Microbiology lab 2014
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.
A 10% per day penalty will be imposed on late assignments. (Weekends will be counted as one
day). If a valid reason, such as a medical 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 2014
Marking rubric for tables
(Each cell is worth 0.25 points)
Presentation
Table 1
Table 2
Table 3
Table 4

Caption, column and row headings and body (with
data) are present
 Appropriate data included
 Table done neatly by computer
 Spaces and/or lines are used judiciously to group data
or separate table components
 Table readability (independent, no redundant text, clear
and simple)
Caption
 Begins with a table number
 First sentence is a specific and complete title
 All nonstandard symbols or abbreviations are explained
in caption
Totals
Converted grade according to points indicated on the
respective assignment
/2.0
/2.0
/2.0
/2.0
Marking rubric for graphs
(Each cell is worth 0.25 marks)
Presentation

Correct Plot type



Appropriate variables plotted
Graph done neatly by computer
Layout on page appropriate – graph filling ½ to 2/3 of the page and
scaled to use most of available space
Caption below graph in remaining 1/3 of page

Data


Axes units and scale (x- and y-axis intervals encompass data range)
Axes labels (appropriate for variables plotted) Units in parentheses
following label
 Curve fitting (best trend is shown)
Caption
 Begins with a figure number
 First sentence is a specific and complete title
Totals
Converted grade according to points indicated on the respective
assignment
Fig 1
/2.5
Fig 2
/2.5
Fig 3
/2.5
Microbiology lab 2014
Marking rubric for PPT presentations
(Each cell is worth 0.25 marks)
Presentation
 Format required was respected and correct number of pictures
provided
 Title slide was included and contained all the information
required.
Data: Quality of images
 Clear image (In focus)
 Good and uniform spread
 Uniform staining (color)
 Correct staining (color)
Caption
 Begins with a figure number
 Provided a specific and complete title
 Type of staining and stain used if appropriate
 Cell shape
 Aggregation
 Magnification
Totals
Converted grade according to points indicated on the respective
assignment
/3.0
Marking rubric for reports on unknowns
Title page: A title and the names of the authors are included.
Intro: The following questions are addressed:
Why is identification necessary?
What are the methods used? (biochemical, immunological and
molecular)
Brief description of the approaches
Results:
Tables with observations and conclusions for each unknown are
included
Flowcharts illustrating identification scheme for each unknown
are included
Discussion: A general review of each of the unknowns
physiology, importance, impact, treatment, is included for each
unknown
References: References were included in an acceptable format
Pictures of the unknowns Gram stains were included in the
report. Graded according to usual criteria
Presentation: Clarity of writing, grammar, spelling, flow,
formatting.
Total
1 point
3 points
1 point
1 point
1 point
4 points
2 points
2 points
6 points
3 points/unknown
2 points
2 points
2 points
20
Microbiology lab 2014
Assignment 1
Due date: Sept. 17
Part I: Problems. Solve the following problems. You are not required to show your calculations.
Only submit your final answers. (1 point/question)
1. What is the molarity of a solution of ammonium chloride prepared by diluting 155.0 mL of a
2.79 M NH4Cl solution to 2.5 L?
2. A student takes a sample of a KOH solution and dilutes it by adding 100.00 mL of water.
The student determines that the diluted solution is 0.016 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 25.0 mL will
produce a solution with a concentration of 0.50 M when added to 100.0 mL of water. What
should the molarity of the stock solution be?
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. 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?
6. A microbiologist has three microbial cultures: E.coli at a density of 1 X 108 cells/mL, B.
subtilis at a density of 5 X 109 cells/mL, and P. notatum at a density 2.5 X 109 cells/mL.
From these, he wishes to prepare a single mixture containing 5 X 106 cells/mL of E.coli,
1.25 X 108 cells/mL of B. subtilis, and 1 X 105 cells/mL of P. notatum in a final volume of
10mL of media. What volume of media and of each of the original cultures should be used
to achieve this?
7. 7 parts of water are added to 1 part of a 3.8 M solution of FeSO4. What is the molarity of the
diluted solution?
8. A microbiologist prepares 480 mL of a 2.50 M solution of K2Cr2O7 in water. A week later, 39
mL of water has evaporated. How many moles of K2Cr2O7 are left and what is the new
molarity of the solution?
9.
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. What is the molar mass of the unknown substance?
10. How many milliliters of a 250mg/mL chloramphenicol solution are needed for a 4 g dose?
11. A pharmacist hands you a 1.5L bottle of a 20% (m/v) NaCl solution and asks that you mix it
with sterile water to make as much of a 0.1M solution as possible. How much sterile water
would you use? (MW of NaCl 58g/mole)
Microbiology lab 2014
12. The following dilutions were performed to determine the concentration of bacteria in a
culture. What was the concentration of bacteria in the stock?
2 mL
1 mL
5 mL 10 mL
0.1 mL
stock 8 mL
4 mL
15 mL
150
colonies
10 mL
13. 52mL of sodium cyanide poison leaked from a bottle, which originally contained 100mL, into
a bucket of water containing 428mL of water. The concentration of poison in the bucket
was found to be 0.85 M after the leak. If the molecular weight of sodium cyanide is
49g/mole, how many grams of sodium cyanide remain in the bottle?
14. Concentrated hydrochloric acid has a concentration of 37.7% (m/m). What is its molar
concentration? (The density of the solution is 1.19 g/mL and MW of HCL: 36g/mole)
15. You wish to prepare a tetracycline solution of 1mg/ml in ethanol from a 10mg/ml stock
solution of tetracycline. Given that you have 10ml of the tetracycline stock solution and 5 ml
of ethanol, what is the maximum volume of the diluted solution that you can prepare?
Part II: Exercise 1.0 (3 points/question)
1. Submit graphs which show the growth yields of yeast as a function of the concentration
(expressed as % m/v) of the different sugars used in the lab.
2. According to your results, what are the optimal concentrations of each of the sugars?
Justify your answer.
3. According to your results, what is the minimum quantity in grams of each of the sugars
required to obtain the maximum yield?
4. The efficiency at which a substrate is used is expressed as the yield of the
microorganism/mole of substrate. According to this definition, which sugar was most
effectively used in your experiments? Justify your answer and show your calculations.
Sugar
Molecular weight (g/mole)
Glucose
180
Fructose
180
Galactose
180
Sucrose
342
Part III: Exercise 1.1 (3 points)
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
Microbiology lab 2014
Assignment 2
Due date: October 1st
Part I (1 point/question)
1. To assess the number of bacteria in ground beef, a 3g sample of meat is homogenized in
6mL of water resulting in a total volume of 9mL. A 1mL sample of the meat suspension is
then diluted by a factor of 104X. 1mL of the final dilution is then added to 2 mL of saline
from which 0.1mL was plated. If 100 colonies were observed on the plate, what was the
original number of bacteria/g of ground beef?
2. You perform the following serial dilutions: 1/10, 1/3, and 1:2. What are your final dilution
and your final dilution factor?
3. 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 for the above problem if the first dilution
was made by adding one mL of sample to 9 mL of diluent? Why or why not?
4. A brand of probiotic yogurt claims to contain approximately 1012 bacteria in 150mL. You
wish to verify this claim by performing a viable count on MRSA plates. If you had only two
plates available, what dilutions would you plate?
Microbiology lab 2014
5. 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
1
0
0
You also perform a viable count from the same suspension. What dilution of the suspension
should you plate to confirm the MPN result; assuming that you plate 0.1mL? Indicate the
dilution and the expected number of colonies.
6. 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 54 bacteria. How many
bacteria are there per mL in the original sample?
7. 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
10-6
10-7
10-8
10-9 10-10 10-11
# 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 between 5-10 cells per the red shaded squares in
the image shown above?
Microbiology lab 2014
8. 0.6mL of a suspension of microscopic beads, at a concentration of 6 X 104 beads/mL, is
added to 3.0mL of water. 0.05mL of this suspension is then deposited onto a slide and
overlaid with a cover slip. The total number of beads observed in the microscope's 40X
objective viewing field was 22 beads. What is the volume of the 40X viewing field?
9. In the above problem, assume that at the same magnification, 63 red blood cells were
observed. According to this information, what is the concentration of red blood cells?
10. A viable count of a bacterial culture of the bacteria shown
in this image was performed in two different ways. In the
first case, 0.1mL of a 10-6 dilution was plated and gave
rise to 115 CFU. In the second case, the culture was first
treated in a homogenizer after which 0.1mL a 10-7
dilution was plated. In this case, 102 CFU were
observed. Briefly explain the difference in the viable
counts obtained.
Part II
Exercise 2.1 (2 points/question)
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 3.0-3.2 (3 points/question)
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).
4. Show your calculation for determining the volumes of the fields of view at 40X and 100X
magnifications.
Part III
Exercises 2.5, 3.0, 3.4, and 3.5 (4 points)
Submit a PPT presentation of the following staining:
 An example of a simple stain with each of the different stains (exercise 2.5; 3 images)
 Gram stain of Lactobacillus (exercise 3.0; 1 image)
 Gram stains of E. coli and S. aureus (exercise 3.4; 2 images)
 Acid fast stain of Mycobacterium and Bacillus (exercise 3.5; 2 images)
 Simple stains of 3 fungal samples (exercise 2.3; 3 images)
Microbiology lab 2014
Assignment 3
Due date: Oct. 22
Part I (1 point/question)
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 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 2014
7.
A.
B.
C.
D.
What does Gram’s iodine reacts with?
Glucose
Starch
Amylase
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
9. You are looking at an insect with a compound microscope. The ocular lens has a
magnification of 8X and the objective lens has a magnification of 2X. What is the total
magnification?
A. 2X
B. 4X
C. 8X
D. 16X
10. Which of the following bacterial species can make heat resistant endospores and be grown
in aerobic conditions?
A. Pseudomonas aeruginosa
B. Bacillus cereus
C. Clostridium tetani
D. Streptococcus pyogenes
Part II (1.5 point/question)
Answer the questions in the PowerPoint presentation accompanying this assignment.
Part III (5 points)
Exercises 4.1 - 4.4
Submit a bar graph illustrating the following data (express data as CFU/g of compost):








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 2014
REPORT ON THE IDENTIFICATION OF GRAM POSITIVE UNKNOWNS
Due date: October 29th
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 2 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 (including 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 pictures if available
Pictures of Gram stains accompanied with a description
Tables of what tests were done, what do these test for, what observations were
made and what conclusions were drawn
Flowcharts illustrating how some or all of these results allowed to identify the
each of 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 2014
Assignment 4
Due date: Nov. 5
Part I (3 points/question)
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)
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. According to the toxic doses given, what are the therapeutic indexes for each antibiotic with
respect to each of the bacteria tested?
4. Submit a table for the results with the antibiotic combinations. Your table must include the
following data:



Concentration of the antibiotics
Estimated MIC
A legend indicating which combinations were additive, synergistic or indifferent.
Part II (3 points/question)
Exercise 6.3
5. Submit a table presenting the results for the hand washing experiment for each of the
different products assayed.
 Average CFU/cm2 for the hand (0)
 Average CFU/cm2 following 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.
Microbiology lab 2014
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.
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 2014
Assignment 5
Due date: Nov. 19
Part I
Exercises 7.0 - 7.2 (10 points)
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 (10 points)
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
Exercises 8.0
1. Indicate the identity of the Gram negative rod provided; including the family, genus and
species. (3 points)
2. Submit tables of the testes performed to confirm the identity of the Gram negative bacteria
provided. Your table should include what tests were done, what do these test for, what
observations were made, what conclusions were drawn and the expected (theoretical
results for the bacteria provided. (5 points)
3. Submit the completed Enteropluri test chart indicating the code generated and the
corresponding microorganism. (2 points)
Microbiology lab 2014
REPORT ON THE POTATO EPIDEMIC
Due date: November 26th
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 2-3 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 was the initial evidence for 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 stain 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 the identification
of 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 if any would be 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.