Topic 6.1 Microbial techniques

Question 1

Why are aseptic techniques important when culturing microorganisms?

Answer 1

To produce uncontaminated culture so results are reliable and repeatable

Question 2

List the basic aseptic techniques

Answer 2

• Wipe surfaces with antibacterial cleaner
• Set up bunsen burner nearby. Convection currents prevent microbes from enetering culture
• Flame innoculating loop and neck of bottles before use
• Minimise time that vessels containing bacteria are open
• Sterilise all equipment e.g. using an autoclave
• Wear protective clothing

Question 3

Outline how to culture microorganisms

Answer 3

1. Transfer bacteria to agar plate using sterile inoculating loop or pipette
2. Tape lid on at 2 ends then invert the dish and incubate. In school lab conditions, ensure dish is not airtight and do not incubate above 25^oC to avoid growth of pathogens

Question 4

Explain the difference between a spread plate and a streak plate

Answer 4

Spread plate: ditrubute microorganisms evenly with a sterile spreader

Streak plate: aim to obtain single colonies by rotating the plate to build layer of the culture on at least 3 separate streaks

Question 5

Describe 3 types of nutrient medium

Answer 5

Usually contain nitrogen carbons and minerals. Often enriched with protein from extract of yeat, blood or meat. May be liquid broth or solid agar.

Selective mediums only contain highly specific nutrient balance. Only certain microorganisms can grow.

Question 6

Give the advantages of using a broth medium

Answer 6

• Can provide anoxic and oxic conditoins depending on depth, which helps to identify microbes/ determine thier optimum conditions.
• Can grow a very large volume of bacteria.

Question 7

Give the advantages of using a broth medium

Answer 7

• Can provide anoxic and oxic conditoins depending on depth, which helps to identify microbes/ determine thier optimum conditions.
• Can grow a very large volume of bacteria.

Question 8

Give the advantage of using agar as the medium

Answer 8

Can obtain a single discrete pure colony for study.

Question 9

Name the 4 phases of a bacterial growth curve

Answer 9

1. Lag phase
2. Log phase
3. Stationary phase
4. Death phase

Question 10

What happens during the lag phase?

Answer 10

Microorganisms need to adjust to the enviroment before reproducing so population size only increases slowly.

Question 11

What happens durong the log phase?

Answer 11

After every round of division population size doubles (exponential growth).

Question 12

What happens during the stationary phase?

Answer 12

Reproduction rate = death rate, so population size stabilises at its maximum.

Question 13

What happens during the death phase?

Answer 13

Microorganisms die due to buildup of toxic waste and lack of nutrients.

Question 14

Name three methods to estimate the growth of a bacterial culture

Answer 14

• Cell count (using a haemocytometer)
• Turbidity meaurement (type of colorimetry to measure opacity)
• Dilution plating

Dry mass could also be used

Question 15

Explain how to conduct a cell count

Answer 15

1. Dilute broth sample with equal volume of trypan blue stain to stain dead cells blue
2. Use a calibrated haemocytometer with volume 0.1mm³. Count the cells in each of the sets of squares and calculate mean
3. Number of bacterial cells = number counted x 10⁴ per cm³

Question 16

Suggest an advantage of using a cell count

Answer 16

Only counts living cells

Question 17

Suggest disadvantages of using a cell count

Answer 17

• Slow
• Expensive equipment
• Large margin for human error

Question 18

Explain how to conduct a turbidity measurement

Answer 18

1. Use a colorimeter. measure absorbance of % transmission of smaples with known microorganism count
2. Plot calibration curve: absrobance/ % transmission (y-axis), number of microorganisms (x-axis)
3. Record absorbance/ % transmission of unknown sample. Interpolate graph.

Question 19

Suggest advantages of using turbidity measurement

Answer 19

• Quick
• Can be conducted in the field

Question 20

Suggest disadvantages of using turbidity measurement

Answer 20

• Expensive equipment
• Counts both living and dead cells
• Requires calibration curve from known samples
• Assumes equal density of cells across culture

Question 21

Explain how to conduct dilution plating

Answer 21

1. Grow a colony from a single microorganisim
2. Perform serial dilution with distilled water to see single colonies
3. Prepare a lawn plate and count colonies
4. Number of cells = number of colonies x dilution factor

Question 22

Suggest advantages of using dilution plating

Answer 22

• No complex or expensive equipent needed
• Only counts living cells

Question 23

Suggest disadvantages of dilution plating

Answer 23

Incubation period needed (slow)

Question 24

Outline the calculation for an exponential rate constant

Answer 24

Arrhenius equation: k = Ae^-EA/RT

Where:
* k = rate constant
* A = frequency factor
* e = mathematical number with the value 2.718…
* E_A = activation energy
* R = gas constant
* T = temperature