Microbiology

Question 1

how is bacteria distinguished

Answer 1

size
shape
staining characteristics
metabolic antigenic
and genetic features

Question 2

What are bacterial cell walls made up of?

Answer 2

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

Question 3

what is gram staining

Answer 3

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

Question 4

outline the process of gram staining

Answer 4

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 5

define gram positive bacteria

Answer 5

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

Question 6

Why do Gram positive bacteria appear purple following Gram staining?

Answer 6

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

Question 7

Define Gram negative bacteria.

Answer 7

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

Question 8

Why do Gram negative bacteria appear red following Gram staining?

Answer 8

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 9

What is an obligate aerobe?

Answer 9

An organism that requires oxygen for
metabolism.

Question 10

What is an obligate anaerobe?

Answer 10

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

Question 11

Define facultative anaerobe

Answer 11

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

Question 12

What are aseptic techniques?

Answer 12

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

Question 13

List the basic aseptic techniques

Answer 13

● 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 14

Outline how to culture microorganisms

Answer 14

● 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 15

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

Answer 15

● 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 16

What is nutrient media?

Answer 16

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

Question 17

Describe the composition of nutrient media

Answer 17

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

Question 18

Describe the conditions used when culturing microorganisms.

Answer 18

● Optimum temperature
● Constant pH
● Nutrient supply
● Aerobic conditions

Question 19

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

Answer 19

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 20

Describe how a viable cell count is conducted.

Answer 20

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

Question 21

What is assumed when conducting a
viable cell count?

Answer 21

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

Question 22

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

Answer 22

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 23

What is a serial dilution?

Answer 23

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

Question 24

what are the three main shapes of bacteria

Answer 24

coccus- spherical shape
bacillus- rod shaped
spirillum- spiral shape or corkscrew