2.9 Culturing Microorganisms

Question 1

Conditions needed for bacterial growth (4)

Answer 1

Under optimal conditions, bacteria can multiply via binary fission as often as once every 20 minutes
- Temperature - most bacteria grow fastest in warm environments.
- Nutrient availability - bacteria need a good supply of nutrients in order to grow rapidly.
- Moisture - most bacteria grow fastest in moist conditions.
- Oxygen - different types of bacteria either need the presence or absence of oxygen for growth.

Question 2

What mixture of nutrients do bacteria need in order to reproduce?

Answer 2

Carbohydrates (for energy), nitrogen compounds (for protein synthesis), vitamins, and minerals.

Question 3

What 2 different culture mediums can be used when culturing microorganisms?

Answer 3

• Nutrient broth - a liquid medium (like water)
• Solid agar jelly - a gel medium (like firm jelly)

Question 4

How does bacteria appear when grown on agar plates? (2)

Answer 4

• Visible colonies
• Spread out to give an even covering of bacteria

Question 5

How is an agar plate made?

Answer 5

Hot agar jelly is poured into a shallow round plastic dish called a Petri dish.

Question 6

In the lab at school, why are cultures of microorganisms not kept above 25 degrees?

Answer 6

Harmful pathogens are more likely to grow above this temperature

Question 7

In industrial conditions, why are cultures incubated at high temperatures?

Answer 7

So that they can grow a lot faster

Question 8

What are aseptic techniques used for when culturing microorganisms?

Answer 8

To prevent contamination of cultures by unwanted microorganism. This would affect your results and could potentially result in the growth of pathogens.

Question 9

Aseptic techniques (7)

Answer 9

• Cleaning surfaces with disinfectant - e.g. cleaning the work area before and after use with alchohol
• Washing hands with antiseptic soap and warm water before handling microorganisms
• The Petri dishes and culture medium must be sterilised before use (e.g. by heating to a high temperature) to kill any unwanted organisms
• If an inoculating loop is used to transfer the bacteria to the culture medium, it should be sterilised first by carefully passing it through a hot flame.
• Creating a sterile field using a Bunsen burner - a sterile field is a sterilised area created by the updraft of the flame
• After transferring the bacteria, the lid of the Petri dish could be lightly taped on - to stop microorganisms from the air getting in
• The Petri dish should be stored upside down - to stop drops of condensation falling onto the agar surface

Question 10

What is inoculation?

Answer 10

Inoculation is the process of transferring bacteria from a broth to an agar plate

Question 11

Describe the process of inoculation (10)

Answer 11

• The inoculating loop is sterilised before use by being placed in a Bunsen burner flame until it is red hot
• The inoculating loop is then held in the air in the sterile field area, and allowed to cool
• Lid of the bacterial culture bottle is removed and the neck of the bottle is placed in the Bunsen burner flame. This moves air out of the bottle and prevents other microorganisms from entering the bottle and contaminating the bacterial solution
• The sterilised inoculating loop is dipped into the flash containing the bacterial culture
• The neck of the bottle is flamed again and the cap is replaced
• The Petri dish lid is lifted slightly
• Zig-zag streaks are made gently and carefully with the inoculating loop across the agar
• The Petri dish is quickly replaced
• Lid of the Petri dish is secured to prevent contamination
• The Petri dish is then stored upside down in an incubator

Question 12

Describe the process of investing the effect of antibiotics on bacterial growth (5)

Answer 12

1) Place paper discs soaked in different types (or different concentrations) of antibiotics on an agar plate that has an even covering of bacteria. Leave some space between the disks
2) The antibiotic should diffuse (soak) into the agar jelly. Antibiotic-resistant bacteria will continue to grow on the agar around the paper discs, but non-resistant strains will die. A clear zone will be left where the bacteria have died.
3) Make sure you use a control. This is a paper disc that has not been soaked in an antibiotic. Instead, soak it in sterile water. You can then be sure that any difference between the growth of the bacteria around the control disc and around one of the antibiotic discs is due to the effects of the antibiotic alone, and not the paper.
4) Leave for 48 hours at 25 degrees.
5) The more effective the antibiotic is against the bacteria, the larger the clear zone will be.

Question 13

What are the zones called in which the bacteria in a medium are killed by the disc soaked in antibiotic?

Answer 13

Zone of inhibition/clear zone

Question 14

When bacteria are grown on agar plates, each visible ‘group’ of bacteria is referred to as a:

Answer 14

Colony

Question 15

What is the term for the procedures used to prevent contamination when culturing microorganisms?

Answer 15

Aseptic techniques