Culturing micro-organisms

Question 1

What are the aims of the practical for culturing microorganisms practical?

Answer 1

.to investigate the effects of antiseptics or antibiotics on bacterial growth

Question 2

What is a culture medium?

Answer 2

It contains the carbohydrates, minerals, proteins and vitamins the bacteria need to grow.

Question 3

What two culture mediums can be used?

Answer 3

Agar jelly and nutrient broth solution

Question 4

How do you uses nutrient broth solution to culture microorganisms?

Answer 4

It involves making a suspension of bacteria to be grown and mixing with sterile nutrient broth stoppering the flask with cotton wool to prevent air from contaminating it and shaking regularly to provide oxygen for the growing bacteria.

Question 5

What happens when we use agar jelly?

Answer 5

The bacteria will form visible colonies on the surface of the jelly or spread out to give an even covering of bacteria.

Question 6

Aseptic techniques

Answer 6

1. Wash hands and clean surfaces
2. Sterilise petri dishes and culture mediums
3. Sterilise inoculating loop
4. Seal the lid of the petri dish
5. Incubate petri dish upside down
6. Incubate at 25ºC

Question 7

Why should you wash your hands and clean the work surfaces before beginning?

Answer 7

To prevent contamination

Question 8

How do you sterilise the petri dishes and culture mediums?

Answer 8

by heating to a high temperature) often done by an autoclave (oven) or UV light. If it’s a glass petri dish you need to sterilise it or you can just buy pre-sterilised plastic ones.

Question 9

Why should you sterilise the petri dishes and culture mediums before use?

Answer 9

If this step doesn’t take place they can be contaminated with other microorganisms. These could be harmless and compete with the desired bacteria for space. They can also be harmful and potentially produce a new pathogen.

Question 10

How do you sterilise the inoculating loop?

Answer 10

By passing it through a hot flame.

Question 11

Why do we need to sterilise the inoculating loop?

Answer 11

Kills unwanted microorganisms.If this step doesn’t take place they can be contaminated with other micro-organisms. These could be harmless and compete with the desired bacteria for space. They can also be harmful and potentially produce a new pathogen

Question 12

How do you seal the lid of the petri dish?

Answer 12

Seal it with tape but not completely

Question 13

Why do we need to seal the petri dish in this way?

Answer 13

This stops airborne microorganisms from contaminating the culture. But it shouldn’t be sealed all the way to prevent harmful anaerobic bacteria from growing due to the lack of oxygen.

Question 14

Why should the petri dish be stored/incubated upside down?

Answer 14

This is to prevent condensation from the lid landing on the agar surface and disrupting growth

Question 15

When/where do we usually incubate the culture at 25ºC?

Answer 15

in school labs

Question 16

Why do we incubate the culture at 25ºC?

Answer 16

This is because, if it were incubated at a higher temperature closer to 37ºC (human body temp) it’s be more likely that bacteria that could be harmful to humans could grow as this is their optimum temperature. At lower temperatures (25ºC), colonies of such harmful bacteria wouldn’t be able to grow.

Question 17

When are cultures incubated at higher temperatures?

Answer 17

in industrial conditions

Question 18

Investigation

Question 19

Step 1

Answer 19

Pour hot agar jelly into a sterile petri dish and leave it to cool and set and

Question 20

Step 2

Answer 20

Then, use a flamed inoculated loops to transfer microorganisms to the culture medium by making streaks on the surface of the agar jelly (alternatively, a sterile dropping pipette can be used to get an even covering of bacteria)

Question 21

Step 3

Answer 21

Soak paper discs in different types (or concentrations) of antibiotics for the same length of time and place them (evenly distributed) on an agar plate using flamed forceps that has an even covering of bacteria. But, leave some space between the discs. Separate the lid of the petri dish into 4 quadrants and label them with the antibiotic.

Question 22

Step 4

Answer 22

The antibiotic should diffuse into the agar jelly

Question 23

Step 5

Answer 23

Also, place a disc that has been soaked in sterile water on one of the quadrants of the agar as a control. So that you can be sure that any difference between the growth of the bacteria around the control disc and around one of the antibiotics is due to the effect of the antibiotic alone

Question 24

Step 6

Answer 24

Tape the lid onto the petri dish and incubate it upside down for 48 hours at 25 degrees celsius.

Question 25

Results

Question 26

A

Answer 26

There should be clear inhibition zones surrounding some of the discs which is where the bacteria have been killed by the antibiotic.

Question 27

B

Answer 27

The more effective the treatment is the larger the inhibition zone. A higher concentration will also result in a larger inhibition zone.

Question 28

C

Answer 28

Some bacteria may be antibiotic resistant and will therefore be able to grow in the presence of bacteria and so there will be no inhibition zone.

Question 29

D

Answer 29

There won’t be an inhibition zone around the control disc which shows that only the antibiotic/antiseptics kill the bacteria.

Question 30

Risk Assessment

Question 31

Hazards

Answer 31

Control measures

Question 32

Contamination - altering experimental results and potentially introducing pathogens.

Answer 32

Aseptic techniques - washing hands and sterilising equipment and cleaning surfaces

Question 33

Disinfectant - flammable

Answer 33

keep away from naked flame

Question 34

Naked flame

Answer 34

Keep away from flammable materials

Question 35

Pathogen Exposure - could cause illness

Answer 35

Aseptic techniques