Unit 6- Microbial Techniques

Question 1

What are the different types of mixtures the microbes are cultured on?

Answer 1

• agar plate (made of agar and a nutrient broth)

- selective media

Question 2

What is the agar plate?

Answer 2

A solid medium of agar mixed with a nutrient broth in a Petri dish

Question 3

What is a a selective media?

Answer 3

A medium made of a specific recipe to allow the growth of only one group of microbes, it may include antiobiotics if the wanted bacteria is antibiotic resistant

Question 4

How is equipment sterilised?

Answer 4

Using ultra violet
autoclave (large pressure cooker)
Using virkon, bleach or ethanol
Flaming

Question 5

What conditions does a culture provide?

Answer 5

• nutrients
• suitable temp and pH
• usually access to O2

Question 6

Aseptic techniques are used to avoid contamination, give some examples of aseptic techniques:

Answer 6

• all equipment should be sterile
• flame the appropriate equipment in Bunsen burner before and after
• replace lids as quickly as possible

Question 7

What are the problems with culturing microbes?

Answer 7

• pathogens may enter the culture and grow risking disease
• safe organisms may mutate to dangerous strains
• contamination can spoil an investigation

Question 8

What precautions can be taken to reduce the risk of culturing microbes?

Answer 8

• adequate O2 supply as pathogens prefer to grow in anaerobic conditions
• culture the medium at about 20 degrees for a few days as body temperature encourages the growth of potentially dangerous human pathogens

Question 9

What 4 different techniques can be used to measure microbial growth?

Answer 9

• cell counting with haemocytometer
• turbidity with a colorimeter
• dilution plating until the microbes can be spread on an agar plate and counted
• weighing dry mass of a known volume

Question 10

How can we measure the number of microbes in a culture broth by dilution plating?

Answer 10

• dilute the culture until the number of cultures can be counted when spread on an agar plate
  (Dilution plating is the same as serial dilution)

Question 11

How can you count the number of bacteria in a culture using a haemocytometer?

Answer 11

• Pipette the culture onto the haemocytometer
• the volume of the haemocytometer is 0.1mm
• count the number of cells in a 0.2mm box
• calculate the number of cells per mm^3

Question 12

What is the convention when counting using a haemocytometer

Answer 12

Count the cells overlapping the north west boundary and discard the cells over the south east boundary

Question 13

How do you only count living cells using a haemocytometer?

Answer 13

• add a vital stain (trypan blue) which only stains dead cells blue

Question 14

What does turbidity mean?

Answer 14

Cloudiness

Question 15

How can we measure cell density using a colorimeter?

Answer 15

• dilute the culture broth until it has an absorbable of below 0.5
• the greater the concentration of cells the more turbid the dilution giving a higher absorbance

Question 16

What is an issue of using a colorimeter?

Answer 16

• must be calibrated regularly

- reading must be taken quick before cells settle at bottom of the curettes

Question 17

How can we count cells using dilution plating?

Answer 17

• take a small sample from a culture broth using aseptic techniques and a sterile pipette
• spread the sample evenly over the agar plate using a sterile spreader
• the agar plate is incubated at 30degrees for a day, each living cell will form a colony
• count the number of individual colonies, and this is the number of colonies in the dilution

Question 18

What are the 4 stages of a bacterial growth curve?

Answer 18

• lag phase
• log phase
• stationary phase
• death phase

Question 19

What occurs in the lag phase?

Answer 19

• cell division and cell death rates are low so there is little change
• the microbes are preparing to use the nutrients and adjusting to the new conditions
• switching on genes and mostly synthesising proteins

Question 20

What happens during the log phase?

Answer 20

There are no limiting factors of growth
Growth is exponential due to rapid doubling
Scale is log so graph appears linear

Question 21

What occurs during stationary phase?

Answer 21

Cell division rate slows down and cell death rate increased
Until the rates are equal
Due to a build up of metabolic waste, lack of space or nutrients

Question 22

What occurs during the death rate?

Answer 22

Population of living cells falls due to less nutrients, space available and more toxic metabolic waste