Cognitive Apprenticeship (L04)

Question 1

What is the purpose of applying aseptic techniques?

Answer 1

Prevent infection of the handler and others who might be exposed to the microbial samples

Question 2

How to enumerate bacteria?

Answer 2

Plate count method and by measuring turbidity

Question 3

What are the advantages of using plate count method rather than measuring by turbidity?

Answer 3

Plate count method only counts living bacteria and allows further characterization of isolated bacteria

Question 4

How to enumerate bacteria by measuring turbidity?

Answer 4

A spectrophotometer is used to measure turbidity of a culture
Measures the amount of light that pass through a suspension of bacterial cells
Higher bacterial concentration, higher turbidity, less light passing through the bacterial cells, higher optical density of broth

Question 5

What are the limitations of turbidity measurement?

Answer 5

Requires a high concentration of bacteria in the sample
Measures both dead and living bacteria
Presence of particles can interfere and contribute to OD reading

Question 6

Differences between spread plate and pour plate

Answer 6

Spread plate:

• requires pre-prepared agar plates
• colonies on the surface can be isolated and further characterized
• aerobic bacteria will grow, obligate anaerobes will not

Pour plate:

• pre-prepared agar plates are not required
• colonies within the agar cannot be isolated
• reduced growth rate of obligate aerobes

Question 7

Difference between solid and liquid culture media

Answer 7

Liquid culture media:
For rapid culturing of high concentration of microorganisms

Solid culture media:
For culturing of microorganisms to obtain isolated colonies

Question 8

What is the purpose of working near the flame?

Answer 8

Air convection currents are formed
Creating an aseptic area around the flame
Lowering the risk of contamination

Question 9

What are the steps that needs to be taken to prepare LB agar media?

Answer 9

1. Label a Duran bottle using masking tape
2. Weigh out the components of media, using a set of weighing boat and spatula
3. Pour ingredients into the Duran bottle
   Fill to 80-90% of the desired volume to dissolve the components
   Top up with RO water to the desired volume, using the graduated volume scale on the bottle
4. Tighten the cap and mix well by shaking the bottle
5. Prepare for autoclaving
6. Autoclaved LB agar media to be kept warm in molten state and ready to be used for pouring of agar plates

Question 10

What are the steps that needed to be taken for pouring of agar plates?

Answer 10

1. Keep LB molten agar warm in the water bath at 50°C till ready to pour
2. Mix LB molten agar well
3. Remove the Duran bottle cap containing LB molten agar
   Pass the rim of the open bottle through the flame
   Place cap down with the inside facing downwards
4. Lift the lid of the empty petri dish up slightly
   Pour 20-25ml of LB molten agar into each plate, close the lid immediately after
5. Keep 50ml of molten agar warm in the water bath at 50°C for pour plate
6. Allow the agar in the plates to cool and solidify

Question 11

What is the importance of serial dilutions?

Answer 11

Plating high concentration of bacteria will make it impossible to count the bacteria accurately
Performed for samples with suspected high concentrations of bacteria

Question 12

What is the purpose of duplicate plating?

Answer 12

To improve the accuracy of bacterial count

Average CFU count can be obtained from the duplicated plates for calculations

Question 13

What is the importance of experimental controls?

Answer 13

To validate the experimental procedure
Positive control: ensure all reagents are working
- known to produce the positive effect

Negative control: ensure the substance used is not contaminated (diluent)
- known to produce the negative effect

Question 13

What is the importance of experimental controls?

Answer 13

To validate the experimental procedure
Positive control: ensure all reagents are working
- known to produce the positive effect

Negative control: ensure the substance used is not contaminated (diluent)
- known to produce the negative effect

Question 14

What are the steps we need to take when aliquoting diluent?

Answer 14

1. Open the top end of the serological pipette
2. Fit in the pipette aid securely and pull the serological pipette out of the sleeve
3. Press the top button to aspirate till __ml
4. Press the bottom button to dispense
5. Place the serological pipette back into the sleeve before discarding it into biohazard bin

Question 15

What are the steps we need to take when performing a dilution?

Answer 15

1. Pick up a new pipette tip
2. Pick up the 1st tube, flame, open, flame
3. Mix well by pipetting up and down
   Aspirate __ml of neat sample
4. Flame, close, flame, replace the tube back to the rack
5. Pick up the 2nd tube, flame, open, flame
6. Dispense neat sample along the wall of the tube
7. Discard pipette tip
8. Flame, close, flame and replace the tube back to the rack

Question 16

What are the steps we need to take when performing spread plate?

Answer 16

1. Dispense __ micrometre of sample in the center of the agar plate
2. Spread inoculum over surface evenly, using disposable spreader (right hand)
   Rotate agar plate (left hand)

Question 17

What are the steps we need to take for pour plate?

Answer 17

1. Inoculate empty plate
2. Add molten agar
3. Swirl to mix
4. Colonies grow in and on solidified medium