4-4-6-required-practical-aseptic-techniques

Required practical: aseptic techniques

• When investigating the effect of antimicrobial substances on microbial growth, aseptic techniques must be used

• Aseptic techniques ensure the microbes being investigated don’t escape or become contaminated with another unwanted, and possibly pathogenic, microbe

Examples of aseptic technique

• Sterilise Petri dishes

  • This involves heating equipment to a high temperature to kill microorganisms 

• Sterilise the nutrient agar

  • This should be carried out before the agar is poured into the sterile Petri dishes

• Disinfect work surfaces with disinfectant or alcohol before inoculation

• Work next to a lit Bunsen burner

  • This creates convection currents that stop contaminants from falling onto the growth media

• Use flamed inoculating loops or sterile swabs when transferring cultures 

  • Heating loops until red-hot kills any unwanted microorganisms

  • This should be done both before and after inoculation

• Keep the lid on Petri dishes when not in use, and lift the lid at an angle during inoculation

  • This reduced the contamination risk from airborne microorganisms

• Tape the Petri dish lids closed after plating

  • Loss of Petri dish lids could lead to the escape of potentially harmful microorganisms

• Incubate plates at a safe temperature of 25 °C

  • This prevents the growth of pathogenic microorganisms which are more likely to grow at human body temperature

• Sterilise or dispose of all equipment after use

Use of aseptic techniques to investigate the effect of antimicrobial substances on microbial growth

• This method is referred to as a disc diffusion experiment and is used to test antibiotic effectiveness

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 can divide and form a colony on an agar plate

• Multiple different antibiotic solutions of a standard concentration

• Paper disks

• Pipettes

• Spreaders

• Bunsen burner

• Gloves

• Goggles

• Incubator

Method

1. 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

2. Create a lawn of bacteria by spreading a sample of the diluted bacterial broth onto the surface of the sterile agar plate

3. Lightly press the paper discs onto the surface of the agar

   • Make sure the discs are evenly distributed on the surface of the plate

   • They should not be touching the edges of the plate or any other discs

4. Keep the agar plate in the incubator overnight

   • The incubator maintains an optimum temperature for bacterial growth

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

6. Measure the clear areas around each paper disc to compare the effectiveness of antibiotics

   • The clear area (also referred to as the ‘zone of inhibition‘) can be calculated by measuring the diameter or radius and calculating the area of a circle using the equation: Area = π × r²

Results

• The antibiotics will diffuse outwards from each paper disk so that a gradient of antibiotics forms. The antibiotic is most concentrated where the paper disk is located

• If the bacteria being investigated are vulnerable to an antibiotic, then a clear area will be visible around the disc

  • There are no bacteria present in the zone of inhibition

• The clear area will end when the concentration of the antibiotic reaches a level at which 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 the bacteria are completely resistant to an antibiotic, then there will be no clear zone around that particular paper disk

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