UNIVERSITY OF THE FREE STATE

BLOEMFONTEIN CAMPUS

DEPARTMENT: MICROBIAL, BIOCHEMICAL & FOOD BIOTECHNOLOGY

MCBE 3714

SEMESTER TEST: 1

THURSDAY, 05 MARCH 2020

ASSESSOR: PROF OM SEBOLAI

MODERATOR: PROF CH POHL

TIME: 1 h 20 min MARKS: 60

Students are allowed to use pocket calculators. The test is closed book.

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SECTION 1 (30)

1.1 Define the following terms (11)

1.1.1 Guild.

 species exploiting the same kind of resources in a similar manner

()

1.1.2 Realised niche.

 “where the species actually lives” ()

1.1.3 Phylotype.

 organisms that share the same or have closely related orthologous genes

()

1.1.4 High throughput culturing assay.

• Allows for the detection of expression of thousands of genes at the same time

()

 a photosynthetic organism (such as a green plant or a cyanobacterium) that utilizes

energy from light to synthesize organic molecules.1.1.5 Photoautotroph.

()

1.1.6 Metatranscriptomics.

•  the science that Analyses community RNA

• Reveals which genes in a community are actually expressed

• Transcripts (mRNA)

• Reveals the level of gene expression

()

1.1.7 Biofilm.

 Microbial cells attached to a surface and enclosed in an adhesive matrix

()

1.1.8 Species richness. ()

1.1.9 “Living” microsensor. ()

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 1.1.10 Isotopic fractionation. ()

1.1.11 Diel cycle. ()

1.2 Multiple choice questions (11)

1.2.1 Winogradsky columns are useful in enriching for ________ microorganisms.

a. aerobic

b. anaerobic

c. facultative anaerobic

d. All of the above choices are correct. ()

1.2.2 What biomolecule type do phylogenetic FISH probes OFTEN target?

a. 5S rRNA.

b. 16S rRNA. ()

c. 30S rDNA.

d. all of the above.

1.2.3 For microbial biodiversity studies, it is common to identify the ________ rather than the

________ as a measure of biodiversity.

a. organisms themselves / genes

b. genes / organisms themselves ()

c. cell types / genes

d. cell types / organisms themselves

1.2.4 How is CARD-FISH an improvement over traditional FISH approaches?

a. It allows for the targeted quantification of several taxa simultaneously.

b. It allows for single-cell sorting of fluorescently-labelled populations.

c. It increases fluorescence detection. ()

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 d. It requires fewer cells (i.e., decreased sample volume).

1.2.5 The separation of nucleic acids in a DGGE gel is based on

a. GC content.

b. melting profiles.

c. taxonomic identity.

d. all of the above. ()

1.2.6 Simultaneous determination of microbial species and their metabolic activity may be

accomplished using

a. isotopically fractionated cells.

b. phylogenetic markers.

c. MAR-FISH. ()

d. single-cell genomics.

1.2.7 The elemental and isotopic composition and species identity of a single microbial cell

may be determined using

a. microautoradiography.

b. flow cytometry.

c. FISH-SIMS. ()

d. single cell genomics.

1.2.8 Which technique does NOT involve PCR?

a. MDA. ()

b. Metatranscriptomics.

c. T-RFLP.

d. SIP.

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1.2.9 Microbial metabolic activity can be measured over very short distances using

a. FISH.

b. flow cytometry.

c. microsensors. ()

d. gene sequencing.

1.2.10 Cellular carbon of living organisms is ________ 12C and ________ 13C.

a. enriched in / depleted in ()

b. depleted in / enriched in

c. present only as / is devoid of

d. devoid of / present only as

1.2.11 The 13C/12C isotopic ratio of geological rocks of different ages has been used to examine

whether the previous biological activity occurred because compounds of biological

origin

a. do NOT contain 13C.

b. have a higher proportion of 13C that compounds of geological origin.

c. have a lower proportion of 13C that compounds of geological origin. ()

d. are not well preserved in geological samples.

1.3 Complete the following Table 1. List the answers in your answer sheet from 1.3.1 to

1.3.8. (8)

Technique Target Purpose

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 Standard FISH 1.3.1 - rRNA 1.3.2 - Fast growers
CARD-FISH 1.3.3 - mRNA 1.3.4 - Gene expression
Standard DNA-binding dyes 1.3.5 - DNA 1.3.6 - Enumerate all

Standard viability stains
microorganisms
1.3.7 - intergrity cytoplasmic 1.3.8 - Differentiate dead
and live cells based on
membrane
membrane integrity

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SECTION 2 (30)

2.1 What is the challenge that is presented by enrichment bias? (1)

 Fast growers (“weed” species) can dominate

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2.2 How can the challenge mentioned above be reduced?

 Dilution of inoculum, eliminate weed/fast growers

(1)

2.3 Work out the following problem. You are asked to enumerate bacteria present in a

seawater sample. You first dissolved 1 ml of the sample in 9 ml of sterilised broth and

incubated the sample at 28oC for 24 h. You then carried out a serial dilution and found

that the 10-4 plate produced 65 colony forming units (CFU). (4)

2.4 A student wants to confirm the purity of a culture of Bacillus cereus. Thus, he/she

decides to perform a Gram staining on a microscope slide. Under the microscope,

she/he observes that both Gram-positive and Gram-negative bacteria are present.

She/he concludes that the culture is contaminated. Briefly detail an experimental

procedure you would perform to evaluate the validity of the conclusion. (4)

2.5 Using DGGE for the 16S rRNA genes, you are analysing four environmental samples

with an unknown number of bacterial species (phylotypes). The image of the gel is

shown below.

2.5.1 Where do you think (top, middle or bottom of the gel) you will find the 16S

rRNA of Frankia, a bacterium with high GC content?

(1)

2.5.2 Explain your answer. (3)

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2.6 Tables of “samples by species, such as the one below, are commonly used to quantify

microbial diversity. Assuming that the numbers in bold represent the number of cells

and that all samples were taken from the same region, answer the following:

Species #1 Species #2 Species #3

Sample #1 20 0 80
Sample #2 0 100 0
Sample #3 40 30 30
Sample #4 50 0 50

2.6.1 What is the species richness in sample #1? (1)

2.6.2 What is the abundance of species #2 in sample #3? (1)

2.6.3 What is the population of species #1 in the region? (1)

2.7 Explain what the principle of stable isotope probing (SIP) is. (2)

2.8 Briefly detail an experimental procedure, wherein SIP can be used to identify a

microbe. (6)

2.9 Why do bacteria form biofilms? Give two reasons. (2)

- self defense: resist physical forces that sweep away unattached cells,

- phargocytosis by immune system cells and penetration of toxins ie antibiotics.

- allows cells to remain in favorable niche (biofilms trap nutrients for microbial

- growth and help prevent detachment of cells in flowing systems). allows bacterial

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 - cells to live in close association with one another and establish cooperative

interactions

2.10 List three methods that are used to control biofilms. (3)

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