3.4 Microbiology 3

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

Gram positive bacteria??

Answer 4

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

Question 5

Why do Gram postitive bacteria appear purplr following Gram staining?

Answer 5

The thick peptidoglycan wall retains crystal violet when rinsed with alcohol

Question 6

Gram negative bacteria??

Answer 6

Bacteria that have a thin peptidoglycan wall with an outer lipopolysaccharide membrane and 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

Obligate aerobe??

Answer 8

Bacteria that require oxygen for metabolism

Question 9

Obligate anaerobe??

Answer 9

Bacteria that can only survive in environments which lack oxygen

Question 10

Facultative anaerobe??

Answer 10

Bacteria that normally respires aerobically but is capable of switching to anaerobic respiration in the absence of oxygen

Question 11

Aseptic techniques??

Answer 11

A range of techniques ised to culture microorganisms under sterile conditions to minimise contamination

Question 12

Basic aseptic techniques

Answer 12

Wipe surfaces with antibacterial cleaner
Convection currents of Bunsen Burner prevent microbes from entering culture
Flame innoculating loop and neck of bottles before use
Minimise time that vessels containing bacteria are open
Sterilise all equipment using autoclave
Wear protective clothing

Question 13

Outline how to culture microorganisms

Answer 13

Transfer bacteria to an agar plate using sterile inoculation loop or pipette, make sure lid is kept at an angle to prevent contamination of agar from the air. Tape lid at two ends invert dish and incubate above 25C to avoid pathogen growth.

Question 14

Difference between spread plate and streak plate

Answer 14

Spread plate - microorganisms distributed evenly with sterile spreader
Streak plate - aims to obtain single colonies by rotating 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 nutriet rich medium used in the cultivation of mocroorganisms

Question 16

Composition of hutrient media

Answer 16

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

Question 17

Conditions when culturing microorganisms

Answer 17

optimum temperature
constant pH
nutrient supply
aerobic conditions

Question 18

Difference between total cell count and viable cell count

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

How is viable cell count 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 ‘one cell one colony’ assumption?

Answer 21

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

Question 22

WHat is serial dilution?

Answer 22

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

Question 23

circular/ spherical bacteria

Answer 23

Coccus

Question 24

rod shaped bacteria

Answer 24

Bacillus

Question 25

spiral/ corkscrew shaped bacteria

Answer 25

Spirillum