Microbiology lab 2013
Assignments
General Directives
The first page must include the following information:
 The assignment number
 Course code :BIO3126
 Your name or names
 Your group number
 The date
Assignments may be done and handed in individually or in groups of two (you and your partner).
Assignments must be typewritten except for calculations which can be done by hand.
Tables and graphs must be computer generated, clear and concise.
Tables and graphs must have an appropriate title and a caption if appropriate
Only hard copies of the assignments will be accepted. DO NOT EMAIL assignments.
Assignments must be handed in to the appropriate teaching assistant at the indicated due date
BEFORE you leave the lab.
No late assignments will be accepted independent of the reason given. If a valid reason is
given, you will be exempted from doing the required assignment.
PowerPoint presentations:
Use the following black and white template format for your PPT presentations
Pictures must be in color
Save as a PDF file with one slide per page
Submit by email to the following address: microassignment@yahoo.ca
The subject line of the email should indicate: Assignment (number)
Do not send other emails to this address. These will not be read or answered.
Microbiology lab 2013
Assignment 1
Due date: September 18th
Part I: Problems. Solve the following problems. You are not required to show your calculations.
Only submit your final answers.
1. What is the molarity of a solution of ammonium chloride prepared by diluting 50.0 ml of a
3.79 M NH4Cl solution to 2.0 L?
2. A student takes a sample of KOH solution and dilutes it by adding 100.00 ml of water. The
student determines that the diluted solution is 0.046 M KOH. Given that the concentration of
the original solution was 2.09 M. What was the volume of the original sample?
3. A microbiologist wants to prepare a stock solution of H2SO4 so that samples of 20.0 ml will
produce a solution with a concentration of 0.50 M when added to 100.0 ml of water.
a. What should the molarity of the stock solution be?
b. If the microbiologist wants to prepare 5.0 L of the stock solution from
concentrated H2SO4, which is 18.0 M, what volume of concentrated acid should
be used?
4. What volume of water should be added to 1.19 ml of an 8.0 M acetic acid solution in order
to obtain a final concentration that is 1.5 M acetic acid?
5. What volume of a 5.75 M formic acid solution should be used to prepare 2.0 L of a 1.0 M
formic acid solution?
6. Three solutions "A", "B", and "C" are mixed to obtain the following ratio: A:B:C = 1:2:27.
What are the dilution factors for each of these compounds?
7. A solution of concentrated nitric acid is at a concentration of 71% HNO3 (m/v). What volume
of concentrated HNO3 would be needed to prepare 10.0 L of a 2.00 M solution of HNO3?
(M.W. of HNO3: 63g/mole)
8. 7 parts of water are added to one part of a 3.8 M solution of FeSO4. What is the molarity of
the diluted solution?
9. A microbiologist prepares 480 ml of a 2.50 M solution of K2Cr2O7 in water. A week later, 39
ml of water has evaporated. What is the new molarity of the solution?
10. A chemical test has determined the concentration of 100 ml of a solution of an unknown
substance to be 2.41 M. The solution is totally evaporated, leaving 9.56 g of crystals of the
unknown solute. Calculate the molar mass of the unknown substance.
Microbiology lab 2013
Part II: Exercise 1.0
1. Submit a table which presents the following data for the aerobic and anaerobic conditions:
Volume (mL) of 0.2%
glucose or lactose
Volume (mL) of
water
Final conc. of glucose
or lactose %(m/v)
Yield
O.D. 600
Glucose
Lactose
+O2
-O2
2. Submit graphs which show the growth yields as a function of the concentration of glucose
and lactose aerobically and anaerobically.
3. According to your results, what are the optimal concentrations of glucose and lactose under
aerobic and anaerobic conditions? Justify your answer.
4. According to your results, which carbon source and under which condition is used the most
effectively? Justify your answer
Part III: Exercise 1.1
Submit a PPT presentation of the following images:




Letter "e" at 10X, 40X and 100X
Cheek cells at 10X, 40X and 100X - localize and identify the cytoplasm, cell membrane
and the nucleus on the image for the 100X objective
Yeast cells at 10X, 40X and 100X
Yogurt bacteria at 10X, 40X and 100X
Microbiology lab 2013
Assignment 2
Due date: October 9th
Part I
1. If you add 2 ml of a bacterial suspension at a concentration of 2 x 109 cells/ml to 13 ml of
water what would be the new cell concentration?
2. Draw a schematic illustrating how to make a 1/10,000 dilution. Maximum and minimum
volumes are 10 ml and 0.1 ml respectively.
3. Draw a schematic illustrating how to make a 1/7.2 dilution. Maximum and minimum
volumes are 10 ml and 0.1 ml respectively.
4. You perform the following serial dilutions: 1/10, 1/3, and 1:2. What is your final dilution?
What is the final dilution factor?
5. After diluting your culture 1/2,500, you plate 0.1 ml and get 154 colonies. What was the
initial concentration?
6. Consider the following dilution scheme:
a. Report the total number of CFUs in the entire 100 ml amount of the original lake water
sample. (TNTC=too numerous to count.)
b. Would you expect any difference in the answer of the above problem if the first dilution
was made by adding one ml of sample to 9 ml of diluent? Why or why not?
7. A brand of probiotic yogurt claims to contain approximately 106 bacteria in 150ml. You wish
to verify this claim by performing a viable count on TSA plates. If you had only two plates
available, what dilutions would you plate?
Microbiology lab 2013
8. You setup an MPN to determine the number of bacteria per gram of Camembert cheese.
To do so, 10g of cheese are homogenized in a final volume of 100ml. The suspension is
then used to perform a three tube MPN assay. You obtain the results indicated in the
table below.
Dilution
10-2
10-3
10-4
10-5
10-6
10-7
# of positive tubes
3
1
2
0
1
0
You also perform a viable count from the same suspension. What dilution of the above
suspension should you plate to confirm the MPN result; assuming that you plate 0.1ml.
Indicate the dilution and the expected number of colonies.
9. One ml of E. coli is mixed with 3ml of dye. A drop of this dilution is placed on a
haemocytometer slide's counting chamber. Three large squares (Yellow coloured square
on image below) are counted giving the following results: 46, 50, and 58 bacteria. How
many bacteria are there per ml in the original sample?
10. It is strongly recommended that honey not be fed to young children below the age of one
because of the potential presence of spores from the bacteria Clostridium botulinum which
causes food infections. To verify for the presence of this bacterium in a new batch of honey
you perform an MPN count as follows. A solution of 1% honey (v/v) is used as a starting
solution to carry out duplicate MPN assays. In one case, before the incubation of the
broths, the tubes are pre-treated by heating them at 95oC for 15 minutes and then
incubated at 37oC for 48 hours. In the second case no pre-treatment was done. The
following results were recorded in both assays:
Dilution
10-3
10-4
10-5
10-6
10-7 10-8
# of positive tubes (Untreated)
3
3
3
3
2
0
# of positive tubes (Treated)
2
1
2
1
0
0
You also perform a direct count on the 1% (v/v) honey solution. What dilution should you
apply to the counting chamber to obtain an average of 15 cells per the red shaded
squares in the image shown above?
Microbiology lab 2013
Part II
Exercise 2.1
1. Have your single colony isolation evaluated.
2. Show your calculation for the viable count of E. coli. Indicate the original concentration
(CFU/ml).
Exercises 3.0-3.1
1. Show your calculation for the MPN of bacteria of the genus Lactobacillus in yogurt. Indicate
the original concentration (cells/g).
2. Show your calculation for the viable count of bacteria of the genus Lactobacillus in yogurt.
Indicate the original concentration (CFU/g).
3. Show your calculation for the direct count of yeast. Indicate the original concentration
(cells/ml).
Part III
Exercises 2.5, 3.0, 3.4, and 3.5
Submit a PPT presentation of the following staining:
 An example of a simple stain with each of the different stains (exercise 2.5)
 Gram stain of Lactobacillus (exercise 3.0)
 Gram stains of E. coli and S. aureus (exercise 3.4)
 Acid fast stain of Mycobacterium (exercise 3.5)
Part IV
Exercises 4.1 - 4.4
Submit a bar graph illustrating the following data:








Aerobic heterotrophic count
Anaerobic heterotrophic count
Gram positive bacterial count
Gram negative bacterial count
Count of bacteria which do mineralization
Count of denitrifying bacteria
Count of bacteria which degrade starch
Count of bacteria which degrade gelatine
Microbiology lab 2013
Assignment 3
Due date: November 6th
Part I
Choose the best answer for each of the following multiple choice questions:
1.
A.
B.
C.
D.
Which of the following best describes TSA medium (Tryptone soya agar)?
A general purpose medium.
A selective medium.
A differential medium.
A selective and differential medium.
2.
A.
B.
C.
D.
Which of the following best describes Columbia CNA medium?
A general purpose medium.
A selective medium.
A differential medium.
A selective and differential medium.
3.
A.
B.
C.
D.
What is the selective agent in MacConkey agar?
Methylene blue.
Colistin.
Malachite green.
Crystal violet.
4.
A.
B.
C.
D.
What carbon source is common to CNA, MacConkey and TSA media?
Proteins.
Glucose.
Lactose.
None of these carbon sources are common to all three media.
5.
A.
B.
C.
D.
Which of the following bacteria would not grow on CNA of Columbia agar?
Clostridium perfringens.
Proteus vulgaris.
Lactobacillus casei.
Micrococcus luteus.
6.
A.
B.
C.
D.
Which type of microorganism would be expected to grow only in an anaerobic chamber?
Decomposers
Nitrogen fixing bacteria
Nitrifying bacteria
Denitrifying bacteria
Microbiology lab 2013
7.
A.
B.
C.
D.
Gram’s iodine is used on the starch plates for what purpose?
To detect the presence of glucose
To detect the presence of starch
To detect the presence of amylase
To detect the presence of cellulose
8. In order to be metabolized, gelatine must be degraded outside of the cell into simpler units
that can be transported inside the cell. These units would be…?
A. Amino acids
B. Carbon
C. Glucose
D. Carbohydrates
Part II
Answer the questions in the PowerPoint presentation accompanying this assignment.
Microbiology lab 2013
Part III
Exercises 6.0 - 6.2
1. Submit a table presenting the results from the Kirby Bauer assay for the three bacteria
assayed. Your table should include the following information:





Name of bacteria
Name of antibiotic
Susceptibility to the antibiotic (resistant, intermediate or sensitive)
The data (from the table on Pg 73) which allowed you to reach your conclusion as to the
susceptibility to each of the antibiotics tested.
A caption indicating which antibiotics would be recommended, if any, to treat an
infection with the bacteria assayed.
2. Submit a table presenting the MICs and MBCs (if applicable) of the three bacteria to each
of the antibiotics assayed in the class.
3. Assuming that the toxic doses of all the antibiotics assayed were the same, which antibiotic
would have the highest and lowest therapeutic index. Show your reasoning.
4. Submit a table of the qualitative results of the bioluminescence assay. Your table must
include the following data:



Concentration of the antibiotics
Estimated MIC (Lowest light emission)
A legend indicating which antibiotic combinations would be recommended, if there
are any, to treat an infection with the bacteria being evaluated.
Exercise 6.3
5. Submit a table presenting the results for the hand washing experiment.



Average CFU/cm2 for the hand (0)
Average CFU/cm2 for each of the wash treatments used (0, 1, and 2)
Average percent reduction (between 0 and 1 as well as 0 and 2)
6. In a caption to the above table write a brief conclusion of the results obtained.
Read the research article available on this course’s web site and answer the
following questions:
7. Several conditions are known to influence the effectiveness of a disinfectant; name two.
8. If the initial bioburden of a table surface was 3 x 107 CFUs of Bacillus subtilis, how much
time would be required to reduce the population to 3 x 10-6 CFUs using a 0.025% (m/v)
solution of sodium hypochlorite? Show your calculation.
Microbiology lab 2013
9. Heat was used to follow the rate of mortality of two bacteria, Bacillus subtilis and
Escherichia coli. The data obtained is available in an Excel file available on this course’s
web site. Using the information you’ve acquired from the research article, calculate the D
values for each bacteria.
10. Which of the two bacteria is most probably Bacillus subtilis?
Microbiology lab 2013
REPORT ON THE IDENTIFICATION OF BACTERIAL UNKNOWNS
Due date: November 20th
Each person or teams of two will write and hand in a paper reporting their findings. The paper
should include the following:
A. Title page : Title and authors names
B. Introduction: This section should be at most a couple of paragraphs long and treat the
following topics:




What were the goals of these experiments
Why are isolation and identification of bacterial organisms important
What was the general approach used to achieve your goals
Compare and contrast standard techniques used in this course to non
microbiological techniques (example: Molecular and immunological approaches)
C. Results: Divide this section into different sub sections; one for each family investigated.
Each section should include in a clear and organized fashion:




Colony appearance and picture if available
Pictures of Gram stains accompanied with a description
What tests were done, what do these test for, what observations were made and
what conclusions were drawn (use tables as much as possible)
Flowcharts illustrating how some or all of these results allowed to identify the
unknown organism
D. Discussion: In this section you should write (at most half a page for each organism) a
detailed description of the organisms you identified. You should find information
regarding their general characteristics, physiology, environmental impact (if any), uses,
clinical significance, and treatment.
E. References: List all references used according to the style recommended by the Journal
of Bacteriology.
F. General format: Reports should be typed, 1.5 lines spacing, using a font of Times New
Roman of 12 pts.
Microbiology lab 2013
Assignment 4
Due date: November 27th
Part I
Exercises 7.0 - 7.2
1. Submit a table summarizing the results for the microbiological quality of the different water
samples supplied to the class. Your table must present the following information:





Water source
Presumptive MPN for coliforms
Confirmed MPN for coliforms
Presumptive MPN for Enterococci
Whether each water sample is suitable or not for drinking or recreation
Exercises 7.4 - 7.6
2. Submit a table summarizing the results for the microbiological quality of the chicken and the
lettuce. Your table must present the following information:




Average counts (CFU/g): aerobic, E. coli and specific pathogens for the fresh and
stored chicken samples distributed in the class (use the confirmed counts whenever
available)
Average counts (CFU/g): aerobic, E. coli and specific pathogens for the fresh and
stored lettuce samples distributed in the class (use the confirmed counts whenever
available)
To which class does each of the food products belong too
Justify your conclusions in an accompanying legend
Microbiology lab 2013
Exercise 9.0
3. Complete the following tables:
Identity of partners exchanges were
performed with
First
Second
Third
Group
number
Number of
exchanges
1
3
J
G
2
2
C
F
3
2
A
B
4
3
D
E
5
1
E
6
2
B
7
1
I
8
3
H
Group
number
Number of
exchanges
9
3
T
Q
10
2
M
P
11
2
K
L
12
3
N
O
13
1
O
14
2
L
15
1
S
16
3
R
ELISA result
Positive or
Negative
F
B
I
C
G
Identity of partners exchanges were
performed with
First
Second
Third
P
L
S
M
Q
ELISA result
Positive or
Negative
Microbiology lab 2013

Indicate for each of the two groups in the class (1-8 and 9-16) which individuals were
initially infected

Propose a possible path illustrating the transmission of the pathogen in one of the two
groups in the class.

Indicate the number of individuals to which each student of your group was directly and
indirectly exposed too.
4. Consider the following hypothesis: The risk of an infection by a sexually transmitted
pathogen increases with the greater the number of unprotected sexual relationships that he
has and if the partners of this individual have had unprotected sexual relationships with
multiple partners.
Evaluate this hypothesis by comparing the results predicted from this hypothesis to the
results observed.
Exercise 9.1
5. Indicate the following data for each of the scenarios performed with the computer simulated
epidemic:



After how many days does the number of afflicted people become epidemic?
What percentage of the population must have contracted the disease before the
effects of herd immunity are observed?
How long does the epidemic last?
6. Briefly explain how and why each of the following factors influence the progression of the
epidemic:



Virulence
Transmission
Duration of the disease
Microbiology lab 2013
REPORT ON THE POTATO EPIDEMIC
Due date: December 3rd
Each person or teams of two will write and hand in a paper reporting their findings. The paper
should include the following:
A. Title page : Title and authors names
B. Introduction: This section should be at most a couple of paragraphs long and treat the
following topics:



What were the goals of these experiments
Which of Koch's postulates were tested
What were the approaches used to achieve your goals
C. Results: Divide this section into different sub sections; one for each goal. Each section
should include in a clear and organized fashion:






What were the initial evidence of the presence of an infection. Provide a detailed
comparative description of the observations collected
What approaches were used to isolate and confirm the presence of the potential
pathogen
What was the range of susceptible hosts to the pathogen
Identification of the pathogen : Gram stained accompanied by a description
What tests were done, what do these test for, what observations were made and
what conclusions were drawn (use tables as much as possible)
A Flowchart illustrating how some or all of these results allowed to identify the
unknown organism
D. Discussion: In this section you should write (at most one page) a detailed description of
the pathology associated with the infection by the organism you have identified. You
should find information regarding its general characteristics, physiology, environmental
impact (if any), and which preventive measures are recommended.
E. References: List all references used according to the style recommended by the Journal
of Bacteriology.
F. General format: Reports should be typed, 1.5 lines spacing, using a font of Times New
roman of 12 pts.