Question

Write a step by step protocol of how to transfer a culture from a petri dish to a broth and name three practices that are part of Aseptic Technique that prevents your culture from getting contaminated by outside microbes?

Answer 1

Ans. It’s assumed that you have your culture and sterilized broths ready in the LAF hood.

Protocol:

1. Make sure all the materials, including culture, sterile broth, 70% ethanol, inoculation loop, burner, etc. are aseptically placed in the pre-cleaned LAF hood.

3. Wash your hands properly and wipe it with suitable (sterile, single use) paper towel. Go to the LAF.

4. Clean your palms with 70% ethanol, let it air dry. Be cautious not to take your hands near the flame (if any), it would catch fire if ethanol is not completely dried off from your hands.

Take care not to take out your arm outside the LAF hood. If it happens, repeat the steps 3 and 4.

5. Light the burner. Clean your palms (arms) with 70% ethanol. Let it air dry.

6. Incinerate the inoculation loop till red hot up to its metal part. Keep it apart for cooling in the LAF with precaution not to touch the metal part with LAF surface. Better, you can use loop sterilizer.

7. Hold the culture petriplate in your left hand. Open it slight near the flame using your left thumb. Take a loopful of culture using the previously incinerated inoculation loop. Be careful not to touch the inoculation LOOP or its part anywhere else except desired colony in the culture plate.

8. Carefully remove the cotton plug from the broth tube with your little finger (or, with little finger and ring finger) of right hand while holding the tube in left hand (or vice-versa as comfortable).

Flame the rim of the tube in 360⁰ direction.

9. Transfer the culture (inoculum) from loop to the broth by dipping the loop in broth. Be careful not to touch the inoculation LOOP or its part anywhere except the broth. That is, only the LOOP shall be dipped in broth but not the wire above it.

10. Flame the rim of tube, plug it with the cotton plug. Gently shake for mixing the inoculum into broth. Keep it in the test tube rack.

11. Incinerate the inoculation loop that was used to transfer culture. Keep is aside for cooling.

# The broth is ready to be transferred to the incubator.

## The three practices that are part of ascetic techniques are-

            1. Incineration, 2. Cleaning the arms (palms) with 70% ethanol.

            3. Avoiding the contact of inoculation loop at all places except the colony or broth.

            4. Flaming the rim of test tubes, etc.