Microbiology - Aseptic technique Flashcards
What is aseptic technique used for?
Prevent contamination when working with microorganisms.
What are the two main methods of culturing microorganisms?
In solid agar or in a liquid broth.
How do you ensure you’re working in a sterile bench space.
1) Disinfect work surfaces before and after use
2) Hands washed with antibacterial soap before and after work
3) Any transfer of microorganisms must be done close to a bunsen burner
What is the reason for transferring microorganisms near a bunsen burner?
Warm air currents draw any airborne microorganisms upwards.
What precautions are taken when accessing the glass container?
1) The neck should be quickly passed through a bunsen burner to kill any surface microbes
2) The lid of the culture bottle should be held in the same hand that holds the bottle with the lid held with the pinky finger curled towards your palm.
3) It should not be allowed to touch the bench.
How is an agar plate prepared?
Pouring liquid sterile nutrient agar (sterilised by autoclaving) into a sterile petri dish, having lifted the lid only at one side to allow access.
How do you add things to the surface of the agar?
Only open the petri dish at one side for a minimal amount of time
How do you sterilise a metal inoculating loop?
Heating in the hottest part of the bunsen flame until red hot, you then allow it to air cool as it would kill any microorganisms it came into contact with. After transfer of microorganisms you must re-flame the loop.
What is used for the transfer of antimicrobial discs?
Forceps - which are sterilised in the same was as the inoculating loop.
Describe the spread plate method and describe what instruments are used?
Cells in suspension are used in inoculation. An L-shaped spreader is used to spread the inoculating bacteria over the surface of the agar. Plastic sterile disposable spreaders are a suitable alternative to glass spreaders.
How do you secure a petri dish?
Fixed two or four strips of tape at opposite ends, they should be labelled on the outside of the base.
How do you incubate them?
Incubate upside down at an appropriate temperature for the microorganism. Incubated upside down to lessen the risk of contamination from airborne particles setting on them and to prevent the accumulation of any water condensation that may or otherwise disturb the culture.
How do you investigate the effect of different antibiotics on bacteria using discs.
1) A sterile petri dish containing sterile nutrient agar is used.
2) A sample is removed from a broth culture using a sterile dropper pipette, few drops are placed onto the surface of the agar.
3) A sterile spreader is used to spread the inoculum over the entire surface of the agar - allowed to dry.
4) Sterile discs of filter paper are soaked in different antibiotics.
5) Using forceps discs are placed suitably spread apart.
(A mast ring may be used instead)
6) After incubation, the plate will be opaque where bacteria has grown but clear where the antibiotic has worked - zones of inhibition.
7) Measure diameter in mm - compare to see which is the most effective.
Investigating the effective concentration of an antibiotic
E-strips are prepared strips that contain a concentration gradient of antibiotic. Place an E-strip on the agar of a petri dish that has been inoculated with bacteria. Following incubation (18-72 hours) and depending on the E-strip and bacterium used you will see a gradient of inhibition zones.
What is the minimal inhibitory concentration (MIC) ?
The lowest concentration of an antibiotic that inhibits bacterial growth.
