RHODES UNIVERSITY

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

EXAMINATION: JUNE 2022

MICROBIOLOGY 201
PRACTICAL PAPER 2

Examiners: Prof Dames, Dr Abrahams, MARKS: 50

Dr Fogel, Prof Knox DURATION: 1.5 hours
COURSE SUB-
MINIMUM: 40 %

Answer ALL questions

GENERAL INSTRUCTIONS TO CANDIDATES:

1. Answer ALL questions.

2. Time management is very important. The value of the mark for each question should be
used as a rough guide to the amount of time allocated to answer the question (50 marks in
90 minutes).

3. It is in the candidate’s interest to write legibly.

4. At the end of the examination, place all answer books inside answer book 1.

5. The Oxford Concise English dictionary may not be used during this examination.

PLEASE DO NOT TURN OVER THIS PAGE UNTIL TOLD TO DO SO.

Microbiology 201 Paper 2 Page 1 of 6

 QUESTION 1
Figure 1 below illustrates a commonly used method in Microbiology. Answer the related
questions.

Figure 1

a) What method is being illustrated? 1

b) The bacterial loads in each of the tubes (a to d) are how much less than the Sample? 4

c) The bacterial load in tube d is how much less than in tube a? 1

d) What is the total volume (in ml) in the tube marked c? 1

e) An 100 µl aliquot from tube d was spread plated onto Nutrient Agar. After incubation,
79 colonies were counted. Calculate the number of colony forming units (CFUs/ml).
Show all calculations. 3

10

QUESTION 2
You have been given a nutrient agar culture of Escherichia coli. Answer the following
questions.

a) With the aid of a diagram, describe how you would conduct a Gram Stain. 6

b) What is the purpose of the Gram staining reagents in differentiating between Gram-
positive and Gram-negative bacteria? 4

c) Describe what you would expect to see on examination of your Gram stain under
the compound microscope at 1000 x magnification. 2

12

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 QUESTION 3
While on a field trip, you are tasked with isolating a thermophilic chemolithotroph that
uses hydrogen sulphide as its only source of energy and electrons.
a) Describe how you would go about isolating such an organism. In your answer, include
a description of the environment you would obtain your samples from, the media
components that you would use to cultivate the microbe(s), and the environmental
conditions you would use to maximise the growth of your microbial isolates. 3

b) What alterations to your incubation system would you make to isolate microbes that
utilise hydrogen sulphide as their source of electrons during anaerobic respiration? 1

c) How could you tell which process (anaerobic respiration or fermentation) is taking
place through an analysis of the end-products present in the spent growth medium? 2

d) Would your incubation system need to be placed in a lighted growth room or

incubator? Why? 1

e) Which metabolic pathway(s) do you think your organism would use to generate ATP?
Explain your answer. 2

f) Is your system more likely to yield archaeal or bacterial isolates? Why? 1

10
QUESTION 4
EcoPlates are used to test the metabolic capabilities of microbial strains and mixtures, such
as microbial communities found in soil. An EcoPlate is a 96-well plate (Figure 2). Within
the wells are three repeated sets of 31 different carbon sources, along with a tetrazolium
dye. Samples of cells are then incubated inside the wells of the EcoPlate in order to
determine which carbon sources can be used by the sample.

Figure 2. Example of an EcoPlate after adding a microbial sample and incubating it

for several days.

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 a) Describe how the tetrazolium dye used in the EcoPlate works to identify potential
sources of organic carbon that can be catabolised by your sample and why we
measure the responses of the EcoPlate as the absorption of light at a wavelength of
590 nm. 2

b) Your team dilutes your sample 10-3-fold, as recommended, and adds the diluted
microbial sample to the Ecoplate. Just as you start incubating your EcoPlate, your
practical partner confesses that they accidentally prepared a 10-6 dilution of the
sample to add to the EcoPlate, instead of a 10-3 dilution. Describe how this change
will impact the results you obtain, why it will impact the results, and suggest changes
to the experiment to address this change so that you can still generate data. 3

c) You start processing the raw absorbance data from your EcoPlate experiment (a
section of this data appears in Table 1). 1

Table 1: EcoPlate absorbance values (After five days’ incubation, with the
Day 0 values already subtracted)

Plate Well Carbon Source A590nm

A1 - (control) 0.150
A5 - (control) 0.168
A9 - (control) 0.142
E1 Tween®80 0.458
E5 Tween®80 0.550
E9 Tween®80 0.492

Use the data in Table 1 to calculate the average metabolic response of your microbial
sample when supplied with Tween®80. Show all necessary calculations.

d) If the average response of your sample when supplied with sucrose was an
absorbance of 0.550 [after you’ve calculated the response for this carbon source as
you do for Tween®80 in QUESTION 4 (c)], what does this suggest about the ability
of the sample to metabolise sucrose compared to Tween®80? 1

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 e) Part of your experiment aimed to compare the growth and metabolic activity of a
single strain of bacteria, compared to a mixed community of microbial organisms.

To compare the growth, you prepare various types of solid media and spread-plate
50 µl of the 10-3-fold diluted samples onto the different media. The results for this
are summarised in Table 2 below:

Table 2: Observed colonies for the 50 µl of 10-3-fold diluted samples

CFU/ml observed on the media

Media type Single-strain sample Mixed community sample
Nutrient Agar 4.23 × 105 X
(830 colonies observed)
5
MacConkey Agar 3.93 × 10 4.10 × 105

You forgot to convert the counted colonies for the mixed community grown on
Nutrient Agar (X in Table 2) sample. Calculate the CFU/ml from this plated sample.
Show all necessary calculations. 1

f) What can you deduce about the outer wall structure of the single-strain sample,
given the information in Table 2? 1

g) When comparing the EcoPlate responses between the two samples, would you
expect the single-strain sample to use a wider range, or a narrow range, of carbon
sources compared to the mixed community? Why? (2 sentences, maximum). 1

10
QUESTION 5
You are a virologist working in a pathology laboratory. You receive a nasal swab sample
from a patient who has suffered from a viral respiratory infection, probably the “flu”. Your
task is to perform a plaque assay to estimate the number of infectious virus particles in the
sample. Answer the following questions:

a) To go about performing the assay, you need to choose a host cell type. What cells
do you think would be appropriate and why? 1

b) You then plan your experiment and decide to perform the following steps:

1. Enrichment of the nasal sample

2. Serial dilution of the virus suspension
3. Mixing of viral dilutions with a culture of host cells and incubation for 30
minutes
4. Pouring of virus/host cell/agar samples onto a fresh lawn of host cells and
incubation overnight

Describe why you have carried out each of steps 1-4. 4

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 c) After performing the plaque assay, you obtain the interesting results shown in
Table 3 below. Do you think these results are expected? Explain your answer. 3

Table 3. Results of Plaque Assay

Virus dilution Observations

10-1 A lawn of host cells
10-2 About 600 plaques
10-3 About 60 plaques
10-4 About six plaques
10-5 Too many plaques to count

50

END OF EXAMINATION PAPER

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