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http://mysite.science.uottawa.ca/jbasso/microlab/home.htm
Microbiology Lab-2016
Contents
GENERAL DIRECTIVES ............................................................................................................................................. 4
GRADING SCHEME ................................................................................................................................................. 5
SCHEDULE .............................................................................................................................................................. 6
O
LAB N 1 ................................................................................................................................................................ 7
DILUTIONS AND CONCENTRATIONS ..........................................................................................................................7
PERCENTAGE .............................................................................................................................................................7
MOLARITY .................................................................................................................................................................9
WEIGHT/VOLUME .....................................................................................................................................................9
RATIOS ......................................................................................................................................................................9
DILUTIONS...............................................................................................................................................................10
EXERCISE 1.0: GENERATING A STANDARD CURVE AND DETERMINING AN UNKNOWN CONCENTRATION OF
METHYLENE BLUE (Groups of 2).........................................................................................................................13
DIFFUSION...............................................................................................................................................................15
OSMOSIS .................................................................................................................................................................15
TONICITY .................................................................................................................................................................15
OSMOLARITY ...........................................................................................................................................................16
OSMOLARITY VS TONICITY ......................................................................................................................................16
EXERCISE 1.1: DIFFUSION, OSMOSIS AND TONICITY IN RED BLOOD CELLS (Groups of 2) ..................................17
MICROBIAL GROWTH IN THE LAB ............................................................................................................................18
MICROBIOLOGICAL MEDIA .....................................................................................................................................18
INOCULATING SOLID MEDIA: SPREADING ...............................................................................................................19
EXERCISE 1.2: VIABLE COUNTS OF A SOIL SAMPLE (Groups of 2) .......................................................................20
INOCULATING SOLID MEDIA: STREAKING ...............................................................................................................20
INOCULATING SOLID MEDIA: STREAKING FOR SINGLE COLONIES ...........................................................................25
EXERCISE 1.3: STREAKING FOR SINGLE COLONIES (Individually) ........................................................................27
O
LAB N 2 .............................................................................................................................................................. 28
DETERMINING THE NUMBER OF MICROORGANISMS – VIABLE COUNTS ................................................................28
VIABLE COUNTS OF A SOIL SAMPLE .........................................................................................................................28
EXERCISE 2.0: BACTERIAL COUNTS IN SOIL (Groups of 2) ..................................................................................28
EXERCISE 2.1: COUNTS OF ACTINOMYCETES IN SOIL (Groups of 2) ...................................................................29
FUNGI ......................................................................................................................................................................29
EXERCISE 2.2: COUNTS OF FUNGI IN SOIL (Group of 2) ......................................................................................29
MOST PROBABLE COUNTS ......................................................................................................................................27
EXERCISE 2.3: MPN OF BACTERIA IN SOIL (Groups of 2) ....................................................................................24
DIRECT COUNTS (HAEMOCYTOMETER SLIDE) .................................................................................................................31
EXERCISE 2.4: DIRECT COUNT OF A YEAST SUSPENSION (Groups of 2) ..............................................................32
VIEWING MICROORGANISMS .................................................................................................................................33
MACROSCOPIC VISUALIZATION – COLONY MORPHOLOGIES ..................................................................................33
EXERCISE 2.5: COLONY MORPHOLOGIES (Groups of 2) ......................................................................................34
MICROSCOPIC VISUALIZATION ................................................................................................................................35
EXERCISE 2.6: FAMILIARISATION WITH THE USE OF THE MICROSCOPE (Groups of 2) .......................................38
MICROSCOPIC VISUALIZATION OF BACTERIA – SIMPLE STAINS ..............................................................................39
EXERCISE 2.7: SIMPLE STAINS (Groups of 2) .......................................................................................................39
MICROSCOPIC EXAMINATION OF FUNGI – SIMPLE STAINS .....................................................................................42
EXERCISE 2.8: SIMPLE STAINING (Groups of 2) ..................................................................................................42
MICROSCOPIC VISUALISATION - GRAM STAINING ..................................................................................................45
EXERCISE 2.9: GRAM STAINING (Groups of 2) ....................................................................................................46
MICROSCOPIC VISUALISATION - ACID-FAST STAINING ............................................................................................46
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Microbiology Lab-2016
EXERCISE 2.10: ACID FAST STAINING (Groups of 2) ............................................................................................47
MICROSCOPIC VISUALISATION – SPORE STAINING .................................................................................................47
O
LAB N 3 .............................................................................................................................................................. 49
GROWTH OF BACTERIA - GROWTH CURVE..............................................................................................................49
EXERCISE 3.0: E.COLI GROWTH CURVE (Groups of 2).........................................................................................49
EXERCISE 3.1: MPN OF BACTERIA IN SOIL - CONTINUED (Groups of 2) ..............................................................30
YEAST FERMENTATION ...........................................................................................................................................50
EXERCISE 3.2: YEAST FERMENTATION BIOASSAY (Groups of 2) .........................................................................51
BIOFILMS .................................................................................................................................................................53
EXERCISE 3.3: EFFECT OF GROWTH CONDITIONS ON BIOFILM FORMATION (Groups of 2)...............................53
O
LAB N 4 .............................................................................................................................................................. 54
BIOFILMS – CONTINUED ..........................................................................................................................................54
EXERCISE 4.0: QUANTIFICATION OF BIOFILMS (Groups of 2) .............................................................................54
CONTROL OF MICROBIAL GROWTH - ANTIBIOTICS .................................................................................................54
KIRBY-BAUER DISC DIFFUSION METHOD.................................................................................................................54
EXERCISE 4.1: KIRBY-BAUER ASSAY (Groups of 2) ..............................................................................................55
E-TEST .....................................................................................................................................................................56
EXERCISE 4.2: SENSITIVITY OF S. FAECALIS TO VANCOMYCIN (Groups of 2) ......................................................56
DETERMINING THE THERAPEUTIC DOSE .................................................................................................................57
EXERCISE 4.3: DETERMINING THE MIC (Groups of 2) .........................................................................................57
DISINFECTANTS & ANTISEPTICS ..............................................................................................................................59
EXERCISE 4.4: EFFECT OF MOUTH WASHES ON BIOFILM OF S. MUTANS (Groups of 2) ....................................60
DEATH KINETICS ......................................................................................................................................................61
EXERCISE 4.5: EFFICACY OF A DISINFECTANT - THE D VALUE (Groups of 2) .......................................................61
O
LAB N 5 .............................................................................................................................................................. 63
CONTROL OF MICROBIAL GROWTH - CONTINUED ..................................................................................................63
EXERCISE 5.0: TTC ASSAY – PENICILLIN CONCENTRATION IN MILK (Groups of 2) ..............................................64
TOXICOLOGY AND INDICATOR MICROORGANISMS ................................................................................................65
EXERCISE 5.1: BIOASSAY FOR THE DETERMINATION OF THE LD50 OF HEAVY METALS (Groups of 2) ...............66
EXERCISE 5.2: KIRBY BAUER DIFFUSION ASSAY .......................................................................................................68
EXERCISE 5.3: SENSITIVITY OF S. FAECALIS TO VANCOMYCIN - E-TEST (Groups of 2) ........................................69
EXERCISE 5.4: DETERMINATION OF THE THERAPEUTIC DOSE – MIC (Groups of 2) ...........................................69
EXERCISE 5.5: EFFECT OF MOUTH WASHES ON BIOFILMS OF S. MUTANS (Groups of 2)...................................70
EXERCISE 5.6: EFFICACY OF A DISINFECTANT - DETERMINATION OF THE D VALUE (Groups of 2) .....................70
O
LAB N 6 .............................................................................................................................................................. 71
BACTERIAL METABOLISM AND DIFFERENTIAL TESTS ..............................................................................................71
UTILIZATION OF COMPLEX CARBON SOURCES: EXOCELLULAR ENZYMES ...............................................................72
EXERCISE 6.0: DEGRADATION OF COMPLEX CARBON SOURCES (Groups of 2) ..................................................72
SUGAR METABOLISM – PHENOL RED BROTH ..........................................................................................................73
EXERCISE 6.1: METABOLISM IN PHENOL RED BROTH (Groups of 2) ..................................................................73
GROWTH IN TSI MEDIUM (TRIPLE SUGAR IRON) .....................................................................................................73
EXERCISE 6.2: GROWTH IN TSI MEDIUM (Groups of 2) ......................................................................................74
USE OF CITRATE AS A CARBON SOURCE ..................................................................................................................75
EXERCISE 6.3 GROWTH ON SIMMON’S CITRATE SLANT (Groups of 2) ...............................................................75
UREA METABOLISM ................................................................................................................................................75
EXERCISE 6.4: GROWTH ON UREA SLANT (Groups of 2).....................................................................................75
DECARBOXYLASES AND DEAMINASES .....................................................................................................................76
EXERCISE 6.5: DECARBOXYLASE AND DEAMINASE ASSAYS (Groups of 2) ..........................................................76
SIM: PRODUCTION OF HYDROGEN SULFIDE, INDOLE AND MOTILITY .....................................................................77
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Microbiology Lab-2016
EXERCISE 6.6: SIM TEST (Groups of 2) ................................................................................................................77
NITRATE AND NITRITE REDUCTION .........................................................................................................................78
EXERCISE 6.7: NITRATE REDUCTION ASSAY (Groups of 2) ..................................................................................78
EXERCISE 6.8: ENTEROPLURI TEST (Groups of 2) ................................................................................................79
THE STREPTOCOCCI AND THE STAPHYLOCCOCI.......................................................................................................81
BLOOD HEMOLYSIS .................................................................................................................................................81
EXERCISE 6.9: THROAT SAMPLING ON BLOOD AGAR PLATES (Groups of 2) ......................................................82
O
LAB N 7 ............................................................................................................................................................... 83
DIFFERENTIAL TESTS –CONTINUED .........................................................................................................................83
EXERCISE 7.0: DEGRADATION OF COMPLEX CARBON SOURCES (Groups of 2) ..................................................83
EXERCISE 7.1: METABOLISM IN PHENOL RED BROTH (Groups of 2) ..................................................................84
EXERCISE 7.2: GROWTH IN TSI (Groups of 2) .....................................................................................................85
GLUCOSE FERMENTATION; PRODUCTION OF MIXED ACIDS OR ACETOIN ..............................................................87
EXERCISE 7.3: METHYL RED - VOGUES-PROSKAUER TEST (MRVP) (Groups of 2) ...............................................87
EXERCISE 7.4 GROWTH ON SIMMON’S CITRATE AGAR SLANT (Groups of 2) .....................................................87
EXERCISE 7.5: GROWTH ON UREA SLANT (Groups of 2).....................................................................................88
EXERCISE 7.6: DECARBOXYLASE AND DEAMINASE ASSAYS (Groups of 2) ..........................................................88
EXERCISE 7.7: SIM TEST (Groups of 2) ................................................................................................................89
EXERCISE 7.8: NITRATE REDUCTION ASSAY (Groups of 2) ..................................................................................90
EXERCISE 7.9: ENTEROPLURI TEST (Groups of 2) ................................................................................................91
EXERCISE 7.10: BLOOD HEMOLYSIS (Groups of 2) ..............................................................................................94
EXERCISE 7.11: CATALASE (Groups of 2) ............................................................................................................94
BILE-ESCULIN...........................................................................................................................................................95
BACITRACIN, OPTOCHIN AND NOVOBIOCIN SENSITIVITY .......................................................................................95
MANNITOL + SALTS AGAR .......................................................................................................................................95
TELLURITE AGAR OR BAIRD PARKER AGAR ..............................................................................................................96
PYR TEST ..................................................................................................................................................................96
EXERCISE 7.12: DIFFERENTIAL STAINS AND STERILIZATION (Groups of 2) .........................................................97
O
LAB N 8 .............................................................................................................................................................. 99
IMMUNOLOGY ........................................................................................................................................................99
LYSOZYME .............................................................................................................................................................101
EXERCISE 8.0: PURIFICATION OF LYSOZYME FROM EGGS (Groups of 2) ..........................................................101
EXERCISE 8.1: LYSOZYME ASSAY (Groups of 2) .................................................................................................102
IMMUNOLOGICAL DIAGNOSTIC ............................................................................................................................103
ELISA .....................................................................................................................................................................103
EXERCISE 8.2: ELISA OF LYSOZYME (Groups of 2) .............................................................................................104
EXERCISE 8.3: LEUKOCYTE COUNTS (Groups of 2) ............................................................................................106
METRIC UNITS.................................................................................................................................................... 107
GROWTH MEDIA COMPOSITION ........................................................................................................................ 108
3
Microbiology Lab-2016
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
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.
4
Microbiology Lab-2016
GRADING SCHEME
Quiz
2 bonus points for 100% on 4/8 quizzes
Assignments
20%
Midterm Exam
30%
Practical Final Exam
10%
Final Exam
40%
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 lab includes 4 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.
Midterm: A midterm exam will be given during lab hours at the date specified in the schedule
on page 6. The midterm will consist of 30 short answers and multiple choice questions given
over a 2 hour period. You will be allowed to bring a single one sided cheat sheet (8 1/2 X 11 in.),
scrap paper, and calculators.
Practical final exam: For this exam, you will have to come on an individual basis for a 2.5 hour
period to perform techniques commonly used during the semester. The tasks you will have to
perform include a Gram stain and the identification of an unknown, a streaking for single
colonies, a viable count and a direct count. This is an open book exam and you are therefore
allowed any printed resource.
Theoretical final exam: A final exam, which is cumulative, will be given during lab hours at the
date specified in the schedule on page 6. The final exam which is cumulative will consist of 40
short answer and multiple choice questions given over a 3 hour period. You will be allowed to
bring a single one sided cheat sheet (8 1/2 X 11 in.), scrap paper, and calculators.
5
Microbiology Lab-2016
SCHEDULE
Date
Lab 1
Sept. 12 (sec. A) or 14 (sec. B)
Lab 2
Sept. 19 (sec. A) or 21 (sec. B)
Lab 3
Sept. 26 (sec. A) or 28 (sec. B)
Lab 4
Oct. 3 (sec. A) or 5 (sec. B)
Thanksgiving
Oct. 10 – 14 NO LABS
Midterm exam
Oct. 17 (sec. A) or 19 (sec. B)
Study break
Oct. 24 - 28
Lab 5
Oct. 31 (sec. A) or Nov. 2 (sec. B)
Lab 6
Nov. 7 (sec. A) or 9 (sec. B)
Lab 7
Nov. 14 (sec. A) or 16 (sec. B)
Lab 8
Nov. 21 (sec. A) or 23 (sec. B)
Practical exam
Nov. 28 (sec.A) or 30 (sec. B)
Theoretical final exam
Dec. 5 (sec.A) or 7 (sec. B)
6
Due dates
Assignment 1
Assignment 2
Assignment 3
Assignment 4
Microbiology Lab-2016
LAB NO 1
DILUTIONS AND CONCENTRATIONS
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
7
x 100
Microbiology Lab-2016
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.
8
Microbiology Lab-2016
MOLARITY
Another way of expressing concentrations is called
molarity. Molarity is the number of moles of solute
moles of solute
dissolved in one liter of solution. The units, Molarity =
liter of solution
therefore are moles per liter, specifically it's moles
of solute per liter 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 1 L, 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.
9
Microbiology Lab-2016
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.
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 liter 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 or dilution.
10
Microbiology Lab-2016
Performing serial transfers: When performing serial transfers as in serial dilutions use a
different pipette for each dilution. Follow the following directives:
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). Using a new pipette, pipette
the desired volume from this tube (“A”) and dispense into the next tube (“B”). Repeat the
process from “A” to “B” with the new pipette.
A
B
C
Dispense and rinse pipette in this tube before changing
pipettes for the uptake of the desired volume from this new
solution.
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.
11
Microbiology Lab-2016
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 the final concentration you
want is (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.
12
Microbiology Lab-2016
EXERCISE 1.0: GENERATING A STANDARD CURVE AND DETERMINING AN
UNKNOWN CONCENTRATION OF METHYLENE BLUE (Groups of 2)
Materials
Methylene blue solution (0.26% m/v, M.W. 320g/mole)
Methylene blue solution of unknown concentration
100 mL of water
Test tubes
Method
1. From the stock solution of methylene blue, prepare 5 mL of a methylene blue solution at a
final concentration of 0.4 mM. Label this tube No1. Make sure to write down how this
solution was prepared.
2. Add to a test tube, labelled No2, 1.2 mL of water and 4.8 mL of solution No1.
3. Add to a test tube, labelled No3, 3.0 mL of water and 2.5 mL of solution No2.
4. Add to a test tube, labelled No4, 1.5 mL of water and 2.0 mL of solution No3.
5. Add to a test tube, labelled No5, 0.5 mL of water and 0.8 mL of solution No4.
6. Add to a test tube, labelled No6, 1.0 mL of water and 1.5 mL of solution No5.
7. From the stock solution of methylene blue of unknown concentration, prepare dilutions in a
final volume of 5 mL of 1/4.5 and of 1/8. Label these test tubes UNK 1 and UNK 2
respectively. Make sure to write down how these dilutions were prepared.
8. Transfer 0.1 mL of each of the solutions to the wells of a 96 well plate as indicated below.
9. Obtain the absorbance readings at 550 nm.
Layout of 96 well plate (one plate/table)
Water
Blank
Soln.
No1
Soln.
No2
Soln.
No3
Soln.
No4
Soln.
No5
Soln.
No6
UNK 1
UNK 2
Water
Blank
Soln.
No1
Soln.
No2
Soln.
No3
Soln.
No4
Soln.
No5
Soln.
No6
UNK 1
UNK 2
Water
Blank
Soln.
No1
Soln.
No2
Soln.
No3
Soln.
No4
Soln.
No5
Soln.
No6
UNK 1
UNK 2
Water
Blank
Soln.
No1
Soln.
No2
Soln.
No3
Soln.
No4
Soln.
No5
Soln.
No6
UNK 1
UNK 2
13
Group 1
Group 2
Group 3
Group 4
Microbiology Lab-2016
10. Complete the following table and have it signed by your teaching assistant before the
end of this lab period.
Solution
Volume of
methylene
blue (mL)
Volume
of water
(mL)
Total
volume
(mL)
No1
No2
No3
No4
No5
No6
UNK 1
UNK 2
14
Final
dilution
factor
Abs
550nm
Final concentration
of methylene blue
(% m/v)
Microbiology Lab-2016
DIFFUSION
The internal environment of any organism consists mainly of water-based solutions. Many
solutes may be dissolved in these solutions. Since the movement of compounds across cell
membranes is strongly influenced by the differences in concentrations and by the permeability of
the lipid bilayer, it is important to understand how differences in concentration of a solute
influence passive membrane transport.
The particles in a solution are generally free to move randomly throughout the volume of a
solution. If there is a difference in the concentration of a solute between a region of a solution
and another, the substance will tend to spread to the area where it is more concentrated to the
location where it is less concentrated until such time that the distribution of the compound is
uniform throughout the volume of the solution. Thus the net distribution occurs from a high
concentration area to a low concentration area, until an equilibrium state is reached. The simple
diffusion represents any non-assisted movement of any compound down the concentration
gradient. In the case of cells, solutes which can readily cross the lipid bilayer (such as small
uncharged molecules) are transported across the cell membrane by simple diffusion.
OSMOSIS
The concentration of water in a solution is inversely proportional to the solute concentration - the
higher is the total concentration of solutes in a solution the smaller is the number of water
molecules per unit volume of the solution. By this fact, the water will diffuse from the area with
the lowest solute concentration to the area with the higher concentration of solutes. Thus, the
diffusion of water in or out of the cell is driven by the differences in the total concentration of
solutes that cannot cross the plasma membrane. This movement of the water by diffusion is
referred to as osmosis.
If water flows from one solution to another, the volume of the second solution will tend to
increase, while that of the first will decrease. In the case of a cell, this change in volume will
have the effect of forcing the membrane to stretch to accommodate the increase in volume,
causing a pressure increase within the cell. The osmotic pressure is therefore directly related to
the total concentration of non-permeable solutes of a solution.
TONICITY
Living cells have the potential to make or lose water by osmosis relative to the extracellular
environment. The net movement of water into or out of the cell is caused by differences in the
concentrations of impermeable solutes to the cell. Thus the effect that an extracellular solution
has on the osmotic movement of water into or out of the cell is described by the tonicity of the
extracellular fluid. For example, if the concentrations of impermeable solutes in the intracellular
and extracellular fluids are the same, no net osmosis will occur. In this case the extracellular
solution is said to be isotonic ("equal tonicity"). If a cell is placed in a solution with a greater
concentration of impermeable solutes relative to the intracellular fluid (e.g., human blood cells in
seawater), water will flow out of the cell and into the extracellular fluid, causing the cell to
shrink and crenate. In this case, the extracellular fluid is said to be hypertonic ("greater
tonicity"). Conversely, if a cell is placed in a solution with a concentration of impermeable
solutes which is lower (e.g. distilled water) water will flow into the cell, causing it to swell to the
15
Microbiology Lab-2016
point where the cell may undergo lysis. In this situation, the extracellular fluid is said to be
hypotonic.
OSMOLARITY
Osmosis is driven by differences in the relationship between solutes and solvents that exist
across a semipermeable membrane. The total solute concentration of a solution (and thus the
osmotic concentration) can be quantified by the osmolarity of the solution. Osmolarity is the
ratio of total moles of solute particles per liter of solution. The unit for the concentration
measurement is osmolality (OsM), where 1 OsM is equal to one mole total of solute particles per
liter of solution. If the solution contains a single solute that cannot dissociate, such as glucose,
the osmolarity of the solution is equal to the molarity of the solution. However, if the solution
contains an ionic solute that can dissociate, such as NaCl, this will have a considerable influence
on the osmotic concentration. E.g. NaCl readily dissociates in water into Na + and Cl-. Thus, for
each mole of NaCl in a solution, there will be two moles of solute particles (1 mole of Na + and
1 mole of Cl). Thus the osmolarity of a solution containing 1 mole of NaCl represent a solution
of 2 osmoles.
OSMOLARITY Vs TONICITY
In contrast to osmolarity, tonicity is only influenced by the concentrations of impermeable
solutes. For example, a glucose solution of 300 mM, a urea solution of 300 mM, and a NaCl
solution of 150 mM all have the same osmolarity; 300 mOsM. But if a cell with an internal
concentration of 300 mOsM was placed in each of these solutions, it would behave very
differently. In a 150 mM NaCl solution, the cell would be isotonic and iso-osmotic with respect
to its environment. The osmotic pressure would be equal on both sides of the cell and it would
maintain the same volume. However, in the case of urea, which is highly permeable through
most membranes, the extracellular environment would be iso-osmotic, but hypotonic in relation
to the interior of the cell. Consequently, the cell would quickly swell as a result of the rapid entry
of urea and water.
16
Microbiology Lab-2016
EXERCISE 1.1: DIFFUSION, OSMOSIS AND TONICITY IN RED BLOOD CELLS
(Groups of 2)
Materials
Sheep’s blood- 3 mL
50% (v/v) Glycerol
1 M NaCl
50% (m/v) Sucrose
100 mL of water
Test tubes
Method
1. Prepare three series of 5 test tubes; one series for each solute.
2. For each series, prepare from the stock solutions 2 mL solutions representing the following
solute concentrations:
Series 1:
Sucrose: 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M
Series 2:
NaCl: 0.065 M, 0.1 M, 0.l5 M, 0.25 M, 0.3 M
Series 3:
Glycerol : 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M
3. Add to each test tube, 0.1 mL of sheep’s blood and mix.
4. Wait for 15 to 20 minutes.
5. Verify in which test tubes hemolysis occurred.
17
Microbiology Lab-2016
MICROBIAL GROWTH IN THE LAB
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 gelatin. Its use is less common since many
bacteria can digest it.
Liquid culture 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.
18
Microbiology Lab-2016
INOCULATING SOLID MEDIA: 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.
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
19
Spread bacteria
Microbiology Lab-2016
EXERCISE 1.2: VIABLE COUNTS OF A SOIL SAMPLE (Groups of 2)
Materials
1g of soil
Flask with 100 mL of sterile water
100 mL sterile water
3 sterile test tubes
3 TSA plates
3 plates of Sabouraud Dextrose Agar containing 100 µg/mL chloramphenicol
3 glycerol agar plates with yeast extract
Method
1. Transfer 1g of soil to the flask containing 100 mL of sterile water. (this represents a 10-2
dilution) Shake on shaking platform for 10 minutes.
2. Allow the soil to settle for 10 minutes.
3. Prepare 10-3, 10-4 and 10-5 dilutions of the soil suspension in a final volume of 10 mL. (see
figure below)
4. Plate 0.1 mL from each of the three highest dilutions on 3 appropriately labelled TSA plates.
5. Plate 0.1 mL from each of the three highest dilutions on 3 appropriately labelled Sabouraud
Dextrose Agar plates.
6. Plate 0.1 mL from each of the three highest dilutions on 3 appropriately labelled glycerol
agar plates with yeast extract.
7. Incubate the inverted plates at room temperature until next week.
1g of soil
+
100 mL Water
10-3
10-4
10-5
10-2 Dilution
0.1ml
0.1ml
20
0.1ml
Microbiology Lab-2016
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.
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.
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 1 g/100 mL.
1g of soil terre
+
100 mL water
Dilution 10-2
Inoculum:
Results:
-
1mL of 10-2
+++
0.1mL of 10-2 1mL of 10-4
+++
+-+
21
0.1mL of 10-4
1mL of 10-6
+--
--
Microbiology Lab-2016
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.
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 most positive results, as shown in example “c” and “d”. If
after following these rules, there is a series showing positive results in higher dilutions than the
chosen three, add the result to the highest dilution as in example “e”.
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
3.0
d
1/3 1/3 2/3 0/3
1-2-0
11
e
1/3 2/3 0/3 1/3
1-2-1
15
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 9.3/10-2 = 9.3 X 102.
22
Microbiology Lab-2016
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
23
Microbiology Lab-2016
EXERCISE 1.3: MPN OF BACTERIA IN SOIL (Groups of 2)
Materials
Soil suspension at 1g/mL prepared in the previous exercise
6 sterile tubes
12 X 5 mL broths of TSB
Sterile water
Method
1. Prepare a series of dilutions in a final volume of 10 mL of sterile water representing the
following dilution factors: 104X, 105X, 106X and 107X
105X
104X
1g of soil
+
100 mL Water
106X
107X
10-2 Dilution
2. Perform an MPN count as indicated below in 4 sets of 3 tubes containing 5 ml of TSB broth.
3. Incubate at room temperature until the next lab period.
Soil sample
Volume of inoculum
10-4
10-5
10-6
10-7
1.0 mL
1.0 mL
1.0 mL
1.0 mL
24
Vol. of medium
3 tubes
5 mL
5 mL
5 mL
5 mL
Microbiology Lab-2016
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.
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.
Once it has cooled down, grab some bacteria from the plate culture supplied. Be careful not to
pick up too much.
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.
25
Microbiology Lab-2016
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.
26
Microbiology Lab-2016
EXERCISE 1.4: STREAKING FOR SINGLE COLONIES (Individually)
Materials
Mixed broth culture
Agar plate of E. coli
2 TSA plates
Method
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.
27
Microbiology Lab-2016
LAB NO 2
DETERMINING THE NUMBER OF MICROORGANISMS – 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 involves the growth of
microorganisms on a suitable medium to obtain single colonies. These colonies represent a
cluster of cells visible to the naked eye that originate from a colony forming unit (CFU). Since
CFUs may be one or more initial cells, this method only gives an estimate of the number of
microorganisms in the sample being evaluated. 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?
There are several different ways to determine the number of colony forming units. One of the
ways you used last week was to make serial dilutions of a sample to be evaluated and spreading
these on suitable media. This method requires that the CFUs are diluted to a concentration where
the growth of a colony does not interfere with neighboring colonies. In the case of bacteria, this
number is between 30-300 CFU.
VIABLE COUNTS OF A SOIL SAMPLE Last week you performed viable counts of a soil
sample. Among the various microorganisms in the soil, bacteria are the most numerous
organisms that can be cultured (viruses are more numerous, but are difficult to grow, because
they require a suitable host). The predominant genera are Arthrobacter, Bacillus, Pseudomonas,
and Streptomyces. Arthrobacter and Streptomyces are actinomycetes that produce cells similar to
molds. Note that the results you get will be dependent on the medium used. The media for the
isolation of bacteria are usually not ideal for mold growth, while those used for molds often have
antibiotics to inhibit bacterial growth.
EXERCISE 2.0: BACTERIAL COUNTS IN SOIL (Groups of 2)
Materials
Viable counts of soil on TSA plates from last week
Method
1. Obtain your TSA plates on which you spread the different dilutions of the soil sample.
2. Count the number of CFUs observed for each of the dilutions. Record these counts in your
lab note book.
28
Microbiology Lab-2016
EXERCISE 2.1: COUNTS OF ACTINOMYCETES IN SOIL (Groups of 2)
Materials
Viable counts of soil on glycerol with yeast extract agar from last week
Method
1. Obtain your glycerol with yeast extract agar plates on which you spread the different
dilutions of the soil sample.
2. Count the number of CFUs observed for each of the dilutions. Record these counts in your
lab note book.
FUNGI
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.
In contrast to bacterial counts, viable counts of fungi are estimated from plates containing
between 10-50 CFUs.
EXERCISE 2.2: COUNTS OF FUNGI IN SOIL (Group of 2)
Materials
Viable counts on Sabouraud dextrose agar from last week
Method
1. Obtain your Sabouraud dextrose agar plates on which you spread the different dilutions of
the soil sample.
2. Count the number of CFUs observed for each of the dilutions. Record these counts in your
lab note book.
ONCE YOU’VE COMPLETED YOUR COUNTS OF BACTERIA, ACTINOMYCETES,
AND FUNGI, KEEP ONE REPRESENTATIVE PLATE OF EACH OF THE COUNTS
(THAT HAS DISTINCT COLONIES) AND STORE THEM AT 4OC UNTIL NEXT
WEEK.
29
Microbiology Lab-2016
Complete the following table and have your teaching assistant sign it before leaving the lab.
Viable counts from soil
Number of CFUs counted
Microorganism
10-3
10-4
10-5
CFU/mL CFU/g
Bacteria
Actinomycetes
Fungi
Show a sample calculation:
EXERCISE 2.3: MPN OF BACTERIA IN SOIL - CONTINUED (Groups of 2)
Materials
MPN broths from last week
Method
1. Examine your broths for the presence or absence of growth.
2. Fill out the following table to indicate your results.
Dilution
Growth : Tube
2
1
Have this signed by your teaching assistant before leaving the lab.
30
3
Microbiology Lab-2016
DIRECT COUNTS (haemocytometer 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/ 0.1 mm3, or
10 bacteria/ 0.0001 cm3 or 10 bacteria/0.0001 mL.
Therefore, the concentration of bacteria in the sample being examined is 1 X 105/mL.
Haemocytometer
Y
Y
31
Microbiology Lab-2016
EXERCISE 2.4: DIRECT COUNT OF A YEAST SUSPENSION (Groups of 2)
Materials
Yeast suspension
Haemocytometer slide
Sterile water
Tubes
Pasteur pipettes/micropipette
Method
1. Prepare the following dilutions of the yeast suspension: 10-1, 10-2, and 10-3. Make sure to
thoroughly mix the yeast suspension before sampling.
2. Fill the haemocytometer counting chamber with a sample from the highest dilution (see
image below or ask a teaching assistant to show you). Make sure to thoroughly mix the
yeast suspension 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.
5. Record the following information in your lab note books: Number of cells counted per
square, dimensions of the chosen squares, dilution used.
32
Microbiology Lab-2016
LAB NO 3
VIEWING MICROORGANISMS
Microbiology is the study of very small organisms, microorganisms that cannot be seen with the
naked eye. In order to observe and study their morphological characteristics, it is necessary to
examine clusters of cells - macroscopic viewing of colonies or visualizing individual cells using
a microscope. There are several groups of organisms that fall into this category, including
bacteria, algae, fungi and protists. In this group, there are several species of human interest
because of their ability to cause disease or their use in the food industry. The emphasis during the
semester will be on bacteria.
MACROSCOPIC VISUALIZATION – COLONY MORPHOLOGIES
Preliminary identification of microorganisms can be based on colonial morphology. Each single
colony represents a population of bacterial cells originating from a single cell that, after multiple
rounds of cell division, generated a colony of cells stacked on top of each other in a characteristic
shape according to the bacterial type (just as a cluster of bananas has a different appearance of a
heap of potatoes). The morphology of a bacterial colony is a function of several factors such as
the shape of the cell, its size and physiology. It is also important to note that the colonies of a
particular bacterial type often have colors, textures and distinct odors. Refer to the figure below
to determine the morphologies of the observed colonies.
33
Microbiology Lab-2016
EXERCISE 3.0: COLONY MORPHOLOGIES (Groups of 2)
Materials
Viable counts on TSA and glycerol + yeast extract agar plates from last week
Method
1. Obtain your TSA and glycerol + yeast extract agar plates from last week on which you
spread different dilutions of a soil.
2. Examine the different colony morphologies observed.
3. Record in your lab note book the different numbers of bacteria with distinct morphologies
observed.
34
Microbiology Lab-2016
MICROSCOPIC VISUALIZATION
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.
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.
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Microbiology Lab-2016
Using immersion oil at 100X
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.
1. Be sure that the specimen is in the EXACT CENTER of the viewing field under the 40X
objective.
2. Rotate the 40X objective away from the slide but do not yet click the 100X objective into
place.
3. Put a small drop of immersion oil on the slide directly over the light.
4. Rotate the nosepiece until the 100X oil immersion objective is clicked into place.
5. DO NOT USE THE COARSE ADJUSTMENT KNOB WHEN FOCUSING UNDER
THE 100X OBJECTIVE! ONLY USE THE FINE ADJUSTMENT KNOB!
6. Adjust the light for optimal viewing.
5
4
2
1
3
Taken from: http://coolessay.org/docs/index-32428.htmL
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Microbiology Lab-2016
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-2016
EXERCISE 3.1: FAMILIARIZATION WITH THE USE OF THE MICROSCOPE
(Groups of 2)
Materials
Slide of the letter "e"
Slides
Coverslips
Method
1. To familiarize yourselves with the operation of the microscope, 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?
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Microbiology Lab-2016
MICROSCOPIC VISUALIZATION OF BACTERIA – 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 41)
EXERCISE 3.2: SIMPLE STAINS (Groups of 2)
Materials
Microscope slides
Viable counts of soil samples on TSA and glycerol + yeast extract agar plates from last week
Stains
Method
Prepare smears from three different bacterial colonies from your TSA plates and at least one
from the glycerol + yeast agar plate.
1. 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
Too thick
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.
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Microbiology Lab-2016
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.
11. Prepare simple stains with methylene blue of each of the broth cultures provided. This time it
is not necessary to add water!
12. 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
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Microbiology Lab-2016
BACTERIAL CELL MORPHOLOGIES
Neisseria
Micrococci
Micrococci
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Microbiology Lab-2016
MICROSCOPIC EXAMINATION OF FUNGI – 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.
EXERCISE 3.3: SIMPLE STAINING (Groups of 2)
Materials
Microscope slides
Sabouraud dextrose agar plates from last week
Lactophenol blue
Plate of Penicillium
Plate of Rhizopus
Plate of Aspergillus
Method
1. Prepare wet mounts of one of the fungi growing on your Sabouraud dextrose agar plates and
of each of the fungal cultures supplies 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.
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Microbiology Lab-2016
FUNGAL COLONY AND MICROSCOPIC MORPHOLOGIES
Colonial morphology
Microscopic morphology
White colored fungus. Cottony
Colorless or brown spores.
Rhizopus
and fuzzy
Nonseptae hyphae with root like
hyphae (rhizoids)
Mucor
Colonies similar to Rhizopus
White colonies become greenish
blue, black, or brown as culture
matures
Colorless or brown spores.
Nonseptae hyphae with no rhizoids
Aspergillus
Mature cultures usually greenish
or blue green
Spores developing in chains
(Conidia) arising from terminal
bulb of conidiophore which arise
from septae hyphae
Penicillium
White colored fungus. Cottony
and fuzzy
Spores developing in chains
(Conidia) arising from branching
conidiophore which arise from
septae hyphae
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Microbiology Lab-2016
Microscopic Fungal Structures
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MICROSCOPIC VISUALIZATION - 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-2016
EXERCISE 3.4: GRAM STAINING (Groups of 2)
Materials
Microscope slides
Broth cultures of S. aureus and E. coli
Stains
Method
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.
MICROSCOPIC VISUALIZATION - 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.
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Microbiology Lab-2016
EXERCISE 3.5: ACID FAST STAINING (Groups of 2)
Materials
Microscope slides
Slant cultures of B. subtilis and M. smegmantis
Carbol Fuschin of Kinyoun
Acid alcohol
Method
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.
MICROSCOPIC VISUALIZATION – 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 unfavorable 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 decolorized 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
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Microbiology Lab-2016
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):
Pictures of simple stains of bacteria with each of the different stains including the
following information (4 images).
o Origins (Growth medium)
o Type of staining and stain used
o Cellular morphology
o Cellular aggregation
o Magnification
A Gram stain of each of the broth culture supplied. (2 images)
o Bacterial genus and species
o Type of staining used
o Cell morphology
o Cell aggregation
o Magnification
An acid fast stain of Mycobacterium smegmantis and Bacillus subtilis (2 images)
o Bacterial genus and species
o Type of staining used
o Cell morphology
o Cell aggregation
o Magnification
Lactophenol blue stains of Fungi which includes the following information (4
images)
o Genus
o Type of staining used
o Magnification
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Microbiology Lab-2016
LAB NO 4
GROWTH OF BACTERIA – GROWTH CURVE
Microbial growth is generally defined as the increase in cell number. A growth curve represents
growth as a function of time. Growth can be divided into four different stages: the lag phase,
exponential or log phase, the stationary phase and the death or the phase of decline. When an
inoculum is transferred to a new environment, the lag phase occurs first. This is the time required
by the microbial cells to adapt to new conditions. If they come out of a prolonged stationary
phase, they may have to adjust metabolically. The lag phase may be either very short or very
long. The latency phase is followed by the logarithmic phase. In this phase, the cells divide and
grow most actively. This is the period where the population is growing exponentially. After the
exponential phase, the population enters the stationary phase. The limitation of nutrients or the
accumulation of metabolic waste slows population growth. A prolonged period in the stationary
phase eventually leads to cell death where the population is in decline. This phase can be caused
by the accumulation of toxic waste or by a very low nutrient availability, or damage to cells.
Microbial growth can be measured in different ways. One way is to measure the optical density
(OD) of a culture broth as a function of time. The change in the apparent absorbance (actually
the loss of light due to light scattering by the particles) can be used to obtain an estimate of the
generation time.
EXERCISE 4.0: E.COLI GROWTH CURVE (Groups of 2)
Materials
Each group of 2 will be assigned a 25 mL culture of E. coli in a 250 mL flask. The cultures will
be initiated before the lab period at a time which represents approximately 10h, 11h, 12h and
13h. The time when the inoculation was performed shall be indicated on the bottle.
Two growth conditions will be available
LB medium at 37oC with shaking
LB medium at room temperature with shaking
Spectrophotometer with disposable cuvettes
20 mL LB broth
One test tube
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Microbiology Lab-2016
Method
1. Obtain the culture flask which you were assigned. Record in your note book the following
information: Growth condition and the time at which the inoculation was done.
2. Transfer 2 mL of the culture to a test tube and place on ice. Record the exact time at which
the sampling was done. Immediately return the flask to the shaker at the appropriate
temperature.
3. Blank the spectrophotometer at a wavelength of 600nm with 1 mL of LB.
4. Empty the cuvette in the waste beaker. Transfer 1 mL of the sample you collected to the
cuvette. Obtain the optical density and record it in your note book.
5. Repeat steps 1-4 every 30 minutes until you’ve obtained 8 optical density readings.
YEAST FERMENTATION
Yeasts are facultative anaerobes; that is to say they can grow in the presence or absence of
oxygen by metabolisms involving either respiration or fermentation, respectively. One of the
main differences between respiration and fermentation is the way that the NADH produced
during glycolysis is recycled to NAD +. During aerobic respiration, the hydrogen electrons from
NADH are transferred to oxygen in the electron transport chain generating 3 ATP per NADH
while during fermentation the hydrogen electrons are transferred to acetaldehyde producing
ethanol without ATP generation:
Yeast’s growth rate can thus be assessed by examining the efficiency with which this
biochemical pathway takes place. In the following exercise, you will examine the efficiency of
fermentation as a function of different carbon sources.
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Microbiology Lab-2016
EXERCISE 4.1: YEAST FERMENTATION BIOASSAY (Groups of 2)
Materials
Dry yeast
Sterile water
20% (m/v) Glucose
20% (m/v) Glycerol
20% (m/v) Fructose
20% (m/v) Sodium acetate
Phosphate buffer pH 7
2 Fermentation assemblies
Method
1. Prepare a yeast suspension by adding 10g of dry yeast to 50 mL of water. Rehydrate at room
temperature for 5 minutes.
2. Prepare the following fermentation media in Falcon tubes :
20 % Carbon
1.0 mL Glucose
1.0 mL Glycerol
1.0 mL Acetate
1.0 mL Fructose
3.
4.
5.
6.
7.
8.
Buffer pH 7.0
5.0 mL
5.0 mL
5.0 mL
5.0 mL
Water
3.0 mL
3.0 mL
3.0 mL
3.0 mL
Once the media have been prepared, add 10 mL of the yeast suspension to the first two tubes.
Transfer all the contents to 50cc syringes.
Assemble the apparatus as illustrated on the next page to follow the production of CO2.
Record the volume of water at time 0 in the collection tube.
Record the volume of water every 10 minutes for a total period of 1 hour.
Repeat the experiment with the other two media.
Data for the calculations of ethanol yields:
Molecular weights
Glucose
180g/mole
Glycerol
92g/mole
Acetate
82g/mole
Fructose
180g/mole
Ethanol
46g/mole
CO2
44g/mole (Density: 1.8g/cm3)
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Microbiology Lab-2016
50 mL Falcon tube
Water
CO2 released
20 mL Yeast
suspension
Lid with holes
Water
50 cc syringe
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Microbiology Lab-2016
BIOFILMS
Microbiologists have realized that most microorganisms like to live in the solid-liquid interfaces.
Biofilms are complex aggregations of microorganisms growing on surfaces. They attach to
various substrates such as soil particles, pipes and contact lenses. In fact, in nature microbial
colonization of submerged surfaces is very fast and has important implications for growth and
microbial survival. In addition, many infections are the result of the formation of biofilms in the
body. This is particularly true in the pulmonary infections where biofilm formation in the lungs
is the main cause of death in patients with cystic fibrosis and pneumonia. Biofilms are also the
major cause of infections resulting from medical implants. Microorganisms that thrive in
biofilms may also contain features that are not found in suspension cultures. For example, it has
been repeatedly observed that growth in biofilms greatly increases levels of microbial resistance
to antibiotics.
EXERCISE 4.2: EFFECT OF GROWTH CONDITIONS ON BIOFILM FORMATION
(Groups of 2)
Materials
4 Petri dishes
Plastic coverslips
Sterile water
20% (m/v) Glucose
30% NaCl (m/v)
YT
YT + 0.2% glucose broth of P. aeruginosa
Method
1. Label two Petri dishes as follows:
2
3
4
5
6
7
8
9
Experimental: 0.2% and 0.02% glucose (Even numbered groups)
Experimental : 0.5%, 0.05% NaCl (Odd numbered groups)
Label a third plate: Control.
Deposit 2 coverslips in each of the 3 Petri dishes.
Add 7.5 mL of YT to each of the 3 Petri dishes.
Add the appropriate volumes of glucose or NaCl as well as sterile water to the experimental
plates to obtain the desired concentrations of glucose or NaCl and a final concentration of
0.5X YT in a final volume of 15 mL.
Add 5 mL of sterile water to the control plate.
Inoculate the 2 experimental plates with 0.1 mL of P. aeruginosa broth culture.
DO NOT inoculate the control plate.
Incubate at 28oC until next week.
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Microbiology Lab-2016
LAB NO 5
BIOFILMS – CONTINUED
Last week you began an experiment to determine the influence of different environmental
conditions on biofilm formation. This week you will continue this experiment to determine the
level at which biofilms were established.
EXERCISE 5.0: EVALUATION OF BIOFILMS (Groups of 2)
Materials
Beaker
3 petri dishes
Plates inoculated with P. aeruginosa from last week
Crystal violet
6 Wells plate
Forceps
Method
1. Using forceps grab each coverslip and immerse it in a beaker of distilled water 3 times in
order to withdraw any planktonic bacteria (bacteria in suspension).
2. Deposit coverslips from identical treatments in a new Petri dish, bacteria side up.
3. Add a drop of crystal violet on each of the coverslips and wait 30-60 seconds.
4. Using forceps grab each coverslip and immerse it in a beaker of distilled water 3 times in
order to withdraw any excess dye.
5. Deposit each coverslip (bacteria facing upward) in one of the wells of a six wells plate and
have the plate read at 630nm. These will then be returned to you.
6. Using forceps, grab each coverslip and deposit them on a slide (bacteria facing downward)
Two coverslips per slide.
7. Examine under the microscope at a magnification of 1000X and take pictures. Two pictures
per treatment.
CONTROL OF MICROBIAL GROWTH - 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
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Microbiology Lab-2016
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
EXERCISE 5.1: KIRBY-BAUER ASSAY (Groups of 2)
Materials
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
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.
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Microbiology Lab-2016
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 5.2: SENSITIVITY OF S. FAECALIS TO VANCOMYCIN (Groups of 2)
Materials
5 mL of S. faecalis broth
1 Chocolate agar plate
Sterile swab
Vancomycin E-test
Forceps
Method
1. Use the same approach as that in exercise 5.1 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-2016
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 5.3: DETERMINING MICs (Groups of 2)
Materials
5 mL broth culture of S. aureus
5 mL broth culture of S. faecalis
5 mL broth culture of E. coli
5 mL broth culture of P. aeruginosa
10 mL of assigned antibiotic (1mg/mL in TSB)
Approx. 50 mL TSB
24 well plate
Antibiotic
Ampicillin
Kanamycin
Nalidixic acid
Erythromycin
Class
Beta-lactam
Aminoglycoside
Quinolone
Macrolide
Abbreviation
A
K
N
M
57
LD50 (mg/Kg)
5300
4000
2040
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Microbiology Lab-2016
Method
1. Add 0.5 mL TSB to each of the wells of a 24 well plate.
2. Add 0.5 mL of the assigned antibiotic to each of the wells numbered 1 of rows A-D.
3. Perform serial 2 fold dilutions of the antibiotic by transferring 0.5 mL from wells numbered
1 to wells numbered 2, from wells numbered 2 to wells numbered 3, from wells numbered 3
to wells numbered 4 and from wells numbered 4 to wells numbered 5.
4. Discard 0.5 mL from wells numbered 6.
5. Dilute in TSB each of the provided cultures in order to obtain 5 mL of broth representing a
10 000X dilution factor.
6. Inoculate each of the wells from row A with 0.5 mL of the diluted S. aureus culture.
7. Inoculate each of the wells from row B with 0.5 mL of the diluted S. faecalis culture.
8. Inoculate each of the wells from row C with 0.5 mL of the diluted E. coli culture.
9. Inoculate each of the wells from row D with 0.5 mL of the diluted P. aeruginosa culture.
10. Seal the plate with parafilm and incubate at 37oC until next week.
S. aureus
S. faecalis
E. coli
P. aeruginosa
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DISINFECTANTS & ANTISEPTICS
There are several classes of antibacterial agents such as antiseptics, disinfectants and antibiotics.
They all aim to either reduce the number of bacteria and to eliminate or inhibit their growth.
Some have a therapeutic use, such as antiseptics and antibiotics, while others are used for
preventive or cosmetic purposes, such as disinfectants. The use of antiseptics and disinfectants is
to significantly reduce the number of bacteria in a given sector and prevent their growth.
Antiseptics are chemical compounds for human use, while disinfectants are used on objects.
One of the common uses of antiseptics is the treatment of biofilms found in the oral cavity.
Indeed, these biofilms present on the tooth surface are recognized as the main cause of cavities
and dental plaque. One approach that is used for the control of oral biofilms is the use of mouth
washes. There are many different types of mouthwashes available to the consumer. Usually each
mouthwash contains a main active ingredient, such as alcohol or fluoride. These ingredients are
the main fighters against harmful bacteria that make up plaque and tartar. A predominant
bacterial species in plaque is Streptococcus mutans. S. mutans is an anaerobic, Gram positive
bacterium with the ability to metabolize sucrose and release lactic acid. This causes an acidic
environment on the enamel of the tooth which makes it susceptible to deterioration.
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EXERCISE 5.4: EFFICACY OF MOUTH WASHES (Groups of 2)
Materials
5 mL broth culture of S. aureus
5 mL broth culture of S. faecalis
10 mL Listerine original (methyl salicylate 0.06% m/v)
10 mL Listerine Zero (sodium fluoride 0.02% m/v)
10 mL Scope Outlast (cetylpyridinium chloride 0.045% m/v)
10 mL of 50% ethanol (v/v)
Approx. 50 mL of TSB + 1% glucose and 3% NaCl.
24 well plate
Method
1. Add 0.5 mL of TSB to each of the wells of a 24 well plate.
2. Add 0.5 mL of the assigned mouth wash to each of the wells numbered 1 of rows A-D.
3. Perform serial 2 fold dilutions of the mouth wash by transferring 0.5 mL from wells
numbered 1 to wells numbered 2, from wells numbered 2 to wells numbered 3, from wells
numbered 3 to wells numbered 4 and from wells numbered 4 to wells numbered 5.
4. Discard 0.5 mL from wells numbered 6.
5. Dilute in TSB each of the provided cultures in order to obtain 5 mL of broth representing a
1000X dilution factor.
6. Each person from a given group will also have to collect approximately 3-4 mL of their
saliva.
7. Inoculate each of the wells with 0.5 mL of the appropriate culture (See below)
8. Seal the plate with parafilm and incubate at 37oC until next week.
S. faecalis
E. coli
Saliva person 1
Saliva person 2
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Microbiology Lab-2016
DEATH KINETICS
Different microorganisms have different susceptibilities to treatments. For example, spores are
particularly resistant. As with bacterial growth, mortality occurs exponentially. Consequently,
mathematical functions that describe the profile of cell death under a given condition were made.
Such functions are useful for determining the minimum time required to reduce a microbial
population below a harmful level. The decimal reduction time, called the D value, represents the
period of time under given circumstances necessary to reduce a population of microorganisms by
a value of one log or 90%. In other words, if the value of 1 D of E.coli is 1 minute, this indicates
that an exposure of one minute is required to reduce the bacterial population by 90%. So to
reduce a population of 1 x 106 to 1 x 104 cells of E. coli would take 2D which is equivalent to 2
minutes. The D values are influenced by the bacterial species, form, and the conditions in which
they find themselves. For example, the D value of spores is usually much higher than that of
vegetative cells. The D value is an important parameter used in several areas to evaluate and
compare the effectiveness of different treatments such as the use of disinfectants.
EXERCISE 5.5: D VALUE OF A DISINFECTANT (Groups of 2)
Materials
Suspension of E. coli in physiological saline (approx. 106-107 UFC/mL) (Even numbered groups)
Suspension of B. subtilis in physiological saline (approx. 106-107 UFC/mL) (Odd numbered
groups)
5 mL Clorox (5.25% hypochlorite)
15 mL 10% (m/v) sodium thiosulfate
50 mL Falcon tube
Approx. 75 mL TSB.
2 X 24 well plate
Micropipettor (P20)
Method
1. Setup 4 sterile test tubes containing sodium thiosulfate at a final concentration of 1% in TSB
for a final volume of 9 mL. Label the tubes T0, T5, T10, and T15.
2. Add to the Falcon tube the required volume of hypochlorite required to obtain a final
concentration of 0.05% in 20 mL.
3. Complete the volume to 19 mL with sterile water.
4. Add 1 mL of the E.coli suspension (Even numbered groups) or of the B. subtilis suspension
(Odd numbered groups) to the hypochlorite solution in the Falcon tube. Mix well.
5. Immediately transfer 1 mL of the hypochlorite treated culture to the T0 tube containing
sodium thiosulfate.
6. Repeat step 5 at 5 minute intervals – until T15. Make sure to mix the suspension before each
sampling.
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Microbiology Lab-2016
7. For each sampling, T0 – T15, prepare serial dilutions from 10-2 to 10-8 in a 24 well plate in a
final volume of 1 mL. See image below.
10-2
10-4
10-5
10-6
10-7
10-8
T0
T5
T10
T15
8. Transfer 0.1 mL of each dilution to the appropriate wells as illustrated below.
10-2
10-4
10-5
10-6
10-7
10-8
T0
T5
T10
T15
9. Add approximately 1.0 mL of TSB to each well. The volume does not have to be very
accurate.
10. Seal the plate with parafilm and incubate at room temperature. Your plates will be read the
next day.
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LAB NO 6
CONTROL OF MICROBIAL GROWTH - CONTINUED
Amongst the commonly used antibiotics (β - lactams, tetracycline, aminoglycosides, and
macrolides), beta-lactams are most commonly used for the treatment of dairy cattle. Such use can
cause considerable excretion of these residues in milk. The presence of antibiotic residues in
milk can cause side effects such as allergic reactions, development of resistance and others.
The redox couple tetrazolium / formazan, a redox system, is widely used in microbiology as an
indicator of microbial activity. In the presence of bacteria, the TTC, which is colorless, is
reduced to formazan, a red compound. Therefore, the TTC test method is considered a relatively
fast method for assessing the antibacterial activity of antimicrobial agents.
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EXERCISE 6.0: TTC ASSAY – SENSITIVITY TO BETA-LACTAMS (Groups of 2)
Materials
Nutrient broth
Ampicillin 200 µg/mL in nutrient broth (Even numbered groups)
Penicillin 200 µg/mL in nutrient broth (Odd numbered groups)
2 mL suspensions in nutrient broth of E. faecalis or E. coli
Preparation of suspensions: The morning before the lab (at approx. 10AM), inoculate
10 mL nutrient broth with 1 mL of an overnight culture. At about 1PM, prepare 10 -2
dilutions of cultures and provide 2 mL samples to students.
1% (m/v) TTC
50% ethanol
10 test tubes
Method
1. Label two series of five test tubes as follows: F0, F12.5, F25, F50, and F100 (F for E.
faecalis) and C0, C12.5, C25, C50, and C100 (C for E. coli).
2. Dispense 2 mL of nutrient broth to each of the ten tubes.
3. Add 2 mL of the assigned antibiotic solution to the tube labelled F100. Mix.
4. Transfer 2 mL from the tube labelled F100 to the tube labelled F50. Mix.
5. Transfer 2 mL from the tube labelled F50 to the tube labelled F25. Mix.
6. Transfer 2 mL from the tube labelled F25 to the tube labelled F12.5. Mix.
7. Withdraw and discard 2 mL from the tube labelled F12.5.
8. Repeat steps 2-6 for the second series of tubes (labelled C).
9. Add 0.1 mL of 1% TTC to each of the ten tubes.
10. Inoculate each of the 5 tubes of the “F” series with 0.1 mL of the E. faecalis suspension.
11. Inoculate each of the 5 tubes of the “C” series with 0.1 mL of the E. coli suspension.
12. Prepare a blank (“B”) which contains the following: 2 mL of nutrient broth + 0.1 mL of 1%
TTC.
13. Incubate all tubes at 37oC for one hour.
14. Label 11 microcentrifuge tubes to correspond to the 11 assay tubes.
15. Following the 1 hour incubation period, briefly vortex each of the assay tubes and then
transfer 1 mL from each these to the corresponding microcentrifuge tube.
16. Centrifuge at maximum speed in the microcentrifuge for 1 minute.
17. Decant the supernatants and then add 0.5 mL of 50% ethanol. Mix on the vortex for 30
seconds.
18. Centrifuge at maximum speed in the microcentrifuge for 1 minute.
19. Transfer 150 µL from each tube to the wells of a 96 well plate.
20. Have your plate read at 480nm.
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Microbiology Lab-2016
TOXICOLOGY AND INDICATOR MICROORGANISMS
Pollution by heavy metals is a widespread source of environmental contamination. The sources
include emissions, effluents, solid industry waste products, vehicle emission, metal smelting and
mining, and the use of insecticides, industrial and municipal waste disposal in agriculture, and
the overuse of fertilizers. These chemicals have a major impact on the environment and
municipal drinking water. To evaluate the presence of these contaminants, biological assays have
been developed which utilize indicator microorganisms. A microorganism commonly used is the
ciliated unicellular eukaryote Tetrahymena pyriformis.
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EXERCISE 6.1: BIOASSAY FOR THE DETERMINATION OF THE LD50 OF HEAVY
METALS (Groups of 2)
Materials
20 mL culture of Tetrahymena pyriformis
24 well plate
PP medium (2% proteose peptone, 0.25% yeast extract; pH 6.7)
5% (m/v) CuCl2 in water
5% (m/v) ZnCl2 in water
4% (v/v) formaldehyde
Haemocytometer slide
Method
1. Add 0.5 mL of the Tetrahymena pyriformis culture to each of the wells of the two rows A
and C of a 24 well plate.
2. Add 0.5 mL of the stock solution of CuCl2 or of ZnCl2 to the appropriate wells of column
number 1. (See the schematic below)
3. Perform serial 2 fold dilutions from the wells of column 1 up to the wells of column 6.
4. Incubate at room temperature for 1h.
5. During the incubation period, perform a count of the original culture as follows: transfer an
appropriate volume to a haemocytometer slide. Count the number of dead organisms (those
that do not move) in three independent squares.
6. Transfer 0.5 mL of the original culture to a microcentrifuge tube. Add 100 µL of
formaldehyde, whose purpose is to kill all the organisms. Mix and then count again.
7. To obtain the number of live organisms, subtract the count obtained before the addition of
formaldehyde from the count obtained after its addition.
8. Following the 1 hour period, determine the counts of dead organisms in each well.
Dilutions : 1/2
1/4
1/8
1/16
1/32
1/64
CuCl2
ZnCl2
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Microbiology Lab-2016
Complete the following table and have it signed by one of your teaching assistants.
Final Conc. CuCl2
*Counts
Final Conc. ZnCl2
* Counts: Indicates the average number of live Tetrahymena pyriformis/mL
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*Counts
Microbiology Lab-2016
EXERCISE 6.2: KIRBY BAUER DIFFUSION ASSAY
Materials
Kirby Bauer assay from last week
Method
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
I = mm
range
S = mm
or more
Antimicrobial agent
CODE
Amoxicillin (Staph)
AMC
19
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
68
20
14-17
18
29
23
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Microbiology Lab-2016
EXERCISE 6.3: SENSITIVITY OF S. FAECALIS TO VANCOMYCIN - E-TEST (Groups
of 2)
Materials
E-Test plate from last week.
Method
1. Read the MIC from the strip, which is represented by the concentration indicated on the strip
which gives rise to the smallest diameter for the zone of inhibition.
CMI
EXERCISE 6.4: DETERMINATION OF THE THERAPEUTIC DOSE – MIC (Groups of
2)
Materials
24 well plate used for the determination of the MIC from last week
Method
1. Have the optical densities of your MIC assay plate from last week read at a wavelength of
600nm.
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Microbiology Lab-2016
EXERCISE 6.5: EFFICACY OF MOUTH WASHES (Groups of 2)
Materials
24 well plate used to compare the efficacy of mouth washes from last week
Method
1. Have the optical densities of your MIC assay plate from last week read at a wavelength of
600nm.
EXERCISE 6.6: D VALUE OF A DISINFECTANT (Groups of 2)
Method
1. Obtain the O.D. 600 readings from the 24 well plate you inoculated last week. For each time
point (T0 – T15), determine the lowest dilution (least dilute) where you observe a reading
that is at most 2 times greater than the reading obtained for the blank. Record this dilution
factor in the table below.
Ex.
Readings at T15 were ≤ 2X that of the blank for dilutions of 10-7 and 10-8. Record 107 for this
time point.
Time
Dilution factor
E. coli
B. subtilis
T0
T5
T10
T15
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LAB NO 7
71
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
Nitrate broth
Sim tube
✓
✓
✓
Lysine agar slant
✓
✓
✓
Phenylalanine agar slant
✓
Urea slant
✓
Ornithine decarboxylase broth
✓
✓
✓
✓
Simmon’s citrate slant
✓
✓
✓
TSI slant
✓
✓
✓
MR-VP
DNA agar
✓
✓
✓
Phenol red broths
Spirit Blue agar
Bacteria
B. cereus
✓
B. subtilis
✓
E. coli
✓
E. aerogenes
P. aeruginosa
P. mirabilis
Milk agar
Starch agar
BACTERIAL METABOLISM AND DIFFERENTIAL TESTS
Bacteria are amongst the most diverse organisms on earth; indeed they can be found everywhere.
It is therefore obvious that they must have very different metabolisms that are adapted to the
environments they inhabit. This great diversity is the biochemical basis of the diagnostic
approach that is used by microbiologists to identify microorganisms. The tests used for
identification and diagnosis are called differential or biochemical tests. This week you will use
these tests to examine different biochemical characteristics dependent on specific enzymatic
reactions in certain metabolic pathways. A summary of the different tests that you will perform is
presented below:
✓
✓
✓
Microbiology Lab-2016
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.
EXERCISE 7.0: DEGRADATION OF COMPLEX CARBON SOURCES (Groups of 2)
Materials
B. cereus
B. subtilis
E. coli
3 starch plates
3 milk plates
3 Spirit Blue plates
3 DNA plates
Method
1. Appropriately label the different media plates with the bacteria to be tested. Four
media/bacteria.
2. Streak for single colonies each of the bacteria indicated above on one of each the agar media.
3. Incubate at 28oC.
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SUGAR METABOLISM – PHENOL RED BROTH
The phenol red broth is a differential medium to which a sugar is added. The base medium
contains a source of protein, peptone, and the pH indicator, phenol red. Phenol red turns yellow
in acid medium and turns red in alkaline medium. In addition, an inverted vial is used to detect
the accumulation of gas. Generally, the presence of acid with or without a gas build-up indicates
a fermentative metabolism.
EXERCISE 7.1: METABOLISM IN PHENOL RED BROTH (Groups of 2)
Materials
P. miribalis
P. aeruginosa
E. coli
E. aerogenes
3 phenol red broths of each of the following sugars: glucose, lactose, and sucrose
Method
1. Label each set of 3 phenol red broths (glucose, lactose and sucrose) with one of the bacteria
indicated above.
2. Inoculate each bacterium in the appropriate broths.
3. Incubate at 37oC.
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 sulfur group, such
as methionine and cysteine. Degradation of these amino acids generates as a by-product
hydrogen sulfide, which reacts with ferrous sulfate in the medium, giving rise to a black
precipitate.
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Microbiology Lab-2016
EXERCISE 7.2: GROWTH IN TSI MEDIUM (Groups of 2)
Materials
P. miribalis
P. aeruginosa
E. coli
3 TSI Slants
Method
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-2016
USE OF CITRATE AS A CARBON SOURCE
This test is designed to determine whether a microorganism can use citrate, an intermediate of
the Krebs cycle, as sole source of carbon and inorganic ammonium salts as sole nitrogen source.
Bacteria which possess the enzyme citrate permease can transport the citrate within the cell to
convert it to pyruvate. The use of citrate generates alkali by-products which are identified by the
inclusion of a pH indicator, bromthymol blue, which is green at a pH of 6.8 and blue at a pH of
7.6 and above.
EXERCISE 7.3 GROWTH ON SIMMON’S CITRATE SLANT (Groups of 2)
Materials
E. coli
E. aerogenes
2 Simmon’s citrate slants
Method
1. Streak each of the above indicated bacteria on a Simon’s citrate slant
2. Incubate at 37oC.
UREA METABOLISM
Urea is a product resulting from the decarboxylation of specific amino acids and thus the
degradation of proteins in vertebrates. Some microorganisms can use urea as a nitrogen source.
Urea catabolism requires urease, an enzyme hydrolyzing the urea in ammonia, carbon dioxide
and water. Ammonia released in the culture medium makes it alkaline. A pH indicator in the
culture medium, phenol red, can detect the presence of alkali products, changing the color of the
medium to pink.
EXERCISE 7.4: GROWTH ON UREA SLANT (Groups of 2)
Materials
E. coli
P. mirabilis
2 Urea slants
Method
1. Streak each of the above indicated bacteria on a urea slant
2. Incubate at 37oC.
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Microbiology Lab-2016
DECARBOXYLASES AND DEAMINASES
Deamination and decarboxylation tests are used for differentiating bacteria of the
Enterobacteriaceae family. The majority of these bacteria produce several enzymes necessary
for the degradation of amino acids. The enzymes that remove a COOH group are decarboxylases
while those that remove NH2 groups are deaminases. The media used to verify the presence of
different amino acid decarboxylase contain glucose, a fermentable carbon source, and the desired
amino acid. The acid produced from glucose fermentation reduces the pH of the medium and
changes the color of the pH indicator from purple to yellow. These acidic conditions stimulate
the activity of decarboxylases. The decarboxylation of amino acids causes a rise in pH, which
changes the color of the indicator, Bromcresol purple, from yellow to purple. If the organism
does not produce the appropriate enzyme the medium remains acid (yellow).
One of the deaminases produced by many bacteria allows for the deamination of phenylalanine
generating phenylpyruvic acid. This reaction can be detected in the phenylalanine agar medium
that provides a high source of phenylalanine. After incubation in this medium, phenylpyruvic
acid reacts with a reagent in this medium, ferric chloride (FeCl3) generating a green color.
Deamination and decarboxylation of lysine can be detected in lysine iron agar medium. This
medium contains, amongst other things, glucose, lysine, sodium thiosulfate as a sulfur source
that can be reduced and the pH indicator, bromcresol purple. If an organism that has lysine
decarboxylase is inoculated into this medium, glucose fermentation creates an acidic
environment that induces the production of decarboxylase. The fermentation of glucose initially
causes the medium to turn yellow, but after decarboxylation the medium becomes alkaline and
turns purple. In contrast, if the organism produces lysine deaminase, the generated products
reacts with ammonium ferric citrate and produces a red color.
EXERCISE 7.5: DECARBOXYLASE AND DEAMINASE ASSAYS (Groups of 2)
Materials
E. coli
P. mirabilis
E. aerogenes
3 decarboxylase broths without any amino acid
3 decarboxylase broths with ornithine
3 phenylalanine agar slants
3 Lysine with iron agar slants
Method
1. Inoculate the decarboxylase broths lacking any amino acids with each of the above indicated
bacteria.
2. Inoculate the decarboxylase broths with ornithine with each of the above indicated bacteria.
3. Overlay all the broths with mineral oil to create anaerobic conditions.
4. Inoculate the phenylalanine agar slants with each of the above indicated bacteria.
5. Inoculate the lysine agar slants with each of the above indicated bacteria.
6. Incubate at 37oC.
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Microbiology Lab-2016
SIM: PRODUCTION OF HYDROGEN SULFIDE, INDOLE AND MOTILITY
H2S PRODUCTION
As with the TSI medium, the degradation of sulfur 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).
EXERCISE 7.6: SIM TEST (Groups of 2)
Materials
E. coli
P. mirabilis
E. aerogenes
3 SIM tubes
Method
1. Use an inoculation needle to inoculate each of the above indicated bacteria down to the
bottom of the tube along a straight line.
2. Incubate at 37oC.
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Microbiology Lab-2016
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 7.7: NITRATE REDUCTION ASSAY (Groups of 2)
Materials
E. coli
P. aeruginosa
P. mirabilis
3 nitrate broths
Method
Inoculate each of the above indicated bacteria in nitrate broths and incubate at 37oC.
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Microbiology Lab-2016
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 color 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.
EXERCISE 7.8: ENTEROPLURI TEST (Groups of 2)
Materials
Gram negative unknown
Enterotube
Method
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 (See
figure 5 on next page).
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Microbiology Lab-2016
See the video at the following link:
http://www.youtube.com/watch?feature=player_detailpage&v=l5qHyr1FULE
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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Microbiology Lab-2016
THE STREPTOCOCCI AND THE STAPHYLOCCOCI
As previously mentioned bacteria are ubiquitous and can normally be found on several parts of
the human body. Unless there is an infection, the majority of the human body which is not in
contact with the external environment is sterile. This includes your blood, all your internal fluids,
internal organs, cavities, etc.. In contrast, various microorganisms inhabit every surface of the
human body exposed to the environment. The majority of these bacterial species are harmless
unless their growth becomes uncontrolled.
The human body provides a wide variety of different environments that differ with regards to the
availability of oxygen, pH, water content, etc. Accordingly, various microorganisms have
established niches in different regions of the body. In the following exercise, you will sample
your throat to isolate microorganisms representative of this niche.
The upper respiratory tract is populated by many Gram-positive species, many of which are
potential pathogens if their growth is unchecked. These include Staphylococci and Streptococci.
Amongst the Staphylococci, S. aureus and S. epidermidis, are species of clinical importance. It is
estimated that in 10-40% of the population S. aureus is present in the natural flora. However, this
species is associated with several medical problems, such as abscesses, bacteremia, and
endocarditis. S. epidermidis, one of the resident species of the skin, is an opportunistic pathogen.
These species are not usually pathogenic in healthy individuals, but can cause serious infections
in weakened individuals, such as immunocompromised individuals.
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 Streptococci within
this group are non-pathogenic.
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Microbiology Lab-2016
EXERCISE 7.9: THROAT SAMPLING ON BLOOD AGAR PLATES (Groups of 2)
Materials
6 blood agar plates
20 mL TSB
Cotton swabs
Method
You will have to sample the throat of two different people.
1. Ask someone to use a cotton swab to sample the tonsil area of your throat (See the
illustration below)
2. Drop the swab in a tube containing 1 mL of TSB.
3. After having vigorously mixed, prepare 10-1 and 10-2 dilutions in TSB.
4. Spread 0.1mL of the undiluted sample and each of the two dilutions on blood agar plates.
5. Incubate at 37oC in the candle jar.
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Microbiology Lab-2016
LAB NO8
DIFFERENTIAL TESTS –CONTINUED
EXERCISE 8.0: DEGRADATION OF COMPLEX CARBON SOURCES (Groups of 2)
1. 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.
2. 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-2016
EXERCISE 8.1: METABOLISM IN PHENOL RED BROTH (Groups of 2)
Method
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.
Possible results
Observation
Interpretation
Yellow broth with bubble in vial
Fermentation with acid by-products and gas
Yellow broth without bubble in vial
Fermentation with acid by-products without gas
Orange broth (Original color)
No fermentation
Red broth without bubble in vial
Degradation of peptones with alkali by-products
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Microbiology Lab-2016
EXERCISE 8.2: GROWTH IN TSI (Groups of 2)
Do an analysis of your TSI slants in order to obtain the following information:
What is the reaction of the slant: acid (A), alkaline (K) or neutral (NC)
What is the reaction in the butt : acid (A), alkaline (K) or neutral (NC)
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
Acid butt and slant + accumulation of gas
Alkaline slant and acid butt
Alkaline butt and alkaline slant + H2S anaerobically
Alkaline slant and neutral butt
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D
Microbiology Lab-2016
Possible results:
Results (slant/butt)
Symbol
Interpretation
Red/yellow
K/A
Glucose fermentation only;
Peptone catabolized
Yellow/yellow
A/A
Glucose and lactose and/or
sucrose fermentation
Red/red
K/K
No fermentation; Peptone
catabolized
Red/no color change
K/NC
No fermentation; Peptone used
aerobically
Yellow/yellow with
bubbles
A/A,G
Glucose and lactose and/or
sucrose fermentation; Gas
produced
Red/yellow with bubbles
K/A,G
Glucose fermentation only; Gas
produced
Red/yellow with bubbles
and black precipitate
K/A,G, H2S
Glucose fermentation only; Gas
produced; H2S produced
Red/yellow with black
precipitate
K/A, H2S
Glucose fermentation only; H2S
produced
Yellow/yellow with black
precipitate
A/A, H2S
Glucose and lactose and/or
sucrose fermentation; H2S
produced
No change/no change
NC/NC
No fermentation
A=acid production; K=alkaline reaction; G=gas production; H2S =sulfur
reduction
Typical results
Escherichia coli
Proteus mirabilis
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexneri
Slant
A
K
K
K
K
Butt
A
A
K
A
A
86
Gas
+
-
H2 S
+
+
-
A : Acid (yellow)
K: Alkaline (Red)
Microbiology Lab-2016
GLUCOSE FERMENTATION; PRODUCTION OF MIXED ACIDS OR ACETOIN
Some bacteria ferment glucose generating large quantities of acids that cause a significant
reduction in the pH of their environment. This can be detected by the pH indicator methyl red
which is red at pH 4.4 or less. In contrast, other bacterial species generate only small amounts of
acids, but large amounts of neutral by-products such as ethanol and butanediol. An intermediate
for the production of butanediol is acetoin, which can be detected using two reagents, alpha
naphthol and KOH.
EXERCISE 8.3: METHYL RED - VOGUES-PROSKAUER TEST (MRVP) (Groups of 2)
Materials
Cultures of E. aerogenes and E. coli in MRVP broths
MRVP reagents
4 test tubes
Method
1. To complete the MR and VP tests, transfer 1 mL of the MRVP broth culture indicated above
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.
Negative
Positive
Positive
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Negative
Microbiology Lab-2016
EXERCISE 8.4 GROWTH ON SIMMON’S CITRATE AGAR SLANT (Groups of 2)
Method
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.
EXERCISE 8.5: GROWTH ON UREA SLANT (Groups of 2)
Négative
Positive
Negative Positive
Method
1. Examine your slants for a color change to pink. This color indicates
that alkaline by-products were generated by the action of urease.
EXERCISE 8.6: DECARBOXYLASE AND DEAMINASE ASSAYS (Groups of 2)
ORNITHINE DECARBOXYLASE
Method
1. Obtain your decarboxylase assay broths and compare the color of the broths with and
without ornithine.
1
+O
2
-O
+O
3
-O
+O
-O
+O: with ornithine; -O: Without ornithine
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-2016
PHENYLALANINE DEAMINASE
Method
1. Obtain your phenylalanine slants.
2. Add a few drops of 12% ferric chloride.
3. Note any change in color. (A : Negative, B : Positive)
LYSINE DECARBOXYLASE AND DEAMINASE
Method
1. Obtain your lysine agar slants.
2. Note any color change and the corresponding area.
Color
Purple butt and slant
Yellow butt and purple slant
Yellow butt and red slant
Black precipitate
Interpretation
Lys deaminase negative
Lys decarboxylase positive
Lys deaminase negative
Lys decarboxylase negative
Fermentation of glucose
Lys deaminase positive
Lys decarboxylase negative
Fermentation of glucose
Sulfur reduction
EXERCISE 8.7: SIM TEST (Groups of 2)
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.
89
Motile +H2S
Motile + Indole
Non-motile
No growth
Microbiology Lab-2016
EXERCISE 8.8: NITRATE REDUCTION ASSAY (Groups of 2)
Method
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
90
Results following the addition of
zinc
Microbiology Lab-2016
EXERCISE 8.9: ENTEROPLURI TEST (Groups of 2)
Method
At the end of incubation:
Observe the change in color of culture media in the different sectors and interpret results
using the table below. NOTE: if there is no change in color 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
Biochemical reactions
Glucose fermentation
Gas production
Lysine decarboxylation
Ornithine decarboxylation
Hydrogen sulfide production
Indole test
Adonitol fermentation
Lactose fermentation
Arabinose fermentation
Sorbitol fermentation
Acetoin production
Dulcitol fermentation
Phenylalanine deamination
Urea hydrolysis
Citrate utilization
Sector colors
Positive reaction Negative reaction
Yellow
Red
Lifted wax
Overlaying wax
Violet
Yellow
Violet
Yellow
Black-brown
Beige
Pink-red
Colorless
Yellow
Red
Yellow
Red
Yellow
Red
Yellow
Red
Red
Colorless
Yellow
Green
Dark brown
Green
Purple
Beige
Blue
Green
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 color 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 color develops within 20
minutes.
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Microbiology Lab-2016
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:
Test
Positivity code
Result
Code sum
Numerical code
Group 1
4
2
1
Group 2
4
2
1
Group 3
4
2
Microorganism:
92
1
Group 4
4
2
1
Group 5
4
2
1
Microbiology Lab-2016
DIFFERENTIAL TESTS FOR THE IDENTIFICATION OF GRAM POSITIVE COCCI
Micrococcaceae family
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 Micrococci, 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.
Streptococcaceae family
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-2016
EXERCISE 8.10: BLOOD HEMOLYSIS (Groups of 2)
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. Betahemolysin 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 the genus Streptococcus and some
of the genus Staphylococcus belong to this group. Alphahemolysin 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 the genus 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 Streptococci genera within this
group are non-pathogenic.
Materials
Sampling of the throat on blood agar
Materials
1. Examine your blood agar plates and determine the type of hemolysis observed: alpha, beta
or gamma.
2. Perform a Gram stain on a colony representing each type of hemolysis
EXERCISE 8.11: CATALASE (Groups of 2)
This test represents the first step in the discrimination of bacteria of the Micrococcaceae family
(catalase positive) from the Streptococcaceae family (catalase
negative). 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.
Materials
Gram positive cocci unknown on TSA plate (Labelled 1 or 2)
3% (v/v) Peroxide
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Microbiology Lab-2016
Method
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.
If your unknown is catalase positive, proceed with the PPT presentation « A ». If your
unknown is catalase negative, proceed with the PPT presentation « B ». These
presentations are available on K: /BIO3126. A description of the different tests follows:
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.
BACITRACIN, OPTOCHIN AND NOVOBIOCIN 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. Novobiocin sensitivity allows the
discrimination of S. saprophyticus from other bacteria of the Staphylococcus genus; S.
saprophyticus being resistant.
S. pneumoniae
S. pyogenes
S. saprophyticus
Bacitracin
Resistant
Sensitive
N.A.
Optochin
Sensible
Resistant
N.A.
Novobiocin
Sensitive
Sensitive
Resistant
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 nonfermenters.
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Microbiology Lab-2016
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.
PYR TEST
The PYR test is a qualitative procedure for determining the
ability of streptococci to enzymatically hydrolyze Lpyrrolidonyl-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 β-naphthylamine. Β-naphthylamine, reacts with p-dimethylaminocinnamaldehyde to form a pink/fuchsia precipitate.
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Microbiology Lab-2016
EXERCISE 8.12: DIFFERENTIAL STAINS AND STERILIZATION (Groups of 2)
As we have seen, dyes can be used as an indicator of metabolism. Since metabolism is an
indication of viability, it is possible to use such dyes to assess the viability of microorganisms in
a sample. An example where this would be desired is to assess the effectiveness of different
methods of sterilization.
Sterilization is a term referring to any process that eliminates (removes) or kills all forms of life
(such as fungi, bacteria, viruses, spores, etc.) in a given region, on a surface, a volume of fluid,
drugs or compounds such as culture media. Sterilization may be performed by one or more of the
following processes: heat, chemicals, irradiation, high pressure, and filtration. Sterilization is
distinct from disinfection and pasteurization in that the sterilization kills or inactivates all forms
of life.
In the following exercise, you will evaluate the effectiveness of different sterilization methods on
different yeast preparations. To obtain a measure of the effectiveness, a metabolic dye,
methylene blue, will be used. During respiration and fermentation, hydrogen electrons are
removed from glucose molecules by enzymes called dehydrogenases and given to various
chemical compounds such as NAD +. In this way, substances such as glucose provide energy for
the vital reactions in living organisms. In this exercise we will be using methylene blue as an
artificial electron acceptor (oxidative agent). When methylene blue is reduced, it becomes
colorless. Thus, after staining with methylene blue, yeast cells which are metabolically active are
colorless, while dead cells are blue.
Materials
Dry yeast preparation (1g)
0.01% methylene blue, 2% (w/v) sodium citrate dihydrate in PBS
1% Glucose
4 petri dishes
Haemocytometer slide
Method
1. Distribute approximately 0.1g of dry yeast to each of 4 petri dishes.
2. Add approximately 0.1g of dry yeast to each of two test tubes.
3. Add 5 mL of the glucose solution to two of the plates and one of the test tubes –humid
preparations.
4. Expose one of the plates with the dry yeast as well as one of the plates with the humid
preparation to UV for 2 minutes.
5. Microwave for 1 minute one of the plates with the dry yeast as well as one of the plates with
the humid preparation.
6. Boil the two test tubes for 5 minutes.
7. Add 5 mL of the glucose solution to each of the dry yeast preparations.
8. Transfer 0.5 mL of each of the treated samples to appropriately labelled microcentrifuge
tubes.
9. Prepare an untreated yeast sample as follows: 0.1g in 5 mL of glucose solution, then transfer
0.5 mL to a microcentrifuge tube.
10. Add 0.5 mL methylene blue to each of the tubes. Wait 1 minute.
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Microbiology Lab-2016
11. Examine with the microscope on a haemocytometer slide. Count the number of colorless
cells and the number of blue cells per square. Obtain counts from a number of squares which
represents approximately a total of 50 cells.
12. Determine the percentage of live cells in each sample as follows.
Number of colorless cells
X 100
Total number of cells total
de cellules
13. Determine the percentage reduction of viability for each treatment.
% of viable cells before treatment - % of viable cells after treatment
% of viable cells before treatment
98
X 100
Microbiology Lab-2016
LAB NO 9
IMMUNOLOGY
Jawed vertebrates, including all organisms from sharks to humans, developed a complex system
to treat all potential invaders of their bodies; the immune system. This system has two main
components; the innate and the acquired. In the case of the innate system, the host has a variety
of cells (leukocytes), enzymes and chemical compounds which are intended to prevent the attack
by any entity. This system is always operational and is non-specific. In contrast, the acquired
system includes cells that are educated (lymphocytes) to distinguish "self" from "non-self".
Simply put, after your conception and your birth, your immune system is educated to ignore
everything that is an integral part of your body, "self". Therefore, your immune system will
mount an attack against almost anything that is not an integral part of your body, "non-self". In
immunological terms all entities being recognized as "non-self" are called antigens.
The microorganisms that are encountered every day in the life of a healthy individual
rarely cause disease. Most are detected and destroyed within minutes or hours after their entry in
the body. This is possible due to defense mechanisms of the immediate response of the innate
immunity. When a pathogen manages to violate any anatomical barriers of the host, some innate
immune mechanisms begin to act immediately. These defenses include several classes of
molecules preformed in the blood, extracellular fluid and secretions that can either weaken or kill
the pathogen. For example antimicrobial enzymes such as lysozyme which digest bacterial cell
walls.
LEUKOCYTES
Leukocytes or WBC (white blood cells), which are the cells that defend the body against
infections, are subdivided into five categories: neutrophils, eosinophils, basophils, monocytes
and lymphocytes. These cells play a vital role in immune responses. Some of these cells operate
in the innate immune response (granulocytes leukocytes), while others are specific to the
acquired response (B and T lymphocytes). Each type of leukocytes has specific properties and
can be distinguished after staining (see table on next page).
ANTIBODIES
Among the artillery of the acquired system are the antibodies; proteins capable of specifically
recognizing a single aspect of the invader. The structure of an antibody can be divided into two
parts (see figure on next page). They have a constant domain, which as the name suggests is
relatively the same for all antibodies produced by your immune system. The primary purpose of
this area is to label the target as an entity for destruction. The second area, which is variable, is
the portion of the molecule responsible for conferring the specificity to the antibody's ability to
bind to the foreign entity. Given the diversity of potential entities that differ significantly from
any component of self is almost infinite, this region is extremely variable.
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Microbiology Lab-2016
Name
Granulocytes
Neutrophils
Morphological
characteristics
Granules
Two lobes separated
by thin chromatin
filament
Main function
Phagocytosis of
bacteria
Granulocytes
Eosinophils
Granules
Bilobed nucleus
Destruction of
parasitic worms
Granulocytes
Basophiles
Very abundant
granules which
usually render
nucleus not visible
Release of
preformed mediators
(Inflammatory
response)
B Lymphocytes
T Lymphocytes
Monocytes
Antibody production
No granules
Nuclei occupies most (Humoral response)
of the cytoplasmic
Attack of infected
space
cells
No Granules
Irregular shaped
nucleus
Phagocytosis
(monocytes
differentiate into
macrophages in
tissues)
Antigens
Variable
Region
]
Constant
Region
Antibody
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LYSOZYME
Lysozyme catalyzes the hydrolysis of β-1, 4-linkages between N-acetylmuramic acid residues
and 2-acetamide-2-deoxy-D-glucose in the peptidoglycan polymers which comprises the
bacterial wall. Catalysis of the reaction is illustrated below:
Egg white, breast milk, tears, the spleen, and many other tissues, including some from vegetable
sources, contain this enzyme. Lysozyme acts as an antibiotic to the human body and represents a
first line of defense.
EXERCISE 9.0: PURIFICATION OF LYSOZYME FROM EGGS (Groups of 2)
Materials
1 egg
6 TSA plate
15 mL Falcon tube
10 mL 0.05 M Tris, 0.05 M NaCl pH 8.2
Method
1. Separate the egg white from the yolk.
2. Place a double layer of a 10 cm x 10 cm square of cheese cloth over a 100 mL beaker.
Gently filter the egg white by pushing the egg white in the cheese cloth against the side of
the beaker. Do not force the egg white through.
3. Transfer 4 mL of the egg white filtrate into two 2 mL microcentrifuge tubes.
4. Centrifuge for 4 min at 12000 rpm at 4 °C in the microcentrifuge.
5. Withdraw the supernatant to a Falcon tube.
6. Add 8 mL of Tris-NaCl buffer, mix well by inversion until the mixture is homogeneous. Put
on ice.
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EXERCISE 9.1: LYSOZYME ASSAY (Groups of 2)
Materials
100 mL 0.05 M Tris, 0.05 M NaCl pH 8.2
30 mL solution of Micrococcus lysodeikticus in 0.05 M Tris, 0.05 M NaCl buffer pH 8.2 with an
optical density of 1.0 at a λ of 600nm.
10 mL of lysozyme (0.06 mg/mL) in 0.05 M Tris, 0.05 M NaCl pH 8.2
12 X 4.5 mL disposable cuvettes
15 mL Falcon tube.
Method
A. Standard curve
1. Prepare 5 mL solutions of lysozyme in Tris-NaCl buffer at the following concentrations:
6 µg/mL, 3 µg /mL, 1.5 µg /mL, 0.75 µg /mL and 0.325 µg /mL.
2. Transfer 2.0 mL of the lysozyme substrate (solution of Micrococcus lysodeikticus) to
each of 5 cuvettes.
3. Transfer 4.0 mL of Tris-NaCl buffer to the sixth cuvette (this represents the blank).
4. Add 2.0 mL of each of the lysozyme solutions prepared in step 1 to cuvettes 1-5 at 5
minute intervals.
5. Read the optical density at a wavelength of 540nm of each of the cuvettes after exactly
20 minutes of incubation at room temperature.
B. Assay of lysozyme purified from eggs
1. Prepare the following dilutions in a final volume of 5 mL of the lysozyme you purified
from the egg: 10-1, 10-2, and 10-3 in Tris-NaCl buffer.
2. Transfer 2.0 mL of the lysozyme substrate (solution of Micrococcus lysodeikticus) to
each of three cuvettes.
3. Add 2.0 mL of each of the lysozyme dilutions prepared in step 1 to cuvettes 1-3 at 5
minutes intervals.
4. Read the optical density at a wavelength of 540nm of each of the cuvettes after exactly
20 minutes of incubation at room temperature.
C. Assay of lysozyme from saliva
1. Collect approximately 2-3 mL of your saliva in a 15 mL Falcon tube.
2. Prepare the following dilutions in Tris-NaCl buffer of your saliva in a final volume of 5
mL: 10-1, 10-2, and10-3 in Tris-NaCl buffer.
3. Transfer 2.0 mL of the lysozyme substrate (solution of Micrococcus lysodeikticus) to
each of 3 cuvettes.
4. Add 2.0 mL of each of the dilutions of saliva prepared in step 2 to cuvettes 1-3 at 5
minutes intervals.
5. Read the optical density at a wavelength of 540nm of each of the cuvettes after exactly 20
minutes of incubation at room temperature.
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Microbiology Lab-2016
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-2016
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-2016
EXERCISE 9.2: ELISA OF LYSOZYME (Groups of 2)
Materials
Carbonate buffer: 3.03 g Na2CO3, 6.0 g NaHCO3, 1000 mL distilled water, pH 9.6
PBS
1% BSA
2M Sulfuric acid
Solution of polyclonal anti-lysozyme antibody from rabbit (ab391 from ABCAM) diluted
1/25000 in 1% BSA.
Solution of polyclonal anti-IgG of rabbit from goat conjugated to peroxidase (ab6721 from
ABCAM) diluted 1/120000 in 1% BSA
Substrate: TMB (EN-N301 Pierce Biotechnology Inc.; Fisher)
96 well plate for ELISA (Fisher: 07200105 Corning Incorporated)
Method
A. Antigen preparation (THIS WILL HAVE BEEN DONE FOR YOU)
1. Prepare the following antigen solutions :
a. Lysozyme at the following concentrations in carbonate buffer: 10 ng/mL, 5 ng
/mL, 2.5 ng /mL, 1.25 ng /mL, 0.625 ng /mL, 0.31 ng /mL et 0.15 ng /mL.
b. 10-1, 10-2 and 10-3 dilutions in carbonate buffer of lysozyme purified from eggs.
c. 10-1, 10-2 et 10-3 dilutions in carbonate buffer of human saliva
B. Antigen binding (THIS WILL HAVE BEEN DONE FOR YOU)
1. Transfer 50 µL of the antigen to the appropriate wells of the 96 well plate for ELISA. (See
plan below)
2. Cover the plate and incubate overnight at 4oC.
10
5
2.5 1.25 0.625 0.31 0.15
0
Lysozyme
10-1 10-2 10-3
Lysozyme from eggs
10-1 10-2 10-3
Saliva
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Microbiology Lab-2016
C. Detection
1. Withdraw the antigen by tapping the plate against paper towels.
2. Wash each well 3 times with 200 µL of PBS. Withdraw the wash by tapping the plate against
paper towels.
3. Block the plate by adding 200 µL of 1% BSA (m/v) to each well and incubate for 1h at room
temperature.
4. Wash the plate 2 times as in step 2.
5. Add 100 µL of the primary antibody to each well and incubate for 1h at room temperature.
6. Wash the plate 4 times with PBS as done in step 2.
7. Add 100 µL of the secondary antibody to each well and incubate for 1h at room temperature.
8. Wash the plate 4 times with PBS as done in step 2.
9. Add 100 µL of the substrate (TMB) to each well and incubate for 30 min at room
temperature.
10. Stop the reactions by adding 100 µL of 2M sulfuric acid.
11. Have your plate read at a wavelength of 450nm.
EXERCISE 9.3: LEUKOCYTE COUNTS (Groups of 2)
Materials
Prepared slide of human blood smear
Method
1. Examine the prepared slide of human blood in order to identify the different classes of
leukocytes.
2. Scan the prepared slide (see below) at the lowest magnification (10x) and count the number
of different leukocytes for a total of 100 counted leukocytes.
3. Calculate the percentage of each class of WBC by dividing the number of each type by the
total number of WBCs counted.
Differential white blood cell count
Cell type
# of each % of each
Neutrophils
Eosinophils
Basophiles
Lymphocytes
Monocytes
Total:
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Microbiology Lab-2016
METRIC UNITS
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Microbiology Lab-2016
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
MacConkey Agar
Peptone
Proteose peptone
Lactose
Bile salts
NaCl
Neutral red
Agar
Nitrate Broth
Digested animal proteins
Beef extract
Potassium nitrate
Starch Agar
Meat Extract
Peptic digest of animal tissue
Starch, soluble
Agar
Simmon's Citrate
Magnesium sulfate
Ammonium dihydrogen phosphate
Dipotassium phosphate
Sodium citrate
NaCl
Bromothymol blue
Agar
TSI
Meat extract
Yeast extract
Peptone
Lactose
Sucrose
Glucose
NaCl
Ferric Sulfate
Sodium thiosulfate
Phenol red
Agar
Nutrient Agar (NA)
Peptone
Yeast extract
NaCl
Agar
MRVP
Digested animal proteins
Casein
Glucose
Potassium phosphate
Urea Agar
Gelatin peptone
Glucose
Urea
Potassium dihydrogen phosphate
NaCl
Phenol red
Agar
SIM
Beef extract
Digested animal tissues
Peptonized iron
Sodium thiosulfate
Agar
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Microbiology Lab-2016
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
Ornithine decarboxylase broth
L-Ornithine Monohydrochloride
Yeast Extract
Glucose
Bromo Cresol Purple
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
Lysine Agar
L-Lysine Hydrochloride
Peptone
Yeast Extract
Dextrose
Ferric Ammonium Citrate
Sodium Thiosulfate
Bromcresol Purple
Agar
Phenylalanine Agar
Yeast extract
Sodium chloride
DL-Phenylalanine
Disodium phosphate
Agar
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