When handling micro-organisms, it is important to correctly use aseptic technique - both to prevent your micro-organisms from getting contaminated by outside forces, as well as to protect yourself and the people around you.
Steps
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Protect yourself. Tie up long hair, and wear a lab coat and protective goggles.
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Wash your hands and your workspace. Wash your hands using water and soap, and spray down the bench with 70% ethanol.Advertisement
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Lay out all your equipment. Make sure you have everything ready to go before starting.
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Turn on the bunsen burner as high as possible. You'll need it to sterilise your equipment, and the upward draft prevents outside contaminants from landing on your culture.
- Turn the collar so that the airflow is all the way open.
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Sterilise the inoculation loop. Hold it at a roughly 45° downwards angle and slowly move it through the flame so that it glows red-white hot.
- It's easiest to hold the loop between your thumb and pointer finger, and supporting it with your middle finger.
- The hottest part of the flame is right at the tip of the inner, blue fire cone.
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Use your other hand to grab your original culture.
- If it is a plate, hold it and crank open the lid just enough to get the loop inside.
- If it is a tube with a lid, use the pinky finger of the hand you're holding the inoculation loop with to remove the lid, then quickly drag the opening of the tube through the flame of the bunsen burner twice. This is called flaming and kills off anything that wants to get into the tube, and creates a convection current that forces air out of the tube so nothing can get it. Keep holding on to the lid with your pinky for the following step.
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Inoculate. Touch the inoculation loop to a colony you want to pick up. If you are inoculating from liquid broth, dip the inoculation loop into the tube and shake the loop to make sure you get enough bacteria.
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Close the original culture. If it's a plate, just close the lid and put it back down. If it's a tube, drag the opening through the flame of the bunsen burner twice, then place the lid back on.
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Open the new plate. As before, use your free hand to crank open the lid just wide enough to get the loop in
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Plate the bacteria. Lightly swipe the inoculation loop over the surface, being careful not to penetrate the agar.
- A common streaking pattern is the quadrant method.
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Close the plate.
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Sterilise the inoculation loop. Like before, hold it at a 45° downward angle and slowly move it through the flame so that it grows red-white hot.Advertisement
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Tips
- In general, gloves aren't necessary. All the above steps serve to protect both yourself and your bacteria, so the risk of contamination is very low. Gloves can give nasty burns if you accidentally touch them with the hot inoculation loop.Thanks
- Keep in mind that you're working with fire. It's not a big deal, just be sure you know what to do if you burn yourself; pause the experiment, rinse the burn with water and, if necessary, notify your supervisor.Thanks
- If you're doing multiple runs of having to use aseptic technique, it's not necessary to completely turn the bunsen burner off between runs - just turn it down to the pilot flame and only turn it all the way off when you're completely done.Thanks
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References
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