Microbiology lab 2016
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: [email protected]
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 2016
Grading scheme for tables
(Each cell is worth 0.25 point)
Presentation
Table 1
Table 2
Table 3
Table 4

Legend, column headings, and row headings are
presented
 Appropriate data is presented
 Tables are computer generated
 Spaces and/or lines are used wisely to group
appropriate data or to separate different
components of the table
 Reading of the table (independent, no
unnecessary text, clear and simple)
Legend
 Starts with the number of the table
 The first sentence is a specific and complete title
 All symbols and non-standard abbreviations are
explained in the legend
Totals
/2.0
/2.0
/2.0
/2.0
Grades are converted according to the points
assigned on the respective assignments
Grading scheme for graphs
(Each cell is worth 0.25 point)
Presentation





Data

Fig 1
Fig 2
Fig 3
Correct type of graph
The appropriate variables are presented
The graph was computer generated
Appropriate page layout – the graph occupies 1/2 to 2/3 of the page
The legend occupies about a third of the page and is below the graph
Units of axes (the intervals for the abscissa and the ordinate cover
the data range)
Identification of axes (appropriate for the chosen variables)
Type of curve adjustment (the best trend is illustrated)


Legend
 Starts with the number of the graph
 The first sentence is a specific and complete title
Totals
Grades are converted according to the points assigned on the
respective assignments
/2.5
/2.5
/2.5
Microbiology lab 2016
Grading scheme for microscopic images
(Each cell is worth 0.25 point)
Presentation
 The required format is respected and the correct number
of images is provided
 A slide with the title including all the information
requested is included
Data: Image quality
 Images are in focus
 Good smear, uniform spread
 Uniform staining (color)
 Good staining (correct color)
Legend
 Starts with a figure number
 Provides a specific and complete title
 Staining technique and stain used if appropriate
 Cell shape
 Aggregation
 Magnification
Totals
Grades are converted according to the points assigned on
the respective assignments
/3.0
Microbiology lab 2016
Assignment 1
Part I: Problems. Solve the following problems. You are not required to show your calculations.
Only submit your final answers. Indicate your answers at 2 digits after the decimal. Note: DO
NOT round off the results of your calculations until the final answer. (3 points/question)
1. What is the molarity of a solution of ammonium chloride prepared by diluting 155.0 mL of a
2.15 M NH4Cl solution to 2.5 L? 0.13M
2. A student takes a sample of a 3.1 M KOH solution and dilutes it by adding 100.00 mL of
water. The student then dilutes this solution 3X and determines that the concentration of the
final solution is 0.016 M KOH. What was the volume of the original sample? 1.55mL
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. What
should the molarity of the stock solution be? 3.0M
4. What volume of water should be added to 2.5 mL of a 9.0 M acetic acid solution in order to
obtain a final concentration that is 3.0 M acetic acid? 5.0mL
5. Three solutions "A", "B", and "C" are mixed to obtain the following ratio: A:B:C = 1:2:10.
What are the dilution factors for each of these compounds? 13X, 6.5X and 1.3X
respectively
6. A microbiologist has three microbial cultures: E.coli at a density of 2 X 108 cells/mL, B.
subtilis at a density of 2 X 109 cells/mL, and P. notatum at a density 1 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 107 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?
0.25, 0.63, 0.1 and 9.02 mL respectively
7. The microbiologist mentioned in the previous problem realizes that he only has 5 mL of
media. Given this information, what is the maximum volume of the microbial mixture
previously described that can be prepared? 5.54mL
8. 3 parts of water are added to 2 parts of a 2.5 M solution of FeSO4 and 1 part of a 1.0 M
solution of FeSO4. What is the molarity of the diluted solution? 1.0M (or 0.98M)
9. A microbiologist prepares 150 mL of a 3.5 M solution of K2Cr2O7 in water. A week later, 30
mL of water has evaporated. How much water must the microbiologist add to the
evaporated solution in order to obtain a final molarity of 0.5M K2Cr2O7? 930 mL
10. A chemical test has determined the concentration of a 100 mL solution of an unknown
substance to be 5.0 M. The solution is totally evaporated, leaving 45 g of crystals of the
unknown solute. What is the molar mass of the unknown substance? 90.0 g/mole
11. How many milliliters of a 50mg/mL chloramphenicol solution are needed for a 400 µg dose?
0.008mL
Microbiology lab 2016
12. A pharmacist hands you a 1.0L bottle of a 5.8% (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 58.0 g/mole) 9.0L
13. 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
0.2 mL
stock 6 mL
8 mL
20 mL
150
colonies
2.7 X 105
10 mL
14. 50mL of sodium cyanide poison leaked from a bottle, which originally contained 100mL, into
a bucket of water containing 300mL of water. After the leak the concentration of poison in
the bucket was found to be 0.1 M. If the molecular weight of sodium cyanide is 60g/mole,
how many grams of sodium cyanide remain in the bottle? 2.1g
15. 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)
12.46M
16. What volume of water would be required to prepare a solution containing 300 grams of a
compound (M.W. 50g/mole; density: 1.5g/mL) which was at a final concentration of 3.0 M?
1.8 L
In the following problems, assume that the volume of solvent is equal to the volume of
the solution.
The following diagram represents two solutions (A and B) separated by a water permeable
membrane but which is impermeable to solutes. Use this as the initial condition to answer
questions 17-20.
A.
B.
480 osmoles
320 osmoles
10 litres
6 litres
17. What are the osmolarities of solutions A and B in the original condition? 48 OsM and 53
OsM
18. What is the osmotic relationship of solution A relatively to solution B in the original
condition? hypoosmotic
19. What will be the osmolarity in compartment A once osmosis is complete? 50.0 OsM
20. What volume of water was displaced in or out of the A compartment? 0.40L
Microbiology lab 2016
21. Calculate the osmolarities of the following solutions : (Note: All solutes are impermeable
except for urea)
0.05 M Na2CO3 + 1M urea (1.15 OsM)
0.5 M Al(NO3)3 (2.0 OsM)
0.14 M LiBr (0.28 OsM)
0.15 M glucose (C6H12O6) + 0.2M NaCl (0.55 OsM)
22. The osmolartiy of blood cells is 0.28 OsM. Indicate whether blood cells are isotonic,
hypotonic, or hypertonic relative to each of the solutions indicated in question 21.
(hypertonic, hypotonic, isotonic, hypotonic)
23. What would happen to red blood cells introduced into each of the solutions indicated in
question 21? (Crenation, hemolysis, or nothing) (hemolysis, crenation, nothing, crenation)
24. How many grams of KCl must you dissolve in 1 L of water in order to obtain a solution that
is isosmotic to blood cells? (M.W. of KCl 74.55 g/mole) (10.44 g KCl)
25. What are the osmotic and tonic relationships of a cell with an osmolarity of 300 mOsM
relative to the following solutions:
The cell is
hypo
The cell is
iso
osmotic and
osmotic and
*Note: urea is permeable.
hypo
hyper
tonic relative to 300 mM NaCl.
tonic relative to 300 mM of urea.
Microbiology lab 2016
Part 2: Experimental data and their interpretation (4 points/question)
EXERCISE 1.0: GENERATING A STANDARD CURVE AND DETERMINING AN UNKNOWN
CONCENTRATION OF METHYLENE BLUE
1. Show your calculation as to how 5mL of a 0.4mM solution of methylene blue was prepared
from a stock solution of 0.26% (m/m).
2. Complete the following table :
Solution
Volume of
methylene
blue (mL)
Volume
of water
(mL)
Total
volume
(mL)
Final
dilution
factor
No1
0.3
4.7
5
20.3
0.0013
No2
4.8
1.2
6
25.4
0.0010
No3
2.5
3.0
5.5
55.88
0.00047
No4
2.0
1.5
3.5
97.79
0.00027
No5
0.8
0.5
1.3
158.9
0.00016
No6
1.5
1.0
2.5
264.8
0.000071
Abs
550nm
Final methylene
blue concentration
(% m/v)
UNK 1
UNK 2
3. Submit a standard curve generated with Excel which represents the relationship between
the concentration of methylene blue (% m/v) and the absorbance at 550nm. Present the line
of best fit. Indicate the equation of the line and the R2 coefficient. Check general directives.
1 point for R2 value ≥ 0.9
Microbiology lab 2016
EXERCISE 1.1: DIFFUSION, OSMOSIS AND TONICITY IN RED BLOOD CELLS
4. Complete the following table :
Sucrose
NaCl
Glycerol
Molarity
(M)
0.1
Osmolarity
(OsM)
0.1
0.2
0.2
0.3
0.3
0.4
0.4
0.5
0.5
0.065
0.13
0.1
0.2
0.15
0.3
0.25
0.5
0.3
0.6
0.1
0.1
0.2
0.2
0.3
0.3
0.4
0.4
0.5
0.5
Hemolysis
(+/-)
5. According to your results, what is the approximate internal osmolarity of red blood cells?
Justify your answer.
Value between lysis and plasmolysis.
6. According to your results, which solute is most probably permeable to the cell membrane?
(1 point)
Glycerol
EXERCISE 1.4: STREAKING FOR SINGLE COLONIES
7. Before handing in this assignment, ask a teaching assistant to evaluate your streakings for
single colonies.
Microbiology lab 2016
Assignment 2
Part 1: Problems and theoretical questions. You are not required to show your calculations.
Only submit your final answers. Indicate your answers at 2 digits after the decimal. Note: DO
NOT round off the results of your calculations until the final answer. (3 points/question)
1. To assess the number of bacteria in ground beef, a 5 g sample of meat is homogenized in
45mL of water resulting in a total volume of 50 mL. A 2mL sample of the meat suspension
is then diluted by a factor of 104X. 1 mL of the final dilution is then added to 2 mL of saline
from which 0.1 mL was plated. If 100 colonies were observed on the plate, what was the
original number of bacteria/g of ground beef? 3 x 108cfu/g
2. You perform the following serial dilutions: 1/12, 1/2.5, and 1:4. What are your final dilution
and your final dilution factor? 150X and 1/150
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.) 5.8 x 107cfu/ml (concentration = 5.8 x
105cfu/ml)
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? No, same
dilution or dilution factor
4. A brand of probiotic yogurt claims to contain approximately 109 bacteria in 150 mL. You
wish to verify this claim by performing a viable count. If you had only two plates available,
0.1 mL of what dilutions would you plate? 10-3 and 10-4
Microbiology lab 2016
5. You setup an MPN to determine the number of bacteria/gram of Camembert cheese. To do
so, 10 g of cheese are homogenized in a final volume of 100 mL. The suspension is then
used to perform a three tube MPN assay. You obtain the results indicated in the table
below.
Dilution
# of positive tubes
10-2
10-3
10-4
10-5
10-6
10-7
3
3
1
2
1
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. dilution of 10-3; (needs to be between 30 and
300 CFU)
6. One mL of E. coli is mixed with 3 mL of dye. A drop of this dilution is placed on a
haemocytometer slide's counting chamber. Three large squares (Yellow colored 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? 3.28 x 107 bact./mL
7. 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 of a
10-7 dilution was plated. In this case, 102 CFU were
observed. Briefly explain the difference in the viable
counts obtained.
Before homogenization aggregates represent more than
one original cell/CFU
8. Which objective provides the smallest field of vision?
100X
40X
10X
Microbiology lab 2016
The size of the field of vision is the same with all the objectives.
9. A specimen is examined under the microscope with an ocular of 4X and an objective of
10X. A measurement of the specimen within the field of vision was 0.01 mm. What is the
original size of the specimen? 0.25µm
10. If the ethanol wash was not done in the Gram staining procedure, what color would bacteria
of the genus Bacillus and bacteria of the genus Salmonella be?
Purple and red respectively
Red and purple respectively
Blue and red respectively
Both would be red
Both would be purple
11. Let’s assume that for a prolonged period of time you diet was deficient in niacin; an
essential vitamin which the human body cannot synthesize. What would be the direct effect
of this deficiency on each of the following metabolic pathways? (A decrease in the activity,
an increase in the activity or no effect)
Glycolysis decrease
The Krebs cycle decrease
The electron transport chain no effect
12. A bacterial culture is sampled at two time points representing an interval of 1 hour and 10
minutes. 3.2 x 106 cells/mL were present in the first sample and 5.12 x 107 cells/mL in the
second sample. How many times did the population double within the time span examined?
4 times
13. Cultures from the tissues of a patient with syphilis are done on samples taken at an interval
of one week. The first sample had 5000 CFUs, whereas the second sample had 32000
CFUs. Four days later, a third sample had 58000 CFUs. Is the population in exponential
growth?
No
14. A biologist studies a newly discovered bacterial species. At time t = 0 h, he introduces one
hundred bacteria in a favorable growth media. Six hours later, he counts 450 bacteria.
Assuming exponential growth, what is the growth rate (µ) of these bacteria?
0.25/hr
15. A bacterial population doubles every 3 hours. Given that there were initially 100 bacteria,
how many bacteria would there be after one day and a half? 409600 bact.
Microbiology lab 2016
Part 2: Experimental data and their interpretation
EXERCISE 2.0 – 2.3: BACTERIAL COUNTS IN SOIL (5 points)
1. Submit your calculations for the determination of the number of bacteria per gram of soil.
2. Submit your calculations for the determination of the number of actinomycetes per gram of
soil.
3. Submit your calculations for the determination of the number of fungi per gram of soil.
EXERCISE 2.4: DIRECT COUNT OF A YEAST SUSPENSION (5 points)
4. Submit your calculations for the determination of the number of yeast cells per milliliter in the
original suspension.
EXERCISE 2.6 – 2.10: MICROSCOPY (3 points/section for a total of 15 points)
5. Submit a PPT presentation to the address indicated in the general directives which includes
the images taken for exercises 2.6 - 2.10.
EXERCISE 3.0: E.COLI GROWTH CURVE (5 points/question)
6. Submit a graph with two growth curves (room temperature and 37oC) which respects the
following criteria :
o Changes in optical density as a function of time
o Each curve must have 32 time points representing the interval T0 – T16h
o Consider the cultures inoculated at 10 o clock = T0, those inoculated at 11
o’clock =T1h, those inoculated at 12 o cock = T2h and those inoculated at 1
o’clock = T2h
7. Determine from your profiles the generation time and the growth rate constant of the two
cultures.
EXERCISE 3.1: MPN OF BACTERIA IN SOIL (5 points)
8. Complete the following table and submit your calculations which shows the determination of
the MPN per gram of soil.
Dilution
1
Growth : Tube
2
3
Microbiology lab 2016
EXERCISE 3.2: YEAST FERMENTATION BIOASSAY (5 points/question)
9. Submit a graph with 4 curves illustrating the production of moles of ethanol as a function of
time for each of the carbon sources.
10. Submit a table presenting the rates of ethanol production (moles/min.) and the final yields
after one hour as a function of the carbon source (moles of ethanol/mole of the carbon
source) for each of the carbon sources.
11. Use a schematic to illustrate the pathway used for ethanol production from each of the
carbon sources. Your pathways must show the reducing equivalents and the ATP produced.
Ex.
1 Glucose + 2 NAD+
2 Pyruvate + 2 NADH + 2 ATP (net)
2 Ethanol + 2CO2 + 2 NAD+
1 Glycerol + 2 NAD+
1 Pyruvate + 1 NADH + 1 ATP (net)
1 Ethanol + 1CO2 + 1 NAD+
1 fructose + 2 NAD+
2 Pyruvate + 2 NADH + 2 ATP (net)
2 Ethanol + 2CO2 + 2 NAD+
1 Acetate
Is not fermented, goes directly in Krebs cycle