6.4.10 Culturing Microorganisms Flashcards
What techniques are used for 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
What is the culturing method?
Pour the sterile agar into the petri dish, cover with the lid and leave to cool
Sterilise the inoculating loop in the Bunsen burner flame
Remove the plug and flame the neck of the culture tube
Take a sample from the culture tube using the inoculating loop
Flame the neck of the culture tube again before replacing the plug
Wipe the end of the loop gently on the surface of the agar
Sterilise the loop again
Tape the lid of the petri dish
Incubate at 25°C for 24 hours
Explanation of each aseptic technique:
- WHENEVER WORKING ASEPTICALLY, ALL WORK SHOULD BE CARRIED OUT IN FRONT OF A LIT BUNSEN BURNER WITH A YELLOW FLAME- THE FLAME CREATES A CONVECTION CURRENT ABOVE THE BENCH, PREVENTING CONTAMINATION OF ANY MICROORGANISMS IN THE AIR.
- HOT AGAR JELLY IS POURED INTO A STERILISED PETRI DISH. THE AGAR IS LEFT TO COOL AND SET-THE PETRI DISH AND CULTURE MEDIUM ARE HEATED TO A HIGH TEMPERATURE TO KILL ANY POTENTIAL MICROORGANISMS THAT COULD CONTAMINATE THE EXPERIMENT.
- AN INOCULATING LOOP IS PASSED THROUGH A HOT FLAME BEFORE IT IS USED TO TRANSFER BACTERIA TO THE CULTURE MEDIUM.- ANY MICROORGANISMS ON THE LOOP ARE KILLED TO PREVENT CONTAMINATION.
- PETRI DISHES SHOULD ONLY BE OPENED AS LITTLE AS POSSIBLE, AT THE SIDE FACING THE BUNSEN BURNER.- THIS DECREASES THE RISK OF MICROORGANISMS CONTAMI- NATING THE DISH.
- THE LID OF THE PETRI DISH SHOULD BE SECURED WITH TAPE AT INTERVALS AROUND THE DISH AND STORED UPSIDE DOWN.- THIS PREVENTS DROPS OF CONDENSATION (ANOTHER SOURCE OF CONTAMINATION) FROM DROPPING ONTO THE SURFACE OF THE AGAR.
- THE CULTURES SHOULD NOT BE INCUBATED ABOVE 25°C IN
A SCHOOL LABORATORY- THIS RESTRICTS THE GROWTH OF HARMFUL PATHOGENS (WHICH ARE MORE LIKELY TO GROW AT HIGHER
TEMPERATURES).
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What are the investigations used for 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
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
What are the results of this?
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
What is the minimum inhibitory concentration?
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
