MODULE 4

Question 1

What is the difference between a viable and total count of bacteria?

Answer 1

Total count: all cells, viable count: living cells only

Question 2

What are the advantages and disadvantages of using turbidity as a measure of bacterial growth?

Answer 2

Advantages: rapid, fairly accurate measure of growth

Disadvantages: any particulate matter interferes with optical density readings, does not directly measure cell numbers

Question 3

Why should a standard curve be prepared when using turbidity as a measure of bacterial growth rates?

Answer 3

Turbidity does not measure actual cell numbers. Absorbance is proportional to cell numbers so a standard curve is needed to relate absorbance to numbers.

Question 4

Why does growing in a salty environment pose a challenge?

Answer 4

A salty environment has higher solute concentration than the cytoplasm of a cell. Osmotic pressure is placed on the cell and water flows out. A halophile has strategies to overcome this, usually by increasing internal solute levels.

Question 5

How do complex and defined media differ?

Answer 5

Complex media: precise composition is unknown

Defined media: precise composition is known

Question 6

Give an example of a selective and differential medium. What are the components that make it selective and differential?

Answer 6

There are numerous, one is MacConkeys agar: contains bile salts and crystal violet that inhibit growth of gram-positive bacteria. Gram negative bacteria that ferment lactose produce red/pink colonies while those that do not ferment lactose produce white colonies.

Question 7

How does sterilisation differ to disinfection?

Answer 7

Sterilisation: complete destruction or removal of all viable microbes
Disinfection: use of chemicals to kill or inhibit pathogenic microbes

Question 8

What are the main methods of (i) dry heat and (ii) moist heat sterilisation?

Answer 8

Dry heat: incineration & burning to ashes, hot air oven

Moist heat: autoclaving, boiling/hot water, UHT, pasteurisation

Question 9

Briefly explain the roles of time, temperature, and pressure in the functioning of the autoclave.

Answer 9

High pressure is used in an autoclave so that liquids do not boil at temperatures beyond 100°C. The heat is more important that the pressure in killing the microbes and is usually used for 15 minutes at 121°C as a balance to kill all organisms and not damage items autoclaved. The time must be increased for bulky samples to ensure the objects or liquids are heated with steam to 121°C for at least 15 minutes.

Question 10

The following disinfectants were tested against S.aureus using the tube dilution test. Which is the most effective disinfectant? What is the MIC for each disinfectant?

Answer 10

Dilution Disinfectant A Disinfectant B Disinfectant C
1/20 - - -
1/40 + - -
1/80 + - +
1/160 + + +
1/320 + + +

Most effective: Disinfectant B
MIC: A – 1/20, B – 1/80, C – 1/40

Question 11

What are the advantages and disadvantages of using radiation to sterilize items?

Answer 11

Advantages: If sufficient dosage is used, radiation is highly effective and quick in killing microbes. Very useful for anything effected/damaged by high temperature (e.g. plastics, foods, pharmaceuticals).

Disadvantages: The high costs associated with the specialized equipment make it feasible only for large industrial applications. UV radiation sterilizes only the surfaces of objects. Insufficient dosage can lead to mutations and survival of microbes. Due to the high energy of some radiation procedures, there are many concerns of potential radioactive contamination, production of carcinogenic or toxic products, and alteration of nutritional value.

Question 12

Briefly describe the mode of action of the β-lactam antibiotics.

Answer 12

Target bacterial cell wall. More specifically, β-lactam ring binds to transpeptidase enzyme (penicillin binding protein) in peptidoglycan synthesis. Therefore cross-linking of the peptide chains can’t occur, resulting in degradation of the cell wall and lysis.

Question 13

Growth factor analogs may inhibit microbial metabolism, though only a few are practically useful. Why would this be the case?

Answer 13

Many can be toxic, also inhibit host cell metabolism and not specifically microbes.

Question 14

Where would you best place a sterilization indicator in an autoclave? Explain your reasoning.

Answer 14

Centre of the autoclave. Steam enters from the back and contacts external parts of the items first, so takes longer to reach the centre

Question 15

Explain what is meant by selective toxicity. How does selective toxicity relate to the use of antimicrobial chemicals and the development of chemotherapeutic agents?

Answer 15

Selective toxicity is the idea that only certain organisms are killed while others survive. With antimicrobials, the target is the particular pathogen causing a disease. Ideally other microbes like normal flora are not killed. In developing chemotherapy drugs certain cells are killed (e.g. cancer cells) and healthy cells are not killed

Question 16

How would the decimal reduction time (D-value) spore-forming and non spore-forming bacteria compare?

Answer 16

D-value is the time required to kill 90% of a microbial population at a given temperature. D-value is shorter for non spore-former as the bacteria die off more quickly than the spore former

Question 17

What are advantages and disadvantages of removing microbes by filtration?

Answer 17

Advantages: no damage to treated material, quick & easy, low cost, especially useful for heat labile liquids and large areas (sterile rooms)

Disadvantages: can only be used for liquids & gases, particulate matter larger than microbes will be filtered out, does not kill microbes (important with pathogens), very small bacteria, Mycoplasma and viruses can pass through filters

Question 18

Why is 70% ethanol more effective as a disinfectant than 100% ethanol?

Answer 18

Water is needed to help denature proteins and allows the ethanol to penetrate into cell.

Question 19

What is meant by a broad spectrum antibiotic?

Answer 19

An antibiotic that acts on a wide range of bacteria, usually both gram positive and gram negative.

Question 20

When using a combination of antibiotics to treat a disease, what is the difference between a synergistic and antagonistic effect?

Answer 20

Synergism: when the effect of two antibiotics together is greater than the effect of either alone

Antagonism: when the effect of two antibiotics together is less than the effect of either alone

Question 21

Penicillin and vancomycin both target cell wall synthesis, how does their mechanism of action differ?

Answer 21

Penicillin binds to the transpeptidase enzyme which prevents it attaching to the D-ala-D-ala section of the peptide chain for cross-linking of peptidoglycan sheets.
Vancomycin binds to the D-ala-D-ala section of the peptide chain in place of transpeptidase thus preventing attachment and cross-linking as well.

Question 22

Streptomycin and chloramphenicol both inhibit protein synthesis, how does their mechanism of action differ?

Answer 22

Streptomycin binds and changes shape of the 30S subunit of the bacterial ribosome causing misreading of mRNA resulting in incorrect protein formed.
Chloramphenicol binds to the 50S subunit and blocks peptide bond formation inhibiting protein synthesis

Question 23

In general, list in order of most resistant: Gram positive bacteria, Gram negative bacteria, endospore-formers, viruses with lipid envelope, viruses without lipid envelope, Mycobacteria.

Answer 23

Endospore-formers  Mycobacteria  Gram negative bacteria  viruses without lipid envelope  Gram positive bacteria  viruses with lipid envelope

Question 24

How is the time of treatment determined for a heat sterilization process?

Answer 24

By ensuring that Geobacillus stearothermophilus spores (most resistant) are killed. Either measuring the time taken for the chance of a one in a million (10-6) survivor (from the death curve) or time taken for these spores to be killed in biological indicators

Question 25

What are the key factors involved in the rise of antibiotic resistant bacteria?

Answer 25

Overuse and misuse of antibiotics, enhancement of transfer of resistance genes

Question 26

What tests would you perform to decide whether a chemical agent could be used as an antiseptic? As a disinfectant? As a sterilant? Are there likely to be any chemicals able to be used as two of these or even all three?

Answer 26

Antiseptic: test the ability to kill pathogens (or representatives) over a range of concentrations in a relatively short time and not damage or irritate living tissue.

Disinfectant: test the ability to quickly kill pathogens (or representatives) over a range of concentrations under clean and dirty conditions with no toxic residues.

Sterilant: test the ability to kill all microbes (including spores) safely.

All three: no, antiseptics & disinfectants: yes, antiseptics & sterilants: no, disinfectants & sterilants: yes (but at different concentrations).