6.4.10 Culturing Microorganisms

• When investigating the effect of antimicrobial substances on microbial growth it is essential that aseptic techniques are used
• Aseptic techniques ensure the microbes being investigated don’t escape or become contaminated with another unwanted, and possibly pathogenic, microbe
• Some general aseptic techniques include:
  • Washing hands thoroughly
  • No food or drink allowed in the lab
  • Disinfecting work surfaces with disinfectant or alcohol
  • Wearing gloves and goggles
  • Working close to a lit Bunsen burner (this creates an updraught of air so prevents contamination from airborne fungal spores, for example)
  • Flaming equipment (to kill microorganisms or create updraughts)
  • Sterilising (in an autoclave) or disposing of all used equipment

Culturing method

1. Pour the sterile agar into the petri dish, cover with the lid and leave to cool
2. Sterilise the inoculating loop in the Bunsen burner flame
3. Remove the plug and flame the neck of the culture tube
4. Take a sample from the culture tube using the inoculating loop
5. Flame the neck of the culture tube again before replacing the plug
6. Wipe the end of the loop gently on the surface of the agar
7. Sterilise the loop again
8. Tape the lid of the petri dish
9. Incubate at 25°C for 24 hours

To prepare an uncontaminated culture of microorganisms, this procedure can be followed

Aseptic Techniques Table

Investigations using cultured microorganisms

• It is possible to test the efficacy of different antibiotics and antiseptics using cultured microorganisms
• The disc diffusion method is commonly used to test for antibiotic resistance in bacteria
  • It allows for multiple antibiotics to be tested at once

Apparatus

• Sterile agar plates
  • The agar can be made sterile by boiling

• Diluted bacterial broth with a concentration of 1 x 10⁸ CFU mm^-3
  • Colony-forming unit (CFU): a live bacterial cell that is able to divide and form a colony on the agar plate

• Multiple different antibiotic solutions of a standard concentration
• Paper disks
• Pipettes
• Spreaders
• Bunsen burner
• Gloves
• Goggles
• Incubator
• Autoclave

Method

• Pre-soak paper discs in the different antibiotic solutions
  • The different antibiotic solutions need to be the same concentration so that the effects of the different antibiotics can be compared

• Spread a sample of the diluted bacterial broth onto the surface of the sterile agar plate
• Lightly press the paper discs onto the surface of the agar
  • Make sure the discs are evenly distributed in the plate
  • They should not be touching the edges of the plate or any other discs

• Keep the agar plate in the incubator overnight
  • The incubator maintains an optimum temperature for bacterial growth

• Remove the agar plate from the incubator and examine the results with the petri dish lid on

Results

• Overnight the antibiotics will diffuse outwards from each paper disk so that a gradient of antibiotic forms. The antibiotic is most concentrated where the paper disk is located
• If the bacteria being investigated is vulnerable to an antibiotic then a clear area will be visible around the disc
  • There are no bacteria present in the clear area

• The clear area will end when the concentration of antibiotic reaches a level that the bacteria are no longer susceptible to
• More effective antibiotics require a lower concentration to kill bacteria and so they will produce larger clear zones
• If a bacteria is completely resistant to an antibiotic then there will be no clear zone around that particular paper disk

Image showing the bacterial growth on an agar plate following a disc diffusion experiment. The most effective antibiotics produce the largest clear zones while. The antibiotics that the bacteria are resistant to produce no clear zone.

We can then calculate the area of the zone of inhibition in order to compare the results quantitively

The minimum inhibitory concentration (MIC)

• When antibiotics are used to treat bacterial infections, the dosage used is carefully controlled
• The minimum inhibitory concentration (MIC) is the lowest concentration of a substance that will inhibit the growth of a microorganism