6.1 microbial techniques

Question 1

why are aseptic techniques used?

Answer 1

to prevent the culture becoming contaminated with unwanted organisms

Question 2

what are the different types of nutrient medium?

Answer 2

• nutrient broth
• nutrient agar
• selective medium

Question 3

what is a nutrient broth?

Answer 3

nutrients in liquid form

Question 4

what is nutrient agar?

Answer 4

nutrients in a solid form

Question 5

what is a selective medium?

Answer 5

a medium in or on which only a select group of microorganisms with those particular requirements can grow

Question 6

what conditions will bacteria need for growth?

Answer 6

-nutrients (usually protein such as blood, yeast extract or meat extract)
-suitable temperature and pH
-usually access to oxygen

Question 7

what is getting your culture of bacteria onto your agar or into your broth called?

Answer 7

innoculation

Question 8

what do you use for innoculations?

Answer 8

innoculating loop

Question 9

how do you innoculate solid media?

Answer 9

1) sterilise the innoculating loop by holding it in the Bunsen burner until it glows red and then leave it to cool
2) dip the sterilised loop into the suspension of the bacteria and streak the loop across the agar (streak plate)
3) close the lid and tape leaving gaps to prevent anerobic respiration

Question 10

what are aseptic techniques used?

Answer 10

• use sterile equipment
• flame equipment using Bunsen burner
  -don’t lift off lids completely
• flame the rim of the bottle if using broth
  -do near a bunsen burner to create vacum

Question 11

what are the different methods of measuring the growth of a bacterial culture?

Answer 11

• cell counts
• dilution plating
• mass
• optical methods (turbidity)

Question 12

what can be counted by cell counts?

Answer 12

bacteria and single-celled fungi

Question 13

how can they be counted?

Answer 13

using a microscope and a heamocytometer

Question 14

what is a haemocytometer?

Answer 14

thick microscope slide engraved with a grid with a rectangular chamber that holds a standard volume of liquid (0.1mm^3)

Question 15

how do you measure bacterial cultures with a haemocytometer?

Answer 15

1) sample of nutrient broth is diluted by half with an equal volume of trypan blue which stains dead cells blue (1:1 ratio)
2) each corner of the haemocytometer grid has a square divided into 16 smaller squares which are the four areas normally counted and the mean calculated
3) this number is then timsed by 10^4 to find the number of bacteria per cm^3 of broth
4) repeated at regular intervals througout growth

Question 16

what are the positives and negatives of using cell counts?

Answer 16

• counts only viable cells and is accurate
  HOWEVER
• slow and the equipment is expensive

Question 17

what is turbidimetry?

Answer 17

a specialised form of colorimetry

Question 18

how do you measure bacterial cultures with optical methods such as turbidity?

Answer 18

1) as the number of bacterial cells in a culture increase it becomes increasingly cloudy looking or turbid
2) as the solution get more turbid it absorbs more light
3) a calibration curve is produced by growing a control culture and taking samples at regular intervals
4) turbidity and cell count using a haemocytometer are made for each sample which gives us a relationship between turbidity and number of bacterial cells present

Question 19

what are the positives and negatives of using turbidity?

Answer 19

• quick and can be conducted in the field
  HOWEVER
• equipment is expensive, counts viable and non-viable cells and assumes that agitation and density is equal across the culture

Question 20

what is dilution plating?

Answer 20

a method used to obtain a culture plate with a countable number of bacterial colonies

Question 21

why and when is dilution plating used?

Answer 21

• this works on the principle that every colony is grown from a single, viable microorganism
• used because immediatly after culturing, colonies cannot be counted because a single mass is often present

Question 22

how do you measure bacterial cultures with dilution plating?

Answer 22

1) serially dilute the original culture until you can count individual colonies
2) multiply by the dilution factor and obtain a cell count for the original sample

Question 23

what are the positives + negatives of using dilution plating?

Answer 23

-doesn’t require complex or expensive equipment, only counts viable cells, obtains direct cell count
HOWEVER
-slow as an incubation period is needed and serial dilutions required

Question 24

how do you measure the area and mass of fungi?

Answer 24

measure the diameter of the patches of mycelium

Question 25

what are the phases of bacterial growth?

Answer 25

1- lag phase
2- log phase
3- stationary phase
4- death phase

Question 26

what happens during the lag phase?

Answer 26

the bacteria is adapting to it’s new environment and not yet reproducing at maximum rate

Question 27

what happens during the log phase?

Answer 27

exponential growth where the rate of reproduction is close to theoretical maximum

Question 28

what happens during the stationary phase?

Answer 28

new cells is equal to dying cells as there is a lack of nutrients

Question 29

what happens during the death phase?

Answer 29

reproduction ceases, nutrients run out and waste becomes toxic

Question 30

what is the time between bacterial divisions called?

Answer 30

generation time