6.1 Microbial Technique

Question 1

Aseptic technique

Answer 1

Alcohol
Moist heat (autoclave)
Dry heat
UV
Ionising radiation
Filtration
Chemical agents

Question 2

Alcohol

Answer 2

65-80%
Disinfect hands and equipment
Solubilize lipids and denature proteins

Question 3

Moist heat (autoclave)

Answer 3

Sterilise liquid cultures
Denature nucleic Acids, denature proteins and disrupt cell membrane

Question 4

UV

Answer 4

Sterilises air and exposed surfaces
Damages DNA

Question 5

Ionising radiation

Answer 5

Sterilises antibiotic hormones, sutures and disposable supplies
Damages DNA and oxidises cell content

Question 6

Filtration

Answer 6

Cleaning liquid cultures though sterilised filtration machine
0.2 micro meter pore size strains out bacteria in small volumes

Question 7

Chemical agents

Answer 7

Sterilising work bench and equipment
Stop it slow bacteria growth

Question 8

Media: selective inhibition

Answer 8

Adding dyes
Antibiotics if specific inhibitors
Couture needs carvin for energy (respiration) and nitrogen to grow (protein)

Question 9

Liquid culture

Answer 9

Method: conical flash allows large SA in contact with the air. Aerobic bacteria cultured sterile air. Brit is sterile and agitated.
Pros:
Culture does not die
Metabolic products can be harvested

Question 10

Measuring growth: direct count

Answer 10

Haemocytometer
Dilution plating
Total or viable used

Question 11

Measuring growth: indirect count

Answer 11

Dry mass
Optical methods (turbidity)
Streak plate

Question 12

Measuring growth: haemocytometer

Answer 12

Direct count
Cell count in a specific volume
NW rule
Viability due (methylene blue)
Limitations:
Needs stain for viability
Cells must be killed/imobilised
. suspension of low density hard to see

Question 13

Measuring growth: dilution plating

Answer 13

Cell count in solid mass.
Diluted and x by dilation factor
Check with haemocytometer
Pros:
Viable cell count
Simple equipment
Con:
Time taken to dilute
need to keep track of volume and dilution
Can’t be used for predictions
Direct count

Question 14

Indirect count

Answer 14

Turbidimetry (optical)
Rapid
measures growth- calibration curve
Done with spectrophotometer

Question 15

Area of fungi

Answer 15

Radius if mycelium in different conditions
Indirect count

Question 16

Mass of fungi/bacteria

Answer 16

Indirect
Sample of broth removed at intervals
Separated by centrifuge or filtration
Dried and weighed

Question 17

Lag phase

Answer 17

Cell active- not reproducing
Adjusting to surroundings (taking in water, growing, making MRNA enzymes etc)
Length depends on conditions

Question 18

Log phase

Answer 18

Bacteria divide by binary fission (max rate)
Size of population doubles at each division (cell growth>cell death)
Most susceptible to antibiotics
Length of time depends on species

Question 19

Stationary phase

Answer 19

Deplete growth medium and release waste products - pH change
Cell growth= cell death
Carrying capacity of an environment in an open system

Question 20

Death phase

Answer 20

Die at an exponential rate
Growth<death
Dead cells become nutrients for growing cells
Most cellular activity stops
In a closed system this causes extinction

Question 21

Causes of inaccuracy with haemocytometer

Answer 21

Can’t distinguish between living and dead cells
Cells are not evenly distributed
Har to tell is cells are touching the side