Aseptic techniques

Question 1

What are aseptic techniques?

Answer 1

• prevents foreign microorganisms from being introduced to a test sample
• ensures that the apparatus and environment stay sterile
• contamination can affect the results and potentially result in growth of pathogens

Question 2

What should you do with the workspace where you’re working?

Answer 2

• wash working areas with alcohol before and after work which ensures that no microorganisms are present within the area
• do it regularly but it can be dangerous as alcohol is flammable

Question 3

What should you do if you’re working with pathogens?

Answer 3

• wear gloves so it prevents no microorganisms passing from sample to skin

Question 4

What should you do when working with glassware and apparatus?

Answer 4

• sterilise all glassware/ apparatus (foreceps) before and after use
• in autoclave (a machine which steams equipment at high pressure)
• prepared agar jelly should also be put through the autoclave

Question 5

Why should you work near a Bunsen burner?

Answer 5

• it prevents microorganisms falling into an open sample

- this is as hot air rises, microbes in the air are drawn away from the culture

Question 6

What should you do to the glass container of bacteria after it’s opened?

Answer 6

briefly flame the neck of the glass container of bacteria just after its opened and just before its closed
- this causes air to move out of the container, preventing unwanted microbes from falling in

Question 7

Why and how should you sterilise a wire loop?

Answer 7

• before using wire loop to transfer microorganisms from one medium to another it needs to be sterilised
• heat in the Bunsen flame until red and then cool it close to the flame, away from the bench

Question 8

How can identify bacterial colonies?

Answer 8

• sterilise loop and take a sample
• make 4 to 5 steaks across one edge
• flame and cool loop to kill off anything still on there
• turn plate and make 4 to 5 new streaks
• repeat 3 and 4 twice more and don’t join the lines together
• incubate the plate
• bacteria can be identified by looking at the colony shape, edge, colour, surface appearance and elevation and further chemical tests can be carried out to identify the colony

Question 9

How can we investigate antimicrobials? (Testing the action of antibiotics)

Answer 9

• pour hot sterilised agar jelly into a sterile Perti dish
• when jelly cooled = inoculating (wire) loops can be used to transfer microorganism into culture medium or a sterile dropping pipette and spreader to get an even covering of bacteria
• take 3 discs of filter paper and soak one is antibiotic A, second in B and C is a control and should be soaked in sterile water
• place discs on jelly using sterile forceps and tape lid into dish to prevent contamination by microbes = antibiotic will diffuse (soak) into agar jelly
• leave space b/w discs and label them and leave dish for 48 hrs at 25 C and the bacteria will multiply and grown into a lawn covering the jelly
• anywhere bacteria can’t grow = clear zone so more effective the antibiotic is against the bacteria = large the clear zone around paper disc

Question 10

Why should you use a control disc?

Answer 10

• so you can be sure that the different between the growth of the bacteria around the control disc and antibiotic discs is due to affect of the antibiotic alone (and not something weird in paper etc)
• need to also control temperature (don’t leave near radiator etc), disc size

Question 11

What temperature is culture of microorganisms are kept at?

Answer 11

• about 25 C because harmful pathogens aren’t likely to grow at this temp
• in industrial = cultures are incubated at higher temp so they can grow a lot faster (no pt too high or enzymes in microorganisms could be denatured)

Question 12

What does no clear zone mean?

What is the jelly?

Answer 12

• that bacteria is resistant to it
• jelly is a culture medium as it contains carbohydrates, minerals, proteins and vitamins that microorganisms need to grow

Question 13

How can you compare antimicrobials?

Answer 13

• can compare effectiveness on bacteria by looking at relative size of clear zones
• large the clear zone = more effective
• can check by eye or for more accurate= calculate area using their diameter (include the area of the disc and don’t open Petri dish to measure clear zone as it should be visible through bottom of dish)
• use area = pi x r (diameter/2) squared (diameter with ruler)