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Practical: Aseptic Techniques

• When investigating the effect of antimicrobial substances on microbial growth it is essential that aseptic (sterile) techniques are used

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

  • This is especially important in preventing the accidental culture of human pathogens

• Aseptic techniques include:

  • Washing hands thoroughly to disinfect them

  • No food or drink allowed in the lab

  • Disinfecting work surfaces with disinfectant or alcohol

  • All apparatus, glassware and collecting loops must be fully sterilised before use

  • The culture medium (either a culture broth or an agar plate) must be sterilised

  • Using flamed loops or sterile swabs when transferring cultures to avoid collecting microbes in the atmosphere

  • Flaming culture bottlenecks to prevent contamination

  • Not allowing the growth of microorganisms at body temperature

  • Only removing petri dish lids when necessary

  • Sterilising or disposing of all used equipment promptly once they are no longer needed

Example of a practical using aseptic techniques: 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

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

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.