optochin sensitivity tests are utilized
to identify Streptococcus pyogenes and Streptococcus
pneumoniae, respectively. Only these two species of
Streptococcus are sensitive to the respective antibiotics
when the test is performed.
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Microbiology Lab-2014
PYR TEST
The PYR test is a qualitative procedure for determining the ability of streptococci to
enzymatically hydrolyze L-pyrrolidonyl-β-naphthylamide (PYR). Most group A streptococci and
group D enterococci hydrolyze PYR. Whereas most group B streptococci and non-group A, B
and D streptococci, yield negative PYR test results.
L-pyrrolidonyl-β-naphthylamide (PYR) is hydrolyzed by bacteria that possess the enzyme
pyrrolidonyl peptidase. Demonstrating PYR hydrolysis involves two reactions. Pyrrolidonyl
peptidase, if present, hydrolyzes PYR to liberate L-pyrrolidone carboxylic acid and βnaphthylamine. β-naphthylamine, in turn, reacts with p-dimethyl-aminocinnamaldehyde to form
a pink/fuchsia precipitate.
Exercise 5.18
Materials (Per groups of 2)
BBL DrySlide PYR having four filter paper reaction areas containing L-pyrrolidonyl-βnaphthylamide. (One dry slide per 4 groups of 2)
BBL DrySlide PYR Color Developer - 0.015% p-dimethyl-aminocinnamaldehyde
Single colonies of catalase negative Gram positive cocci
Method (Per groups of 2)
1. Deposit a loopful (approximately 10 μL) of distilled water onto a reaction area of the BBL
DrySlide PYR.
2. Using a sterile inoculation loop, pick isolated colonies or a sweep of confluent growth from
the culture to be tested and generously smear the specimen onto the moistened reaction area
of the BBL DrySlide PYR.
3. Incubate the slide at room temperature for 2 min.
4. Ask a teaching assistant to dispense 1 drop of BBL DrySlide PYR Color Developer onto the
inoculated area of the slide.
5. Examine for the appearance of a pink/fuchsia color within 1 min.
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Microbiology Lab-2014
DIAGNOSTIC OF THE CATALASE POSITIVE GRAM POSITIVE COCCI
MANNITOL SALTS AGAR
Negative
Positive
TELLURITE AGAR
Verify your plating on Tellurite agar and determine whether colonies typical of Staphylococcus
aureus are observed. If a presumptive positive identification of S. aureus is made, perform a
coagulase test as indicated below.
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Microbiology Lab-2014
COAGULASE
This test is useful in differentiating S .aureus from other coagulase-negative staphylococci. Most
strains of S .aureus produce two types of coagulase, free coagulase and bound coagulase. While
free coagulase is an enzyme that is secreted extracellularly, bound coagulase is a cell wall
associated protein. Free coagulase is detected in a tube coagulase test and bound coagulase is
detected in a slide coagulase test.
SLIDE COAGULASE TEST
Principle: The bound coagulase is also known as clumping factor. It cross-links the α and β chain
of fibrinogen in plasma to form fibrin clots that deposits on the cell wall. As a result, individual
cocci stick to each other and clumping is observed.
Exercise 5.18
Materials (Per groups of 2)
Single colonies of suspected culture of S. aureus on chocolate agar
Citrated rabbit plasma
Sterile water
Method (Per groups of 2)
1. Using a marker, divide a slide into two areas as shown below:
Test
Control
2. Label one side “Test” and the other “Control”.
3. Add a small drop of water to each side.
4. With a sterile inoculation loop, suspend two to three large colonies of the suspected bacteria
in each of the two drops of water. Mix well until a good cloudy suspension is obtained.
5. Add a drop of citrated rabbit plasma to the test area and mix well.
6. Agglutination of cocci observed within 5-10 seconds is taken as positive.
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Microbiology Lab-2014
Note: Some strains of S. aureus may not produce bound coagulase, and such strains must
be identified by the tube coagulase test.
NOVOBIOCIN SENSITIVITY
1. Measure the zone of inhibition. A diameter of 17mm or less indicates resistance, whereas a
diameter greater than 17mm indicates that the isolate is sensitive.
OXIDASE
This test allows the discrimination of bacteria between the genera Staphylococcus and
Micrococcus. In contrast to the staphylococci, micrococci are oxidase positive.
1. Add a drop of the oxidase reagent on the growth of your unknown.
2. Examine whether there is a change in color after 1-2 minutes.
IDENTIFICATION OF YOUR UNKNOWN GRAM POSITIVE COCCI
Use the flow chart on the web page of this course to identify the bacterial genera and species of
your unknowns. (http://mysite.science.uottawa.ca/jbasso/microlab/IDFlowcharts.pdf)
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Microbiology Lab-2014
LAB #6
CONTROL OF MICROBIAL GROWTH
Day 1
ANTIBACTERIAL COMPOUNDS- ANTIBIOTICS
Antibiotics can be synthetic, semi-synthetic or natural compounds, which inhibit the growth or
kill bacteria. Antibiotics can be generally classified according to their mode of action as being
bacteriostatic, bacteriolytic, or bactericidal. Bacteriostatic antibiotics inhibit growth but do not
kill bacteria. Bacteriolytic antibiotics kill bacteria by causing their lysis. Bactericidal antibiotics
kill bacteria without lysis.
KIRBY-BAUER DISC DIFFUSION METHOD
When faced with a newly discovered bacterial pathogen or a new derivative of a known
pathogen, it is essential to assess the antibiotic susceptibility of the isolate. Initially, one must test
the effect of a wide variety of different antibiotics to determine which may be potentially used.
This is usually assessed by a semi-quantitative assay referred to as the Kirby-Bauer disc
sensitivity method. In this test, the sensitivity of a bacterium to a variety of different antibiotics is
tested. This test is performed on a Mueller-Hinton agar plate of which the surface is covered with
an inoculum of the bacterium to be tested. Filter discs containing known amounts of different
antibiotics are then placed on the surface of the plate. Following a suitable incubation period for
optimal growth of the bacterium being tested, the plate is examined for the absence of bacterial
growth in proximity to the antibiotic discs. These zones of inhibition are observed as halos
around the antibiotic discs. The diameter of the halo is used as a measure of the relative
sensitivity of the bacterium to the antibiotic (see figure). Zone size recommendations for the
interpretation of the Kirby-Bauer (resistant, intermediate, sensitive) are established by disease
control organizations.
14mm
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Microbiology Lab-2014
Exercise 6.0
Materials (Per groups of 2)
5 mL broth culture of S. aureus
5 mL broth culture of S. faecalis
5 mL broth culture of E. coli
3 chocolate agar plates
Sterile swabs
Antibiotic discs
Forceps
Method (Per groups of 2)
1. Label three chocolate agar plates according to the bacteria to be tested (Staphylococcus,
Streptococcus and E. coli)
2. Immerse a cotton swab into the broth culture until it is thoroughly wet. Remove surplus
suspension from the swab by rotating against the inside of the culture tube.
3. Spread the entire surface of each plate with the appropriate culture. Even distribution is
essential; spread evenly in three directions so that even, confluent growth will result.
4. With plates covered, allow the inoculums to dry for 3 to 5 min.
5. Deposit the antibiotic discs. Using aseptic techniques, tap each disc gently to ensure full
contact with the agar surface.
6. Incubate the inverted plates at 37°C.
E-TEST
This test combines the principals of the Kirby Bauer assay and the dilution method described in
the next experiment. The E-test is a well-established method for antimicrobial resistance testing
in microbiology laboratories around the world. E-test consists of a predefined gradient of
antibiotic concentrations on a plastic strip and is used to determine the Minimum Inhibitory
Concentration (MIC) of antibiotics.
Exercise 6.1
Materials (Per groups of 2)
5 mL broth culture of S. faecalis
1 chocolate agar plate
Sterile swabs
Vancomycin E-test strip
Forceps
Method (Per groups of 2)
1. Use the same approach as that in exercise 6.0 to inoculate a chocolate agar plate with S.
faecalis.
2. Using forceps, deposit the E-test strip in the center of the plate. Gently tap the strip to ensure
full contact with the agar surface.
3. Incubate the inverted plate at 37°C.
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Microbiology Lab-2014
DETERMINING THE THERAPEUTIC DOSE
When an antibiotic is to be used for therapeutic purposes, it is essential to determine the minimal
concentration of the antibiotic, which is going to be effective. Lack of this information can result
in the prescription of too high or too low a dose. Why would either of these scenarios
represent a problem? The most common way of determining both the minimal inhibitory
concentration (MIC) and the minimal bactericidal concentration (MBC) of an antibiotic is the
broth dilution method. A fixed amount of bacteria is inoculated into broth containing varying
amounts of the antibiotic being tested. The lowest antibiotic concentration at which no bacterial
growth is observed after a suitable incubation period is referred to as the MIC.
Exercise 6.2
Materials (Per groups of 2)
5 mL broth culture of S. aureus
5 mL broth culture of S. faecalis
5 mL broth culture of E. coli
10 mL of assigned antibiotic (1mg/mL in TSB)
Approx. 100 mL TSB
27 Sterile test tubes
Antibiotic
Ampicillin
Kanamycin
Naladixic acid
Tetracycline
Erythromycin
Class
Beta-lactam
Aminoglycoside
Quinolone
Tetracycline
Macrolide
Abbreviation
A
K
N
T
M
LD50 (mg/Kg)
5300
4000
2040
6443
4600
Method (Per groups of 2)
1. Label three sets of 8 test tubes (One set/bacterial species) with the bacterial species being
tested, the antibiotic being assayed and your group number.
2. Add 2.5mL of TSB to each tube.
3. Add 2.5mL of the assigned antibiotic stock solution to the first tube of each set of eight. Mix
well.
4. Transfer 2.5mL from the first tube of each set to the second tube of each set. Mix well.
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Microbiology Lab-2014
5. Transfer 2.5mL from the second tube of each set to the third tube of each set. Mix well.
6. Continue this approach for the 8 tubes. You should therefore have generated 8 serial 2 fold
dilutions of the assigned antibiotic. All the tubes should contain 2.5mL of medium except for
the last one.
7. Withdraw and discard 2.5mL from the last tube of each set.
8. Dilute in TSB each of the supplied cultures in order to obtain 5 mL of broth representing a
100 000X dilution factor.
9. Inoculate each of the eight tubes from a given set with 0.1 mL of the Staphylococcus culture
you diluted previously.
10. Inoculate each of the eight tubes from a given set with 0.1 mL of the Streptococcus culture
you diluted previously.
11. Inoculate each of the eight tubes from a given set with 0.1 mL of the E. coli culture you
diluted previously.
12. Incubate the inoculated broths at 37oC.
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Microbiology Lab-2014
COMBINATION ANTIBIOTIC THERAPY
Combination antibiotic therapy involves the treatment of an individual with two antibiotics
simultaneously. This may be done for several reasons, which include:




To avoid the development of resistance
To provide broad coverage in polymicrobial infection
To treat severe infections where the cause is unknown
To treat infections by pathogens that cannot be killed with a single antibiotic (ex.
Mycobacteria)
Combination antibiotherapy also has several disadvantages, which include:



Increased toxicity
Antagonism - the two agents interact resulting in reduced antibiotic effect
Increased cost
There are three potential effects of using a combination of antibiotics.
Additive effect: the activity of two drugs in combination is equal to the sum (or a partial sum) of
their independent activity when studied separately.
Synergistic effect: the activity of two drugs in combination is at least 4 times greater than the
sum of their independent activity when studied separately.
Antagonistic effect: the activity of two drugs in combination is less than the sum (or a partial
sum) of their independent activity when studied separately.
Exercise 6.3
Materials (Per groups of 2)
5 mL broth culture of E.coli
Ampicillin solution at 1mg/mL in TSB.
Chloramphenicol solution at 1mg/mL in TSB.
Nalidixic acid solution at 1mg/mL in TSB.
Tetracycline solution at 0.02mg/mL in TSB.
TSB
96 well plate
Method (Per groups of 2) (See schematic below)
1. Dispense 50µL of TSB to each of the wells (1-12) of rows “A – H”.
2. Add 50µL of the ampicillin solution (1mg/mL) to wells “A1” and “E1”.
3. Transfer 50µL from well “A1” to well “A2”. Mix by pipetting up and down several times.
4. Transfer 50µL from well “A2” to well “A3”. Mix as previously.
5. Continue this approach up to well “A12”.
6. Withdraw 50µL from well “A12”.
7. Repeat steps 2-6 for row “E”
8. Add 50µL of the chloramphenicol solution (1mg/mL) to wells “B1” and “F1”.
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Microbiology Lab-2014
9. Repeat steps 2-6 for rows “B” and “F”
10. Add 50µL of the nalidixic acid solution (1mg/mL) to wells “C1” and “G1”.
11. Repeat steps 2-6 for rows “C” and “G”
12. Dispense 50µL of the tetracycline solution (0.02mg/mL) to all the wells of rows A, B, and
C.
13. Dispense 50µL of TSB to all the wells of rows E, F, and G.
14. Prepare a 1/100 000 dilution in TSB in a final volume of 5 mL of the E.coli culture
15. Dispense 100 µL of the diluted E.coli culture to each of the wells of rows A-C and E-G.
16. Incubate at 37oC until the next lab period.
17. Dispense 200 µL of TSB in well H12. Have your plate read at a wavelength of 600nm.
Ampicillin
Chloramphenicol
+ Tetracycline
Nalidixic acid
Ampicillin
Chloramphenicol
Nalidixic acid
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- Tetracycline
Microbiology Lab-2014
DISINFECTANTS & ANTISEPTICS (Individually)
There exist several classes of antibacterial compounds, such as antiseptics, disinfectants, and
antibiotics. These all act to either reduce the number of bacteria, eliminate them, or inhibit their
growth. Some have a therapeutic use, such as antiseptics and antibiotics, while others are used
for preventive or aesthetic measures, such as disinfectants. The purpose of antiseptics and
disinfectants is to significantly reduce the number of bacteria within a given area and to prevent
bacterial growth. Antiseptics are chemicals intended for human use whereas disinfectants are
chemicals intended for use on inanimate objects (formites).
THE EFFICACY OF HAND SOAPS
Probably the most commonly used disinfectant/antiseptic in your everyday life is soaps. One of
their most popular uses is to wash your hands, whether it is before you start cooking your meal
or in the case of a surgeon, before he operates on an individual. The objective of washing your
hands is twofold: Eliminate bacteria which you may have acquired from handling different
objects (the transient flora) and to reduce the number of bacteria which normally resides on you
(the resident flora). Obviously, the sterilization of one’s hands is impossible since
microorganisms inhabit not only the surface of the skin, but also the deep layers of the skin. This
natural flora, for the most part is composed of non-pathogenic organisms of the Staphylococcus
genus. In the following exercise, we will evaluate the efficiency of hand washing soaps.
The skin microbiome
Elizabeth A. Grice & Julia A. Segre
Nature Reviews Microbiology 9, 244-253
(April 2011)
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Microbiology Lab-2014
Exercise 6.4
Materials (Per groups of 4)
Purell, Antibiotic soap, Natural soap (glycerine), Water
12 chocolate agar plates
Cotton swabs
Sterile water
Method (Per groups of 4)
1. Assign to each person within a group of 4 one of the treatments indicated above.
2. Each person should obtain 3 chocolate agar plates.
3. Each person should label their agar plates with their name and the treatment number (0, 1 or
2). These are the plates on which you will plate samples from your hands.
4. Moisten a sterile cotton swab in some sterile water. Swab as much of the surface as possible
of the palm of your hand.
5. Now swab as much of the surface as possible of the plate labelled “0” with this sampling.
6. Following your initial sampling, wash your hands for two minutes with the assigned
treatment.
7. Sample the palm of your hands as previously and plate this sampling on the plate labelled
“1”.
8. Repeat the hand washing for an additional two minutes, and then sample the palm of your
hand as previously and plate this sampling on the plate labelled “2”.
9. Incubate the inverted plates at 37oC.
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Microbiology Lab-2014
Day 2
ANTIBACTERIAL COMPOUNDS - ANTIBIOTICS
Exercise 6.0
KIRBY-BAUER DIFFUSION ASSAY
Obtain the diameters of the zones of inhibition for each of the antibiotics with each of the
bacteria tested.
Use the data in the following table to determine the susceptibility of each of the bacteria.
R = mm
or less
19
I = mm
range
S = mm
or more
20
Antimicrobial agent
CODE
Amoxicillin (Staph)
AMC
Amoxicillin (other bacteria)
AMC
13
Ampicillin (Staph)
AM
28
Ampicillin (other bacteria)
AM
11
12-13
14
Carbenicillin (Pseudomonas)
CB
13
14-16
17
Carbenicillin (other bacteria)
CB
17
18-22
23
Cefoxatime
CTX
14
Cephalothin
CF
14
15-17
18
Chloramphenicol
C
12
13-17
18
Erythromycin
E
13
14-22
23
GM
12
13-14
15
M (or DP)
9
10-13
14
Penicillin
P
28
Streptomycin
S
11
12-14
15
SXT-TMP
10
11-15
16
TE
14
15-18
19
Gentamycin
Methicillin (Staph)
Sulfamethoxazole-trimethoprim
Tetracycline
86
14-17
18
29
23
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Microbiology Lab-2014
Exercise 6.1
E-TEST
Read off the MIC from the strip, which is represented
by the concentration on the strip which gives the
smallest diameter for the zone of inhibition.
MIC
Exercise 6.2
DETERMINATION OF THE THERAPEUTIC DOSE
Since most antibiotics are bacteriostatic at low concentrations, the MIC determination does not
allow the determination of the effective dose of a bactericidal antibiotic. Determining the MBC
assesses the effective dose of a bactericidal antibiotic. The method used to determine the MBC is
the same as that used to determine the MIC. Following the MIC determination, subcultures are
made from the dilution tubes in which no growth was observed into fresh broth lacking any
antibiotics. The antibiotic concentration from which no bacterial growth could be rescued is
referred to as the MBC.
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Microbiology Lab-2014
Materials (Per groups of 2)
MIC assays done previously
Tubes containing 5mL TSB
Method (Per groups of 2)
1. Obtain one test tube containing 5mL TSB for each concentration at which no growth was
observed in the MIC determination experiment.
2. Label each tube with the antibiotic concentration at which no growth was observed.
3. Inoculate each tube with 1.0 mL from the corresponding MIC assay tube in which no growth
was observed.
4. Incubate overnight at 37oC.
5. Determine the lowest antibiotic concentration from which growth could not be rescued.
Exercise 6.3
COMBINATION ANTIBIOTIC THERAPY
1. Have your plates read at a wavelength of 260nm to determine the optical densities.
Determine the lowest concentration of each antibiotic which gives the lowest optical
densities. Consider these concentrations to be your MICs.
2. For your assignment, determine the MIC of each antibiotic individually and when it is used
in combination with tetracycline.
Exercise 6.4
EFFICACY OF HAND SOAPS
1. You will want to first determine the approximate surface area of the palm
of your hands. To do so, place your hand face down on a sheet of paper as
illustrated and trace its outline not including the wrist area. Then,
determine the weight of the sheet of paper. After having determined the
weight of the sheet of paper, cut out your hand profile and determine its
weight.
2. A standard sheet of paper (8in X 11in) has a surface area of
approximately 603 cm2. Determine the relationship between the weight
and the area and then use the same relationship to determine the
approximate surface area of your palm.
3. Determine the number of CFU on each of your plates and record your
data as follows on the Excel sheet at the podium: CFU/cm2 for each treatment (0, 1 and 2)
under the appropriate product (Purell, Antibiotic soap, Natural soap (glycerine), or Water).
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Microbiology Lab-2014
KINETICS OF DEATH
Different microorganisms have varying susceptibilities to different treatments. For instance,
spores are particularly resistant. As is the case for the growth of bacteria, death occurs in an
exponential fashion. Consequently, mathematical functions describing the killing profile over
time under a given condition may be derived. Such functions are useful in determining the
minimal time required to reduce a bacterial population below harmful levels. The decimal
reduction time, referred to as the D value, represents the amount of time under given conditions
required to reduce a population of microorganisms by 1 log value or 90%. In other words, if the
D value of E. coli is 1 minute, this indicates that a one-minute exposure is required to reduce the
bacterial population by 90%. Therefore, to reduce a population of 1 x 108 cells to 1 x 106 cells of
E. coli it would take 2 minutes. D values are influenced by the bacterial species, their form, and
the conditions in which they are. For instance the D value of spores is usually much higher than
that of vegetative cells.
Read the research article available on this course's web page under the heading
assignments in order to answer the pertinent questions in the assignment.
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Microbiology Lab-2014
LAB # 7
WATER AND FOOD MICROBIOLOGY
Day 1
MICROBIOLOGICAL QUALITY OF WATER
The importance of potable (drinking) water supplies cannot be overemphasized. With increasing
industrialization, water sources available for consumption and recreation have been adulterated
with industrial as well as animal and human wastes. As a result, water has become a formidable
factor in disease transmission. Polluted waters contain vast amounts of organic matter that serves
as excellent nutritional sources for the growth and multiplication of microorganisms. The
presence and number of coliform bacteria, bacteria that are normally residents of the mammalian
intestine, and other enteric organisms in water is indicative of fecal contamination and may
suggest the presence of pathogens. These pathogens are responsible for intestinal infections such
as bacillary dysentery, typhoid fever, cholera, and paratyphoid fever. Analysis of water samples
on a routine basis would not be possible if each pathogen required detection. Therefore, water is
examined to detect indicator microorganisms such as Escherichia coli. It should be emphasized
that the presence of indicator bacteria does not mean the water contains pathogenic
microorganisms but rather the potential exists for the presence of pathogens since the indicator
bacteria point to the presence of fecal material in the sample.
Both qualitative and quantitative methods are used to determine the sanitary condition of water.
Contamination of either drinking water or natural bodies of water with these indicator organisms
is a good indication of poor waste management. Several different indicator microorganisms are
used to determine the level of contamination with fecal matter.
INDICATOR MICROORGANISMS OF FECAL CONTAMINATION
Total coliforms are small rods of the Enterobacteriacae family that ferment lactose at 37oC with
formation of acid and gas in 48 hours. These include both bacteria native to the mammalian
intestine or the environment such as Escherichia coli, Klebsiella sp., Enterobacter sp.,
Citrobacter sp., Serratia sp., Shigella sp., and Proteus sp.
Fecal coliforms (Escherichia coli): This is the only coliform that is not normally found outside
of the intestinal environment. Its presence is therefore an excellent indication of fecal
contamination. However, because of its relatively low viability outside of its natural
environment, a negative test is not necessarily indicative of the absence of fecal contamination.
Fecal Streptococci: Includes both Streptococci and Enterococci species, of which several are
enteric or fecal in origin. These have the advantage of surviving longer in the environment.
However, it is often difficult to distinguish them from natural soil and water inhabitants.
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Microbiology Lab-2014
STANDARD QUALITATIVE ANALYSIS OF WATER FOR COLIFORMS
The three basic tests to detect the presence of coliform bacteria in water are presumptive,
confirmed, and completed. The tests are performed sequentially on each sample under analysis.
They detect the presence of coliform bacteria (indicators of fecal contamination) which are
Gram-negative, non-spore forming bacilli that ferment lactose with the production of acid and
gas that is detectable following a 48-hour incubation period at 37ºC.
PRESUMPTIVE COLIFORM TEST - DETERMINING THE MOST PROBABLE
NUMBER OF COLIFORMS.
Purpose:
1. To evaluate the presence of coliform bacteria in a water sample.
2. To obtain some index as to the possible number of organisms present in the sample under
analysis.
Principle
The presumptive test is specific for detection of coliform bacteria. Measured aliquots of the
water to be tested are added to a lactose fermentation broth containing an inverted gas vial
(Durham tubes). Because these bacteria are capable of using lactose as a carbon source (other
enteric organisms are not), their detection is facilitated by use of this medium. Tubes of this
lactose medium are inoculated with 10-mL, 1-mL, and 0.1-mL aliquots of the water sample. The
series consists of at least three groups, each composed of three tubes of the specified medium.
The tubes in each group are then inoculated with the designated volume of the water sample.
Development of gas in any of the tubes is presumptive evidence of the presence of coliform
bacteria in the sample. The presumptive test also enables the microbiologist to obtain some idea
of the number of organisms present by means of the most probable number test (MPN). The
MPN is estimated by determining the number of tubes in each group that show gas following the
incubation period.
Lauryl-trypotose broth. This medium contains peptones as a carbon and nitrogen source as well
as other nutrients. In addition this medium contains lactose as a fermentable carbohydrate and
sodium lauryl sulfate which inhibits the growth of organisms other than coliforms. An inverted
vial is included to detect the accumulation of gas. A positive test, gas accumulation following a
48 hour incubation period at 37oC indicates the presumptive presence of coliforms.
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Microbiology Lab-2014
Exercise 7.0
Materials (Per groups of 2)
Water sample
3 double strength lauryl tryptose broths
6 single strength lauryl tryptose broths
Method (Per groups of 2)
1. Label the three tubes of double-strength lauryl tryptose broth "10".
2. Label three tubes of single-strength lauryl tryptose broth "0.1" and the three other "1".
3. Inoculate three double-strength tubes with 10 mL of water to be tested.
4. Inoculate three of the single-strength tubes with 1 mL of water to be tested.
5. Inoculate three of the single-strength tubes with 0.1 mL of water to be tested.
6. Incubate all your tubes at 37oC.
Water Inoculums
0.1 mL
[1X
]
[1X
]
1.0 mL
[1X
]
[1X
]
[1X
]
10 mL
[1X
]
Lauryl Tryptose Broths
92
[2X
]
[2X
]
[2X
]
Microbiology Lab-2014
STANDARD QUATITATIVE ANALYSIS OF WATER
FOR COLIFORMS
Petrifilm assay. This test is a variation of viable counts on
selective and differential media. Petrifilm plates are thin layer of
dehydrated media. These plates have several advantages as
compared to traditional plates; such as the visual biochemical
confirmation, their ease of use, the minimal amount of space
required and the fact that no media preparation is required.
Exercise 7.1
Materials (Per groups of 2)
Water sample
Petrifilm plate
Methods (Per groups of 2)
1. Apply 1 mL of the water to be tested on the Petrifilm as
shown.
2. Overlay with the plastic film and spread the sample by
rolling a 10mL pipette on the surface.
3. Label and incubate at 37oC.
STANDARD QUALITATIVE ANALYSIS OF WATER FOR FECAL STREPTOCOCCI
The feces of humans and animals contain large numbers of streptococcal bacteria that can be
classified as belonging to the fecal streptococci group. There are six species Source Ratio
of streptococci: S. faecalis, S. faecium, S. avium, S. gallinarum, S. bovis, and Man
4.4
S. equinus. These bacteria are Gram positive facultative anaerobes catalase Duck
0.6
negative non spore forming cocci that ferment glucose at 37oC. They are Sheep
0.4
primarily found in the feces of warm-blooded animals. In contrast to Chicken 0.4
coliforms, the numbers of fecal streptococci is generally higher than that for Pig
0.4
fecal coliforms. Furthermore the ratio of their numbers relative to each other Cow
0.2
(FC/FS) in a water sample is an indication of the source of fecal
contamination (human vs. animal).
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Microbiology Lab-2014
ENTEROCOCCI TEST
The enterococci are a subset of the fecal streptococci that include the first four species of fecal
streptococci listed above. Cultural methods analogous to the coliform tests have been developed
to determine the presence and concentration of these bacteria in water samples. As with the
presumptive test for coliforms, measured aliquots of the water to be tested are added to a
selective medium (SF broth). Growth of all other cocci and Gram-negative bacteria is inhibited
in this medium by sodium azide. Fermentation of glucose is indicated by a color change in the
broth. Bromcresol purple is the indicator.
Exercise 7.2
Materials (Per groups of 2)
Water sample
3 double strength SF broths
6 single strength SF broths
Method (Per groups of 2)
1. Label the three tubes of double-strength SF broth "10".
2. Label three tubes of single-strength SF broth "0.1" and the three other "1".
3. Inoculate three double-strength tubes with ten millilitres of water to be tested.
4. Inoculate three of the single-strength tubes with 1 mL of water to be tested.
5. Inoculate three of the single-strength tubes with 0.1 mL of water to be tested.
6. Incubate all your tubes at 37oC.
Water Inoculums
d’eaux
1.0 mL
0.1 mL
[1X
]
[1X
]
[1X
]
[1X
]
[1X
]
SF Broths
94
[1X
]
10 mL
[2X
]
[2X
]
[2X
]
Microbiology Lab-2014
MICROBIOLOGICAL QUALITY OF FOOD
Bacteria in food products contribute to both spoilage and diseases. Spoilage organisms mainly
belong to the Pseudomonacae family. Bacteria of this family contribute to the deterioration of
the organoleptic properties of food. They give off bad odours, off flavours, influence texture, and
may cause discolorations. Species of the Pseudomonacae family comprise short Gram-negative
strict aerobic rods. One species of primary concern is Pseudomonas aeruginosa.
Food-borne diseases fall into two classes: food infections and food intoxications. Food
infections are the result of ingestion followed by the growth of the pathogenic bacteria. The
predominant genera include Gram negative bacteria such as members of the Enterobacteriaceae
and the Campylobacteriaceae families, some Gram positive non-spore forming genera of the
Listeriaceae family, such as Listeria and some Gram positive spore forming species from the
Bacillus genera such as Bacillus cereus.
In contrast, food intoxications result from the ingestion of preformed toxins that may accumulate
in food because of improper or prolonged storage and thus bacterial growth. The predominant
bacterial species responsible for such intoxications belong to the Clostridium genus. Species of
this genus are typically Gram-positive strict anaerobic spore forming short rods.
Guidelines
These guidelines identify five categories of food (table 1). The categories are based on expected
aerobic colony counts, according to the type of food product and the processing it has received.
There are four grades of microbiological quality (box 2) – related to the actual aerobic colony
count, number of indicator organisms, and the presence/ number of pathogens determined by the
microbiological examination of the food.
Aerobic colony count
The term aerobic colony count (ACC) is a count of viable bacteria based on counting of colonies
grown in nutrient agar plate. This is commonly employed to indicate the sanitary quality of
foods.
Indicator organisms
The main objective of using bacteria as indicators is to reflect the hygienic quality of food. E.
coli is commonly used as indicator. Its presence in food generally indicates direct or
indirect faecal contamination. Substantial number of E. coli in food suggests a general lack of
cleanliness in handling and improper storage.
For t h e assessment of hygienic quality, food items are grouped into five categories. The
categorisation of some food products is summarised in the Food category table for ACC
assessment on the next page.
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Microbiology Lab-2014
Food Category for Aerobic Colony Count Assessment
Food group
Food item
Category
Meat
Beef
1
Poultry
2
Preserved meat
4
Seafood
Crustaceans
3
Shellfish (cooked)
4
3
Vegetable
Coleslaw / salads
Fruit and vegetables (dried)
3
Fruit and vegetables (fresh)
5
5
Dairy
Cheese
Yogurt
5
Specific pathogens
These are bacteria that may cause food poisoning. Mechanisms involved may be toxins
produced in food or intestinal infection. The symptoms of food poisoning vary from nausea
and vomiting (e.g. caused by S. aureus), through diarrhoea and dehydration (Salmonella spp.
and Campylobacter spp.) to paralysis and death in the rare cases of botulism.
Classification of Microbiological Quality
The microbiological assessment of food on the above three components leads to the
classification of the food quality into one of the following four classes:
(a)
Class A: the microbiological status of the food sample is satisfactory.
(b)
Class B: the microbiological status of the food sample is less than satisfactory
but still acceptable for consumption.
(c)
Class C: the microbiological status of the food sample is unsatisfactory.
(d)
Class D: the microbiological status of the food sample is unacceptable. The
food sample contains unacceptable levels of specific pathogens that is
potentially hazardous to the consumer.
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Microbiology Lab-2014
Table of microbiological limits
Microbiological limits with respect to the above components and the associated
microbiological quality of the food concerned are summarised in the table below.
Microbiological quality
Colony-forming
unit (CFU) Per gram
Criterion
Class A
Class B
Class C
Class D
Satisfactory
Acceptable
Unsatisfactory Unacceptable
Aerobic colony count (ACC)
Food Category 1
N/A
< 103
103 – < 104
> 104
4
4
5
5
2
< 10
10 – < 10
> 10
N/A
5
5
6
6
3
< 10
10 – < 10
> 10
N/A
6
6
7
7
4
N/A
< 10
10 – < 10
> 10
5
N/A
N/A
N/A
N/A
Indicator organism (applies to all food categories)
E. coli (total)
< 20
20 - < 100
>100
N/A
Pathogens (apply to all food categories)
Not detected
Salmonella
N/A
N/A
Present in 25g
in 25g
S. aureus
< 20
20 - < 100
100 - <104
> 104
B. cereus
< 103
103 - < 104
104 - < 105
> 105
Not detected
Campylobacter
N/A
N/A
Present in 25g
in 25g
L. monocytogenes
Not detected
in 25g
N/A
N/A
Present in 25g
N/A denotes “Not applicable”
In the following experiments you will be testing the microbiological quality of a fresh and a
stored sample of a meat and a vegetable food product; specifically chicken and ready to eat
lettuce.
Exercise 7.3 SAMPLE PREPARATION
Materials (Per groups of 4)
10g of fresh ready to eat lettuce stored in a « Ziploc » bag. (Odd numbered groups)
10g of ready to eat lettuce stored at 4oC for 1 week in a « Ziploc » bag (Even numbered groups)
Fresh chicken drum stick in a « Ziploc » bag (Even numbered groups)
Chicken drum stick stored at 4oC for 1 week (Odd numbered groups)
Sterile water
Method (Per groups of 2)
1. Weigh and record the weight of your food sample in the bag.
2. Add 2 mL of sterile water per gram of food in the bag.
3. Mix by rocking and massaging the food for 5 minutes.
4. Recover and transfer as much of the wash solution to sterile Falcon tubes.
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Microbiology Lab-2014
Exercise 7.4 AEROBIC COLONY COUNT (ACC)
Materials (Per groups of 2)
6 TSA plates
10 sterile test tubes
50 mL sterile TSB
Method (Per groups of 2)
Perform viable counts of the appropriate dilutions of your wash solutions to determine in which
class (A, B, C, or D) your food products fall in.
Exercise 7.5 INDICATOR ORGANISMS – E. coli
Eosin methylene blue (EMB) agar is a differential medium used for the detection and isolation of
Gram negative intestinal pathogens. A combination of eosin and methylene blue is used as an
indicator and allows differentiation between organisms that ferment lactose and those that do not.
Saccharose is also included in the medium because certain members of the Enterobacteria or
coliform group ferment saccharose more readily than they ferment lactose. In addition,
methylene blue acts as an inhibitor to Gram-positive organisms. Colonies of E. coli normally
have a dark center and a greenish metallic sheen, whereas the pinkish colonies of Enterobacter
aerogenes are usually mucoid and much larger than colonies of E. coli.
Materials (Per groups of 2)
4 EMB plates
6 sterile test tubes
50 mL sterile TSB
Method (Per groups of 2)
Perform viable counts for the presence of E.coli of the appropriate dilutions of your wash
solutions to determine in which class (A, B, C, or D) your food products fall in.
Exercise 7.6a SPECIFIC PATHOGENS – Salmonella
Rappaport-Vassiliadis Broth is a selective media used for the enrichment of Salmonella
species. The selectivity of the media is due to the presence of malachite green, high osmotic
pressure, and a low pH. The high concentration of magnesium chloride raises osmotic pressure,
and in combination with malachite green, inhibits bacteria other than Salmonella. The low pH of
the medium increases selectivity by inhibiting accompanying flora, including intestinal bacteria.
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Microbiology Lab-2014
Materials (Per groups of 2)
3 double strength Rappaport-Vassiliadis Broths
6 single strength Rappaport-Vassiliadis Broths
Method – MPN (Per groups of 2)
1. Inoculate the Rappaport-Vassiliadis broths with the wash solution from your food products
as shown below:
Inoculums of the solution
de lavage1.0 mL
0.1 mL
[1X
]
[1X
]
[1X
]
[1X
]
[1X
]
[1X
]
10 mL
[2X
]
[2X
]
[2X
]
Rappaport-Vassiliadis Broths
Exercise 7.6b SPECIFIC PATHOGENS – S. aureus
Materials (Per groups of 2)
6 Mannitol + salts agar plates
6 sterile test tubes
50 mL sterile TSB
Method (Per groups of 2)
Perform viable counts for the presence of S. aureus of the appropriate dilutions of your wash
solutions to determine in which class (A, B, C, or D) your food products fall in.
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Microbiology Lab-2014
Exercise 7.6c SPECIFIC PATHOGENS – Bacillus cereus
Bacillus Cereus Selective Agar (PEMBA) is sufficiently selective to be able to detect small
numbers of Bacillus cereus cells and spores in the presence of large numbers of other food
contaminants. Bromothymol blue is added as a pH indicator to detect mannitol utilisation. The
medium is made selective by addition of Polymyxin B. The primary diagnostic features of the
medium are the colonial appearance, precipitation of hydrolysed lecithin and the failure of
Bacillus cereus to utilise mannitol. The typical colonies of Bacillus cereus have a distinctive
turquoise to peacock blue colour surrounded by a good egg yolk precipitate of the same colour.
Materials (Per groups of 2)
6 PEMBA plates
6 sterile test tubes
50 mL sterile TSB
Method (Per groups of 2)
Perform viable counts for the presence of B. cereus of the appropriate dilutions of your wash
solutions to determine in which class (A, B, C, or D) your food products fall in.
Exercise 7.6d SPECIFIC PATHOGENS - Campylobacter
Campylobacter Agar (Campy-CVA) is a selective medium for the primary isolation of
Campylobacter. This medium supports the growth of Campylobacter species due to its content of
peptones, dextrose, and yeast extract. The peptones supply nitrogenous compounds, carbon, and
sulfur and trace ingredients. Yeast extract is a source of the B vitamins. Dextrose is utilized as an
energy source. The incorporation of the antimicrobial agents, amphotericin B, cephalothin,
polymyxin B, trimethoprim and vancomycin, suppresses the growth of most other microbes,
thereby facilitating isolation of Campylobacter.
Materials (Per groups of 2)
2 Campy-CVA agar plates
Campy Pouch System (Catalog No.: B-260685) For the whole class
Method (Per groups of 2)
1. Perform viable counts for the presence of Campylobacter in your wash solutions to
determine in which class (A, B, C, or D) your food products fall in.
2. Incubate the plates in the anaerobic chamber in an atmosphere consisting of approximately 56% oxygen, 10% carbon dioxide and 84-85% nitrogen for 48 hours at 37°C.
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Microbiology Lab-2014
Day 2
MICROBIOLOGICAL QUALITY OF WATER
Exercise 7.0 and 7.2: QUALITATIVE TESTS OF WATER
1. Obtain all your tubes for the presumptive tests.
2. For each of the assays, determine the number of tubes out of three (for each set of 3) which
are positive for the organism being tested.


Lauryl tryptone broth: Presence of gas (>10%)
SF broth: Acid production
3. Use the formula presented in lab 2 to determine the MPN/100 mL for each microorganism.
4. Obtain the data for the four water samples distributed in the class.
Exercise 7.0b: CONFIRMED COLIFORM TEST
The presence of a positive presumptive test immediately
suggests that the water sample is non-potable.
Confirmation of these results is necessary, since positive
presumptive tests may be the result of organisms of noncoliform origin that are not recognized as indicators of
fecal pollution. The confirmed test requires that selective
and differential media such as endo agar be streaked from
a positive lactose broth. Gram-positive bacteria are
inhibited on this media by desoxycholate and lauryl
sulfate. Lactose fermenting organisms form aldehydes,
which react with Schiff’s reagent (basic fuchsin and
sodium sulfite) to give red colored zones around the
colonies. Coliform colonies are therefore red with a
characteristic metallic sheen.
Method
1. Streak for single colonies a sample from all positive lauryl tryptone broths.
2. Incubate at 37oC for 24 hours.
3. Enter the number of confirmed tubes at the podium computer.
One person from each group will have to come at the assigned time to obtain their results.
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Microbiology Lab-2014
Exercise 7.1 PETRIFILM ASSAY
1. Determine the number/mL of coliforms (red colonies + gas)
+ (Blue colonies + gas) as well as the number of E.coli
(Blue colonies + gas).
2. Obtain the data for the four water samples distributed in
the class.
MICROBIOLOGICAL STANDARDS FOR WATER QUALITY IN ONTARIO:
Total coliform bacteria:
Escherichia coli:
Fecal Streptoccoci:
Drinking water
0/100mL
0/100mL
0/100mL
102
Recreational waters
100/100mL
100/100mL
N/A
Microbiology Lab-2014
MICROBIOLOGICAL QUALITY OF FOOD
Exercise 7.4 AEROBIC COLONY COUNT
1. Obtain the aerobic colony counts for the fresh and stored lettuce and chicken.
Exercise 7.5 INDICATOR ORGANISMS – E. coli
1. Obtain the E. coli counts for the fresh and stored lettuce
and chicken. E. coli colonies are typically green
metallic in color.
Exercise 7.6a SPECIFIC PATHOGENS -SALMONELLA
MPN OF SALMONELLA
1. Obtain all your Rappaport-Vassiliadis broths.
2. Determine the number of positive tubes/ three (for each set of 3) which show growth.
3. Obtain the data for all the chicken samples distributed in the class.
CONFIRMED SALMONELLA TEST
Just as with the water coliform test, the presence of
Salmonella must be confirmed on a selective media such
as Salmonella Shigella agar. Beef Extract, Enzymatic
Digest of Casein, and Enzymatic Digest of Animal Tissue
provide sources of nitrogen, carbon, and vitamins required
for organism growth. Lactose is the carbohydrate present
in Salmonella Shigella Agar. Bile Salts, Sodium Citrate
and Brilliant Green inhibit Gram-positive bacteria and
most coliform bacteria, while allowing Salmonella to
grow. Sodium Thiosulfate and Ferric Citrate permit
detection of hydrogen sulfide by the production of colonies
with black centers.
1. Streak for single colonies a sample from all positive Rappaport-Vassiliadis broths.
2. Incubate at 37oC for 24 hours. (One person from each group will have to come at the
assigned time to obtain their results.)
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Microbiology Lab-2014
Exercise 7.6b SPECIFIC PATHOGENS –S. aureus
1. Obtain the S. aureu counts for the fresh and stored lettuce and
chicken. Colonies of S. aureus are typically yellow.
Exercise 7.6c SPECIFIC PATHOGENS –Bacillus cereus
1. Obtain the B. cereus counts for the fresh and stored lettuce and
chicken. Typical colonies of B. cereus have an irregular form,
are approximately 5mm in diameter and turquoise blue in color
surrounded by an egg yolk precipitate of the same color.
Exercise 7.6d SPECIFIC PATHOGENS –Campylobacter
1. Obtain the Campylobacter counts for the fresh and stored
lettuce and chicken.
2. Perform a Gram stain to confirm the identity of Campylobacter.
Typically, these bacteria are curved Gram negative rods.
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Microbiology Lab-2014
LAB #8
BACTERIAL DIAGNOSTICS –GRAM NEGATIVE BACTERIA
Day 1
The Enterobacteriaceae
Bacteria of the Enterobacteriaceae family are amongst the microorganisms most frequently
found within clinical specimens. The Enterobacteriaceae represent a large and diverse family
commonly referred to as enteric fermenting Gram negative bacilli, indicating that they are Gram
negative rods capable of fermenting sugars. Several members are naturally found within the
natural flora of the intestinal tract of humans and animals. Some infect the intestinal tract.
Members of this family have the following four characteristics in common:
1.
2.
3.
4.
They are Gram negative rods
They are facultative anaerobes
They are oxidase negative
They all ferment glucose but vary greatly with regards to their biochemical characteristics
The Pseudomonaceae
In contrast to the Enterobacteriaceae, the Pseudomonaceae are non-fermenting Gram negative
rods. Non-fermenting Gram negative rods are natural residents of soil and water. They may
cause infections in humans when they colonize immunocompromised individuals or they obtain
access to the interior of the body following a trauma. The most common Gram negative non
fermenting rod which causes infections in humans is Pseudomonas aeruginosa.
The Nesseriaceae
This family includes the genera Neisseria and Moraxella Gram negative diplococci non motile
organisms. All members of this family are oxidase-positive — a key biochemical test to identify
members from this family. The genus Neisseria includes several saprophytic species which are
found in the natural flora of the mucus membranes of the respiratory and genital tracts. Two
pathogenic species are: N. gonorrhoeae (causative agent of gonorrhea) and N. meningitidis
(cause of meningitis).
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Microbiology Lab-2014
DIAGNOSIS OF GRAM NEGATIVE UNKNOWNS
In this exercise, you will have to identify the Gram negative unknown you isolated from the
infected potatoes as well as confirm the identity of a Gram negative bacteria which we supplied.
Exercise 8.0
Materials (Per groups of 2)
TSB culture of a known Gram negative oxidase negative bacterium
Single colonies on a TSA plate of potato isolate
2 MacConkey plates
2 Simmons citrate agar slants
2 Urea agar slants
2 TSI agar slants
2 SIM tubes
2 decarboxylase broths without amino acids
2 decarboxylase broths with ornithine
2 decarboxylase broths with lysine
2 MR-VP broths
2 Phenol red lactose broths
2 Phenol red sucrose broths
2 Phenol red glucose broths
Enteropluri-test
Methods: Perform all of the following tests on each of your 2 Gram negative bacteria (Per
groups of 2)
GRAM STAIN
1. Perform a Gram stain of each of your bacteria.
2. Take a digital picture for your report.
3. Record the Gram reaction, cell shape and aggregation.
OXIDASE
1. Do the oxidase test on the plates of the unknown bacteria you isolated.
GROWTH ON MACCONKEY
1. Streak both of your bacteria for single colonies on MacConkey plates and incubate at 37oC.
USE OF CITRATE AS A CARBON SOURCE
1. Streak both of your bacteria on Simmons citrate agar slants and incubate at 37oC.
UREASE
1. Streak both of your bacteria on urea agar slants and incubate at 37°C.
MR-VP
1. Inoculate each of your bacteria in two MR-VP broths.
2. Incubate at 37°C.
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Microbiology Lab-2014
PHENOL RED BROTHS
1. Inoculate each of your bacteria in a phenol red sucrose, lactose and glucose broth.
2. Incubate at 37°C.
GROWTH IN TSI MEDIUM (TRIPLE SUGAR IRON)
This growth medium is commonly used to obtain a preliminary identification of bacterial
members of the Enterobacteriaceae family. It contains four different potential carbon sources,
glucose, lactose, sucrose, and proteins, as well as a pH indicator that allows one to discriminate
between the use of proteins or sugars. The fermentation of the sugars as carbon source gives rise
to an acidic reaction, whereas the oxidation of proteins results in an alkaline reaction. The
medium is supplied as a slant, which allows the simultaneous observation of growth under both
aerobic and anaerobic conditions. The surface of the slant provides good aerobic conditions,
whereas the butt is essentially devoid of oxygen thus favoring fermentation or anaerobic
respiration.
Amongst the three sugars, glucose is limiting (0.1%) whereas sucrose and lactose are available in
excess (1.0%). Since all the Enterobacteriaceae can metabolize glucose, this metabolism initially
makes the medium acidic (yellow). For continued growth, after the glucose has been exhausted,
one of the other carbon sources must be used. If neither sucrose nor lactose can be used, the
carbon source which will be metabolized will then be proteins, generating alkaline by-products.
The resulting increase in pH will thus change the medium color from yellow to a neutral or an
alkaline (orange or red respectively. However, if sucrose and/or lactose can be used
anaerobically the acids produced will cause the medium to remain acid (yellow).
In addition to allowing the distinction between the fermentation of different sugars, TSI medium
allows one to determine whether a bacterium can degrade amino acids with a sulphur group, such
as methionine and cysteine. Degradation of these amino acids generates as a by-product
hydrogen sulphide, which reacts with ferrous sulphate in the medium, giving rise to a black
precipitate.
1. Inoculate the surface and the butt of TSI slants with your two bacteria. (See image)
2. Incubate at 37oC.
Stab the inoculation loop down into the
bottom of the butt. As you withdraw the
loop, streak the surface of the slant.
Slant
Butt
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Microbiology Lab-2014
SIM; PRODUCTION OF HYDROGEN SULFIDE, INDOLE AND MOTILITY
H2S PRODUCTION
As with the TSI medium, the degradation of sulphur containing amino acids can be determined
by the production of a black precipitate.
MOTILITY
Some microorganisms have the ability to move with the help of flagella. This characteristic can
easily be observed in semi-solid media in which non-motile bacterial growth is restricted to the
site of inoculation whereas the growth of motile bacteria can be observed to spread beyond the
site of inoculation.
INDOLE PRODUCTION: DEGRADATION OF TRYPTOPHAN
Another amino acid that some microorganisms can use as carbon source is the aromatic amino
acid tryptophan. The degradation of tryptophan generates the by-products pyruvic acid and
indole. Pyruvic acid can then be metabolized as a carbon source whereas indole is secreted
within the growth medium as a waste product. Indole production can be detected by its reaction
with the reagent dimethylaminobenaldehyde (Kovac's reagent).
1.
2.
3.
Obtain tubes of solid SIM medium.
Use an inoculation needle to inoculate each of your bacteria down to the bottom of the tube
along a straight line.
Incubate at 37oC.
ORNITHINE AND LYSINE DECARBOXYLASES
The medium used to verify the presence of different amino acid decarboxylases contains glucose
as a fermentable carbon source as well as the desired amino acid, either ornithine or lysine. The
acid produced from the fermentation of glucose reduces the medium’s pH and causes the pH
indicator in the medium to change color from purple to yellow. In addition, these acidic
conditions stimulate the activity of the decarboxylases. Decarboxylation of lysine generates
cadaverine, whereas the decarboxylation of lysine generates putrescine. These products elevate
the pH changing the indicator’s color from purple to violet. The medium remains acid (yellow),
if the organism does not produce the appropriate enzymes
1.
2.
3.
4.
5.
Inoculate each of your bacteria in decarboxylase broths lacking amino acids.
Inoculate each of your bacteria in decarboxylase broths containing lysine.
Inoculate each of your bacteria in decarboxylase broths containing ornithine.
Overlay all the broths with mineral oil to create anaerobic conditions.
Incubate at 37oC.
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Microbiology Lab-2014
ENTEROPLURI TEST: ENTEROBACTERIACEAE SYSTEM
This test is a 12-sector system containing special culture media that permits identification of the
Enterobacteriaceae and other gram negative, oxidase negative bacteria. The system allows the
simultaneous inoculation of all media present in the sectors and the execution of 15 biochemical
reactions. The microorganism is identified by evaluating the colour change of the different
culture media after 18-24 hours of incubation at 36 ± 1 °C and by a code number obtained from
the biochemical reactions. The combination of reactions obtained permits, with the help of the
codebook to identify significant Enterobacteriaceae from a clinical point of view.
Method (This test is to be done only with the known Gram negative bacteria provided)
1. Remove both caps. The tip of the inoculating wire is under the white cap. Do not flame the
wire.
2. Pick a well isolated colony directly with the tip of the Enteropluri inoculating wire (Figure 1
on next page). A visible amount of inoculum should be seen at the tip and the side of the
wire.
3. Inoculate the Enteropluri test by first twisting wire, then withdrawing wire through all
twelve compartments applying a turning motion (Figure 2 on next page).
4. Reinsert wire (without sterilizing) into Enteropluri tube, using a turning motion through all
compartments, until the notch on the wire is aligned with the opening of the tube (Figure 3 on
next page).
5. Break wire at notch by bending. The portion of the wire remaining in the tube maintains
anaerobic conditions necessary for true fermentation of glucose, production of gas and
decarboxylation of lysine and ornithine.
6. With the broken off part of the wire, punch holes through the film covering the air inlets of
the compartments Adonitol, Lactose, Arabinose, Sorbitol, VP, Dulcitol/PA, Urea, and Citrate
in order to support aerobic growth. (Figure 4 on next page). Replace both caps.
7. Incubate at 37° C until the next lab period with Enteropluri test lying on its flat surface
(Figure 5 on next page).
See the video at the following link:
http://www.youtube.com/watch?feature=player_detailpage&v=l5qHyr1FULE
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Microbiology Lab-2014
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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Microbiology Lab-2014
Day 2
GROWTH ON MACCONKEY
Examine the growth on your MacConkey agar plates to determine whether your bacteria ferment
lactose.
Fermenter
Non Fermenter
UREASE
Examine your urea slants to determine whether your bacteria can use
urea as a carbon source.
USE OF CITRATE AS A CARBON SOURCE
Examine your citrate slants to determine whether your bacteria can use
citrate as a carbon source.
111
Negative Positive
Negative Positive
Microbiology Lab-2014
PHENOL RED BROTHS
Determine whether your bacteria can use and ferment either lactose or sucrose.
GROWTH IN TSI MEDIUM (TRIPLE SUGAR IRON)
Do an analysis of your TSI slants in order to obtain the following information:




What is the reaction of the slant: acid, alkaline or neutral
What is the reaction in the butt : acid, alkaline or neutral
Is there H2S production
o if so, is it produced aerobically, anaerobically or both
Is there any gas accumulation
A
A.
B.
C.
D.
B
C
D
Acid butt and slant + accumulation of gas
Alkaline slant and acid butt
Alkaline butt and alkaline slant + H2S anaerobically
Alkaline slant and neutral butt
Interpretation of results:
An acid slant-acid butt (yellow/yellow) indicates fermentation of lactose and/or sucrose.
An alkaline slant-acid butt (red/yellow) indicates fermentation of glucose only.
An alkaline slant-alkaline butt (red/red) indicates neither sucrose nor lactose were fermented
(non-fermenter).
Cracks, splits, or bubbles in medium indicate gas production.
A black precipitate indicates hydrogen sulfide production.
Typical results
Escherichia coli
Proteus mirabilis
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexneri
Slant
A
K
K
K
K
Butt
A
A
K
A
A
112
Gas
+
-
H2 S
+
+
-
A : Acid (yellow)
K : Alkaline (Red)
Microbiology Lab-2014
GLUCOSE FERMENTATION; PRODUCTION OF MIXED
ACIDS OR ACETOIN
1. To complete the MR and VP tests, transfer 1 mL of the MRVP
broth culture you prepared in the previous lab to each of two new
test tubes.
2. For the MR test, add 2 drops of methyl red to one of the two tubes.
Observe the color at the surface of the broth. A red color indicates
the presence of a large quantity of acids whereas a yellow color
indicates the absence of any acids.
Negative
Positive
Positive
Negative
3. To complete the VP test, add 6 drops of alpha-naphtol to the
second tube.
4. Add 3 drops of KOH to the tube.
5. Mix well and allow the reaction to proceed for 10-15 minutes.
6. Observe if there is a color change. If a red color develops it
indicates the presence of acetoin.
SIM: PRODUCTION OF HYDROGEN SULFIDE, INDOLE AND MOTILITY
Do an analysis of your SIM tubes to obtain the following information:



Are your bacteria motile
Is there production of H2S
Is tryptophan used as a carbon source
o Is there indole production? To obtain this result, add a few drops of Kovacs
reagent. If indole was produced the reagent becomes red
A.
B.
C.
D.
A
B
C
113
D
Motile +H2S
Motile + Indole
Non-motile
No growth
Microbiology Lab-2014
ORNITHINE AND LYSINE DECARBOXYLASES
LDB : Medium with amino acid (ornithine or lysine)
DCB : Medium without amino acid
1. Negative result : Alkaline reaction in the presence and absence of amino acids
2. Positive result : Alkaline reaction with the amino acid but an acid reaction in its absence
3. Negative result : Acid reaction with or without amino acids
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Microbiology Lab-2014
ENTEROPLURI TEST: ENTEROBACTERIACEAE SYSTEM
Results and interpretation:
At the end of incubation:
 Observe the change in colour of culture media in the different sectors and interpret results
using the table below. NOTE: if there is no change in colour in the sector Glucose/Gas
while in some other sectors there are color changes, the microorganism being tested does
not belong to the family of Enterobacteriaceae.
 Record the results obtained on the data chart; except for the Indole (sector H2S/Indole) and
Voges-Proskauer tests (sector VP).
Sector
Glucose/Gas
Lysine
Ornithine
H2S/Indole
Adonitol
Lactose
Arabinose
Sorbitol
VP
Dulcitol/PA
Urea
Citrate

Sector colors
Positive reaction
Negative reaction
Glucose fermentation
Yellow
Red
Gas production
Lifted wax
Overlaying wax
Lysine decarboxylation
Violet
Yellow
Ornithine decarboxylation
Violet
Yellow
Hydrogen sulfide production
Black-brown
Beige
Indole test
Pink-red
Colorless
Adonitol fermentation
Yellow
Red
Lactose fermentation
Yellow
Red
Arabinose fermentation
Yellow
Red
Sorbitol fermentation
Yellow
Red
Acetoin production
Red
Colorless
Dulcitol fermentation
Yellow
Green
Phenylalanine deamination
Dark brown
Green
Urea hydrolysis
Purple
Beige
Citrate utilisation
Blue
Green
Biochemical reactions
Perform Indole and Voges-Proskauer tests.
o Indole test
 Lay the EnteroPluri-Test with its flat surface pointing upward and, by
punching the plastic film, add 3 or 4 drops of Kovac’s Indole Reagent in
the sector H2S/Indole. The reaction is positive if a pink-red colour develops
in the added reagent within 10-15 seconds.
o Voges-Proskauer test
 Lay EnteroPluri-Test with its flat surface pointing upward and, by punching
the plastic film, add 3 drops of α-naphtol and 2 drops of potassium
hydroxide. The reaction is positive if a red colour develops within 20
minutes.
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Microbiology Lab-2014

Generate the 5-digit code as follows:
1. The 15 biochemical tests are divided into 5 groups each containing 3 tests and each one is
indicated with a positivity value of 4, 2, or 1.
 Value 4 : first test positive in each group (Glucose, Ornithine, Adonitol, Sorbitol, PA)
 Value 2 : second test positive in each group (Gas, H2S, Lactose, VP, Urea)
 Value 1 : third test positive in each group (Lysine, Indole, Arabinose, Dulcitol, Citrate)
 Value 0 : every test negative
2. Adding the number of positive reactions in each group, you will obtain a 5 digit code which,
by the use of the Codebook, allows the identification of the microorganism under
examination as in the following example:
Group 1
Test
4
2
Group 2
1
4
2
Group 3
1
4
Positivity code
Result
Code sum
Numerical
code
Microorganism:
116
2
Group 4
1
4
2
Group 5
1
4
2
1
Microbiology Lab-2014
LAB # 9
IMMUNOLOGICAL DIAGNOSTIC
Day 1
IMMUNOLOGICAL DIAGNOSTIC
Immunologically based diagnostic methods include both indirect methods based on the search in
a patient's serum for specific antibodies against the microbial agent, whether a bacterium, a virus,
a parasite or a fungus responsible for a pathology as well as direct methods which make use of
antibodies to detect the microorganism of interest. These diagnosis methods are essential for
diagnosing infections by microorganisms that are difficult to identify or grow in the lab; in
particular viruses, strict intracellular bacteria and facultative intracellular bacteria. Technically,
these methods consist of detecting, in the patient's serum, an antigen-antibody reaction in which
the antigen is represented by all or part of the infectious microbial agent to be detected and the
antibody is represented by immunoglobulins specific to the infectious microbial agent. Several
immunological assays are routinely used for this purpose. One of the most widely used is the
Enzyme Linked Immunoabsorbant Assay; commonly called the ELISA.
ELISA
This assay is a two-component system. The first component relies on the use of antibodies that
can specifically recognize and bind to the agent that you want to detect, the primary antibody.
The second component is based on an enzyme conjugated to a second antibody, which will
generate a visible colored product.
There are several variations of the ELISA protocol. In the following exercise we will perform
what is referred to as a direct ELISA to detect the presence of an infectious agent (the antigen).
Briefly, this procedure is performed as follows: First, the proteins within the sample containing
the agent to be detected are fixed on a solid matrix such as plastic. Antibodies that can
specifically recognize and bind the antigen of interest are then added. If the matrix has the
antigen bound to it, then some of the antibodies will attach to the antigen that is fixed onto the
matrix. Thus, both the antigen and the antibody bound to it are now immobilized onto a solid
support. The goal is to then determine whether any antigen specific antibodies are now
immobilized. The detection of the antigen specific antibody is performed using a secondary
antibody (the detecting antibody) that can specifically recognize and bind to the constant region
of the first antibody. This second antibody has an enzyme linked to it. Thus, if the primary
antibody was immobilized as a result of its binding to the antigen, then the second antibody and
the enzyme linked to it will also be immobilized. A colorless substrate is then added that can be
cleaved by the enzyme that is linked to the second antibody. The product resulting from the
enzymatic cleavage of the substrate is colored and can thus be quantified with a
spectrophotometer. The amount of color detected is directly proportional to the quantity of
enzyme that was immobilized.
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Microbiology Lab-2014
Step 1: Binding of antigen to solid support.
Step 2: Binding of primary antibody.
Step 3: Binding of enzyme conjugated secondary antibody.
Step 4: Conversion of colorless substrate to a colored product.
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Microbiology Lab-2014
Exercise 9.0
In this simulation you will evaluate factors associated with the spread of HIV. Each group
begins the simulation with an “uninfected” solution. They sequentially mix their solution with a
variable number of solutions representing potential mates, some of which contain an antigen
used to represent HIV. Groups will then evaluate the final solution with a modified ELISA
procedure to screen for the antigen simulating HIV. These results will then be analyzed to
reconstruct the spread of “HIV” through the population. The results are evaluated in light of the
impact of two risk factors: number of partners, and partners’ sexual history.
Materials (Per groups of 2)
Tube of personal bodily fluids (labelled with group number)
Positive bodily fluids
Negative bodily fluids
Wash bottle (PBS)
Primary antibody (simulated sheep anti-HIV)
Secondary antibody conjugated to horse radish peroxidase (Mouse IgG anti - sheep IgG)
Substrate for peroxidase
96 wells plate
0.2 mL Micropipette
1 mL Micropipette
Method
Simulated Exchange of Bodily Fluids Associated with Sexual Contact
Two sets of labeled tubes (A-J or K-T) have been set out (one at the bench closest to the
windows and one at the front on the bench to the right of the podium) to represent potential
sexual partners; one or two of the tubes in each set contains a protein that we will use to
represent HIV. Each group will be given a solution in a microcentrifuge tube which represents
your initial uninfected body fluid (labelled with your group number).
Groups 1-8 will perform exchanges with the simulated partners in the set of labelled tubes at the
front of the lab and must follow the directives indicated on page 116.
Groups 9-16 will perform the exchanges with the simulated partners in the set of labelled tubes
at the bench closest to the windows and must follow the directives indicated on page 117.
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Microbiology Lab-2014
Directives for groups 1-8
Exchanges will be performed in a specifically designated order according to your group number.
Therefore, group 1 will be the first group to perform their exchanges, followed by group 2 and so
on and so forth. If each group does not exchange fluids in the designated order everyone’s
results will be erroneous and difficult to interpret.
Each group has been randomLy assigned a number of partners with whom they will exchange
bodily fluids. Refer to the table on the next page to determine the number of exchanges your
group has been assigned and the identity of the partners the exchanges are to be performed with.
Identity of partners exchanges were
performed with
First
Second
Third
Group
number
Number of
exchanges
1
3
J
G
2
2
C
F
3
2
A
B
4
3
D
E
5
1
E
6
2
B
7
1
I
8
3
H
ELISA result
Positive or
Negative
F
B
I
C
G
Fluid Exchange
1. When it is your group’s turn to perform the exchange, you and your partner should go with
the tube representing your bodily fluids to the designated area where the tubes of bodily
fluids from different partners are located.
2. Open your microcentrifuge tube and set it in the rack. Using a clean tip, remove 500µL of
solution from your tube; while still holding the pipet with half of your solution ask your
partner to use a second pipettor to remove 500µL of the solution from the randomLy
assigned partner tube indicated in the above table. Dispense your solution into the partner
tube, and the partner’s solution into your tube.
3. Repeat steps 2 as many times as the indicated number of exchanges you are to perform
according to the above table.
4. Once you’ve completed your exchanges, inform the next group in the sequence that they
may proceed.
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Microbiology Lab-2014
Directives for groups 9-16
Exchanges will be performed in a specifically designated order according to your group number.
Therefore, group 9 will be the first group to perform their exchanges, followed by group 10 and
so on and so forth. If each group does not exchange fluids in the designated order everyone’s
results will be erroneous and difficult to interpret.
Each group has been randomLy assigned a number of partners with whom they will exchange
bodily fluids. Refer to the table on the next page to determine the number of exchanges your
group has been assigned and the identity of the partners the exchanges are to be performed with.
Identity of partners exchanges were
performed with
First
Second
Third
Group
number
Number of
exchanges
1
3
T
Q
2
2
M
P
3
2
K
L
4
3
N
O
5
1
O
6
2
L
7
1
S
8
3
R
ELISA result
Positive or
Negative
P
L
S
M
Q
Fluid Exchange
1. When it is your group’s turn to perform the exchange, you and your partner should go with
the tube representing your bodily fluids to the designated area where the tubes of bodily
fluids from different partners are located.
2. Open your microcentrifuge tube and set it in the rack. Using a clean tip, remove 500µL of
solution from your tube; while still holding the pipet with half of your solution ask your
partner to use a second pipettor to remove 500µL of the solution from the randomly assigned
partner tube indicated in the above table. Dispense your solution into the partner tube, and
the partner’s solution into your tube.
3. Repeat steps 2 as many times as the indicated number of exchanges you are to perform
according to the above table.
4. Once you’ve completed your exchanges, inform the next group in the sequence that they
may proceed.
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ELISA
1. Transfer 50 µL of shared fluid into each of 3 wells of an ELISA plate.
2. Transfer 50 µL of the positive control into each of 3 wells of an ELISA plate.
3. Transfer 50 µL of the negative control into each of 3 wells of an ELISA plate.
4. Let the plate sit undisturbed on the bench for 15 minutes.
5. Shake off the fluid into the sink. To empty the wells completely, tap the plate against the side
of the sink.
6. Use the squeeze bottle of wash solution to wash the wells, and then remove the wash solution
as indicated previously.
7. Repeat the washing step two more times.
8. After washing the wells three times, tap the plate firmLy upside down on some paper towels
to remove any remaining fluid. When you finish the wash procedure, there should be no
more liquid appearing on the paper towel when you tap the plate.
9. Using a new pipette tip, add 50 µL of the primary antibody solution to each well. Let the
plate sit on the bench for 15 minutes.
10. Shake off the fluid and wash the plate as in steps 6-7 above.
11. Using a new pipette tip, add 50 µL of the secondary antibody solution to each well. Let the
plate sit on the bench for 15 minutes.
12. Shake off the fluid and wash the plate as in steps 6-7 above.
13. Last step: Using a new pipette tip, add 50 µL of color reagent to each well. Let plate sit on
bench for 10 minutes
14. Observe the results. Negative wells (“uninfected” and the negative controls) will remain
totally clear. Positive wells (“infected” and the positive controls) will turn blue.
Recording results:
Record your result for the shared bodily fluids as being either positive or negative in the table on
the previous page. Then record your results at the computer on the front podium. The results for
the whole class will be made available on this course’s web site.
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Microbiology Lab-2014
Epidemiological Practice Data
Our goal is to use the data summarized in the table below, which includes the results of the
ELISA tests, and the sexual history of the group, in order to track the path of the epidemic. This
data will allow you to uncover some of the steps in the spread of the infection.
Look at the negative results to make a list of exchange partners whom you know were not
initially infected. Now consider the ELISA results of student A3. What does this tell you about
one source of the epidemic? Draw a circle around one source of the infection, and squares
around partners subsequently infected from this source. How many partners did Student A1 and
Student A10 individually have sex with and what was the identity of these partner(s)?
Individual Behavior:
A1:
A10:
Exposure to sexually transmitted diseases is a result of the individual’s sexual history, as well as
the history of all their partners, and their partner’s partners. Compare the exposure to STDs of
Student A1 in contrast to A10. List the number of individuals that each has been directly and
indirectly exposed to.
Exposure Risk:
A1:
A10:
Student ID
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
Exchange partners
ELISA result
1st 2nd 3rd 4th
3
Neg.
6
4
1
Neg.
7
3
Pos.
4
5
3
Pos.
10 7
4
5
Pos.
2
Neg.
7
2
Pos.
8
9
Neg.
10 2
5
7
Pos.
3
Pos.
9
4
Pos.
4
6
Pos.
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Microbiology Lab-2014
IMMUNOCHROMATOGRAPHY
Immunochromatographic assays, also called lateral flow tests, represent a quick qualitative test
used for diagnostic purposes. As with the ELISA, the test is based on the specific antibody
antigen recognition principal. For most of these tests, the binding of a ligand to a visually
detectable solid support, such as dyed microspheres is detected. Some of the more common
lateral flow tests currently on the market are tests for pregnancy, Strep throat, and Chlamydia.
As shown below, the sample to be tested (the anylate) is applied to one end of a chromatographic
strip. The sample then migrates by capillary action through an area containing antibodies
conjugated to colored particles. If the sample contains antigen recognized by the antibodies,
some of these will complex. If no antigen is present, then the antibody will remain unbound. In
either case, the sample then continues to migrate across the membrane until it reaches the capture
zone where a second antibody against the antigen of interest is present. If antibody-antigen
complexes occurred at the first stage, then these will be captured by the second antibody
producing a visible line. If no complex were formed, then the sample will continue its migration
without being captured. The sample then migrates further along the strip until it reaches the
control zone, where excess conjugate will be captured by a second antibody whose antigen is the
antibody conjugated to the colored microparticles and produce a second visible line on the
membrane. This control line indicates that the sample has migrated across the membrane as
intended. Two clear lines on the membrane is a positive result. A single line in the control zone
is a negative result.
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Microbiology Lab-2014
The OSOM Strep A Test uses color immunochromatographic dipstick technology with rabbit
antibodies coated on the nitrocellulose membrane. In the test procedure, a throat swab is
subjected to a chemical extraction of a carbohydrate antigen unique to Group A Streptococcus.
The Test Stick is then placed in the extraction mixture and the mixture migrates along the
membrane. If Group A Streptococcus is present in the sample, it will form a complex with the
anti-Group A Streptococcus antibody conjugated color particles. The complex will then be
bound by the anti-Group A Streptococcus capture antibody and a visible blue Test Line will
appear to indicate a positive result
Exercise 9.1
IDENTIFICATION OF GROUP A STREPTOCOCCI
1. Pick up 1–3 suspect colonies on the chocolate agar plate using a sterile swab.
2. Just before testing, add 3 drops Reagent 1 (pink) and 3 drops Reagent 2 to a Test Tube (the
solution should turn light yellow).
3. Immediately put the swab into the Tube.
4. Vigorously mix the solution by rotating the swab forcefully against the side of the Tube at
least ten (10) times. Let stand for 1 minute.
5. Express as much liquid as possible from the swab by squeezing the sides of the tube as the
swab is withdrawn.
6. Discard the swab.
7. Obtain a Test Stick and place the Absorbent End of the Test Stick into the extracted sample.
8. Read results at 5 minutes or as soon as the red Control Line appears.
Interpretation of results:
Positive
A blue Test Line and a red Control Line is a positive result for the detection of Group A
Streptococcus antigen.
Negative
A red Control Line but no blue Test Line is a negative result.
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Microbiology Lab-2014
Metric Units
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Microbiology Lab-2014
GROWTH MEDIA COMPOSITION
TSA or TSB
Tryptone
Soytone - digested soya proteins
NaCl
Agar (only for TSA)
Phenol Red Broth
Casein
NaCl
Phenol red
Desired sugar
MRS Broth
Peptone
Meat extract
Yeast extract
Glucose
Sodium acetate trihydrate
Polysorbate 80
Dipotassium hydrogen phosphate
Triammonium citrate
Magnesium sulfate heptahydrate
Manganese sulfate tetrahydrate
Nitrate Broth
Digested animal proteins
Beef extract
Potassium nitrate
MacConkey Agar
Peptone
Proteose peptone
Lactose
Bile salts
NaCl
Neutral red
Agar
CNA of Colombia
Casein
Digested animal tissues
NaCl
Yeast extract
Beef extract
Corn starch
Colistin sulfate
Nalidixic acid
Agar
Nutrient Agar (NA)
Peptone
Yeast extract
NaCl
Agar
MRVP
Digested animal proteins
Casein
Glucose
Potassium phosphate
Urea Agar
Gelatine peptone
Glucose
Potassium dihydrogen phosphate
NaCl
Phenol red
Agar
Simmon's Citrate
Magnesium sulfate
Ammonium dihydrogen phosphate
Dipotassium phosphate
Sodium citrate
NaCl
Bromothymol blue
Agar
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Microbiology Lab-2014
DNA Medium
Casein
Proteose Peptone
Desoxyribonucleic acids
NaCl
Agar
Methyl green
Spirit Blue
Casein
Yeast extract
Agar
Spirit Blue
Lipid suspension
Chocolat Agar
Peptone
Meat extract
NaCl
Boiled blood
BloodAgar
Proteose peptone
Liver extract
Yeast extract
NaCl
Whole blood
Agar
Bile Esculine Agar
Meat extract
Meat peptone
Bile
Ferric citrate
Esculine
Agar
Endo Agar
Peptone
Dipotassium hydrogen phosphate
Lactose
Sodium Sulfite
Fuchsine
Agar
Mannitol + Salts Agar
Casein
Animal tissue extract
Beef extract
Mannitol
NaCl
Phenol red
Agar
Tellurite Agar
Proteose Peptone
Beef extract
NaCl
Tellurite
Lauryl Tryptose Broth
Tryptone
Glucose
Dipotassium phosphate
Monopotassium phosphate
NaCl
Sodium azide
Bromcresol purple
EMB Agar
Peptones
Lactose
Methylene blue
Eosin Y
Agar
Buffer
Rapport Vassiliadis Broth
Peptone from soya
NaCl
Potassium dihydrogen phosphate
Dipotassium phosphate
Magnesium chloride
Malachite green
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Microbiology Lab-2014
SS Agar
Peptones
Lactose
Bile
Sodium citrate
Thiosulfate
Ammonium iron(III) citrate
Brilliant green
Neutral red
Bromothymol blue
NaCl
Agar
PEMBA
Casein
Egg yolk emulsion
Mannitol
Polymyxin B
NaCl
Magnesium sulfate
Disodium Phosphate
Monopotassium Phosphate
Bromthymol blue
Pyruvate
Agar
Campy-CVA
Casein
Glucose
Digested animal tissues
Yeast extract
NaCl
Sheep blood
Sodium bisulfite
Agar
Cefoperazone
Vancomycine
Amphotericin B
Decarboxylase broth
Digested animal tissues
Beef extract
Glucose
Pyridoxal
Cresol red
Bromcresol purple
TSI
Meat extract
Yeast extract
Peptone
Lactose
Sucrose
Glucose
NaCl
Ferric Sulfate
Sodium thiosulfate
Phenol red
Agar
SIM
Beef extract
Digested animal tissues
Peptonized iron
Sodium thiosulfate
Agar
129