7.1.8 Practical - Aseptic Technique

• 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
• Aseptic techniques include: 
  • Washing hands thoroughly
  • No food or drink allowed in the lab
  • Disinfecting work surfaces with disinfectant or alcohol
  • Not allowing the growth of microorganisms at body temperature
    • This restricts the types of microorganisms that can be used in experiments in schools to those that won't readily invade human hosts
  • Using flamed loops or sterile swabs when transferring cultures
  • Wearing gloves and goggles
  • Flaming culture bottlenecks to prevent contamination
  • Sterilising or disposing of all used equipment
  • Washing hands thoroughly
  • Having a lit bunsen burner in the room
    • This is a measure taken to stop contaminants falling onto/into the growth media
  • Only removing petri dish lids when necessary
  • Autoclaving of glassware and growth media

Testing for bacterial antibiotic resistance using the disc diffusion method

• The disc diffusion method is commonly used to test for antibiotic resistance in bacteria
• It allows for multiple antibiotics to be tested at once
• This is not a required practical but it helps to illustrate the use of aseptic techniques

Apparatus

• Sterile agar plates
  • The agar can be made sterile by boiling
• Diluted bacterial broth
• Multiple different antibiotic solutions of a standard concentration
• Paper disks
• Pipettes
• Spreaders
• Bunsen burner
• Gloves
• Goggles
• Incubator

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.