Cognitive Apprenticeship (L08)

Question 1

What is generation time?

Answer 1

The time taken for the cell numbers to double during exponential growth

Question 2

Why is an overnight culture diluted into a larger volume for growth?

Answer 2

Stored colonies need to re-acclimatize to growth conditions

Step-wise dilutions are needed to:

1. Reduce metabolic waste
2. Replace nutrients for continued growth
3. Ensure sufficient inoculum size for next scale-up step

Question 3

How to carry out inoculation of overnight cultures?

Answer 3

1. Using aseptic technique, pipette 27ml of minimal medium/LB broth into a sterile conical flask, covered with aluminum foil
2. Inoculate 3.0ml of the overnight culture into the conical flask, with 27ml of minimal medium/LB broth
3. Immediately, mix the inoculated conical flask well and aliquot 1.5ml of the bacterial culture into a sterile microcentrifuge tube and label the tube as “0hr”
4. Place the culture in the conical flask into the 37 degree Celsius shaker incubator set at 120 rpm

Question 4

How to monitor bacterial growth and plot a growth curve?

Answer 4

1. At the start of the experiment, prepare to take visible-light measurements using the UV spectrophotometer. Set the wavelength to 600nm (done only once)
2. Aliquot 1ml fresh medium of the growth medium into a clean cuvette. This will be the blank
3. Place the blank cuvette into the spectrophotometer and set the spectrophotometer to “0” absorbance before reading the experimental cuvette. Use a parafilm to cover the blank
4. At every time point, mix well and transfer 1 ml of the sample from the microcentrifuge tube into a clean cuvette and obtain the absorbance immediately
5. Record the readings obtained and tabulate the readings
6. Plot a graph of OD600nm vs time, using the data obtained
7. Perform serial dilution by diluting 0.1ml of the sample in 0.9ml of sterile saline to respective dilution factors.
8. Perform spread plating, using 0.1ml of the diluted samples stated.
9. Incubate the inverted spread plates at 37 degrees celsius overnight
10. Count the number of colony forming units for each plate, calculate bacterial concentration based on the results obtained
11. Plot a growth curve based on bacterial concentration vs time

Question 5

How to grow a lot of bacterial cells as quickly as possible?

Answer 5

Control physical environment
When microorganisms are exposed to optimal growth conditions, microbial cells will multiply in massive numbers and synthesize large volumes of the desired product

Question 6

What are the types of microbial strains?

Answer 6

Wild-type: prevails in natural conditions
Type strain: usually the first studied strain of a species
Recombinant strain: genetic recombination has occurred

Question 7

Factors affecting the growth of microorganisms

Answer 7

pH
Temperature
Oxygen level
- correlates with the natural habitat of the microorganism

Question 8

Why do we shake/stir the conical flask containing the culture?

Answer 8

Allows aeration of the culture and mixing of microorganisms in the culture media

Question 9

What does the lag phase mean?

Answer 9

• Acclimatization of the bacteria to new growth environment
• Increase in cell size but no ell division
• No increase in the number of living bacterial cells

Question 10

What does the log phase mean?

Answer 10

• Bacteria multiply at maximum rate under the conditions provided
• Cell numbers increase exponentially

Question 11

What does the stationary phase mean?

Answer 11

• Exhaustion of nutrient and accumulation of toxic waste products
• Rate of cell division = rate of cell death
• Many cells undergo sporulation, forming endospores
• Secondary metabolites are being synthesized

Question 12

What does the death phase mean?

Answer 12

• Rate of cell death > rate of cell division
• Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores

Question 13

When are primary metabolites harvested

Answer 13

Primary metabolites are produced during major metabolic pathways and are essential to the microbe’s function
They are harvested during log phase

Question 14

When are secondary metabolites harvested?

Answer 14

Secondary metabolites are by-products of metabolism that may not be critical to the microbe’s function
They are harvested during stationary phase

Question 15

What is a batch culture?

Answer 15

• Closed culture with a single batch of medium
• No fresh medium is provided during incubation
• Nutrient concentration decrease and concentration of waste products increase

Question 16

What is a continuous culture?

Answer 16

• Constant environmental conditions are maintained, through continual provision of nutrients and removal of wastes

Question 17

What is fed-batch culture?

Answer 17

• Semi-continuous culture
• Fresh media is sequentially added to the culture, without removing any cells
• Volume of culture will increase

Question 18

Advantages of the 3 cultures

Answer 18

Batch culture:

• Ease of operation
• Reduced risk of contamination

Continuous culture:

• High and constant productivity
• Allow the cells to achieve a steady state

Fed-batch culture:

• Higher biomass/ higher product yield
• By-product accumulation is low

Question 19

Disadvantages of the 3 cultures

Answer 19

Batch culture:

• Low cell density
• Long downtime between batches

Continuous culture:

• Fail to produce product, due to mutation
• Long growth periods, increase the risk of contamination

Fed-batch culture:
- Long downtime between batches

Question 20

The uses of the 3 types of culture

Answer 20

Batch culture: to optimize conditions in the early stages of experimental design

Continuous culture: grow cells in a constant environment and study basic microbial physiology

Fed-batch culture: used when the product/biomass yield is the highest at low substrate concentration