Microbiology

Question 1

What are bacterial cell walls made up of?

Answer 1

A three-dimensional mesh of peptidoglycan (murein), a polymer of amino acids and sugars.

Question 2

What is Gram staining?

Answer 2

A technique used to differentiate between Gram negative and Gram positive bacteria.

Question 3

Outline the process of Gram staining.

Answer 3

1. Stain culture with crystal violet. Remove and rinse with water
2. Add iodine solution and rinse after 1 minute
3. Alternate washes of alcohol and water for 30 seconds
4. Counterstain with red safranin for 1 minute
5. Dry and examine sample under microscope

Question 4

Define Gram positive bacteria.

Answer 4

Bacteria that have a thick peptidoglycan wall and a purple appearance following gram staining.

Question 5

Why do Gram positive bacteria appear purple following Gram staining?

Answer 5

The thick peptidoglycan wall retains crystal violet when rinsed with alcohol.

Question 6

Define Gram negative bacteria.

Answer 6

Bacteria that have a thin peptidoglycan wall with an outer lipopolysaccharide membrane and a red appearance following gram staining.

Question 7

Why do Gram negative bacteria appear red following Gram staining?

Answer 7

On treatment with alcohol, the lipopolysaccharide layer is lost and the crystal violet washes away. The counterstain safranin stains the thin peptidoglycan layer red.

Question 8

What is an obligate aerobe?

Answer 8

An organism that requires oxygen for metabolism.

Question 9

What is an obligate anaerobe?

Answer 9

An organism that can only survive in environments which lack oxygen.

Question 10

Define facultative anaerobe

Answer 10

• An organism that normally respires aerobically
  It is capable of switching to anaerobic
  respiration in the absence of oxygen

Question 11

What are aseptic techniques?

Answer 11

A range of techniques used to culture microorganisms under sterile conditions in order to minimise contamination.

Question 12

List the basic aseptic techniques.

Answer 12

Wipe surfaces with antibacterial cleaner
Set up Bunsen burner nearby - convection currents prevent microbes from entering culture
* Flame inoculating loop and neck of bottles before use
Minimise time that vessels containing bacteria are open
Sterilise all equipment e.g. use of an autoclave
Wear protective clothing

Question 13

Outline how to culture microorganisms.

Answer 13

Transfer bacteria to an agar plate using a sterile
inoculating loop or pipette. Make sure lid is kept at an angle to prevent contamination of the agar from the air.
* Tape lid at two ends, invert dish and incubate
In the school laboratory, ensure dish is not airtight and do not incubate above 25°C to avoid growth of pathogens

Question 14

Explain the difference between a spread plate and a streak plate.

Answer 14

Spread plate - microorganisms distributed evenly with a sterile spreader
Streak plate - aims to obtain single colonies by
rotating the plate to build layers of the culture on at least three separate streaks

Question 15

What is nutrient media?

Answer 15

A solid or liquid nutrient-rich medium used in the cultivation of microorganisms.

Question 16

Describe the composition of nutrient
media.

Answer 16

Contains a carbon source, nitrogen
source, water and growth factors (e.g. salts and vitamins).

Question 17

Describe the conditions used when culturing microorganisms.

Answer 17

Optimum temperature
Constant pH
* Nutrient supply
Aerobic conditions

Question 18

What is the difference between total cell
count and viable cell counts?

Answer 18

In a given area or volume, total cell count is the total number of cells (both living and dead) whereas viable cell count is the total number of living cells.

Question 19

Describe how a viable cell count is
conducted.

Answer 19

• Add a known volume of organisms to an agar plate
• Incubate the plate
• Count the number of colonies

Question 20

What is assumed when conducting a viable cell count?

Answer 20

It is assumed that one cell gives rise to a single colony.

Question 21

What is the problem with the ‘one cell one colony’ assumption?

Answer 21

It does not account for clumping of cells in the original inoculum. This may result in a lower estimate of the number of cells

Question 22

What is a serial dilution?

Answer 22

A sequence of dilutions, in which the dilution factor is constant, used to dilute a
stock solution.