Aseptic techniques in microbial culture

Culturing bacteria on an agar plate

Step through the method. Each stage tells you what to do and why.

1. 1Sterilise firstBefore anything else, sterilise all the equipment, for example in an autoclave. It steams and heats the equipment to very high temperatures, which kills microorganisms. The bacteria will grow on an agar plate: a Petri dish of growth medium set with agar jelly.+
2. 2Clean hands, clean benchWash your hands to remove microorganisms from your skin. Spray and wipe the work surface with disinfectant to kill the microorganisms on it. Safety rule: the disinfectant can be flammable, so never spray it near a lit Bunsen burner.+
3. 3Work close to the flameLight the Bunsen burner and work close to it. Doing this reduces contamination. The next block shows you exactly how.+
4. 4Flame the loopPass the wire inoculating loop through the flame until it glows red. That sterilises it. Then let it cool before you use it.+
5. 5Open up carefullyOpen the bottle of bacteria and pass its neck through the flame, which stops bacteria escaping and contaminating the air. Lift the plate's lid only to about 45 degrees, not right off, so unwanted microorganisms from the air don't settle on the agar. Streak the bacteria gently so the agar surface isn't damaged, and replace the lid quickly.+
6. 6Close downPass the neck of the bottle through the flame again before you replace its lid, for the same reason as before. Then flame the loop again until it glows red, so any bacteria left on it are killed.+
7. 7Tape, label, incubateAttach the lid with two pieces of tape, not sealed all the way round. Label the underside of the plate with your initials, the date and the name of the bacteria. In school, incubate the plate upside down at a maximum of 25 °C (usually for 24 hours) to prevent the growth of pathogens.+

Have a go: the tape

Sealing the plate right round feels like the safest option. Is it?

The lid is attached with two pieces of tape, not sealed all the way round. What is the reason for NOT sealing it?

Biology · Aseptic technique

What is each step protecting?

Pick a step, then pick the job it does. Commit first, then read why.

10 of 10 still to sort.

Ask which way the microorganisms would be travelling.

What you need to know

• Aseptic technique stops unwanted microorganisms contaminating you, your work area or your samples.
• Bacteria are cultured on an agar plate in a warm incubator, and all equipment is sterilised first, for example in an autoclave.
• Wash your hands and wipe the bench with disinfectant, but never spray it near a lit Bunsen, because it can be flammable.
• Have a goYour lab partner sprays the bench with disinfectant right next to a lit Bunsen burner, announcing: "Extra clean!" What do you say?

  Stop! Disinfectant can be flammable, so it must never be sprayed near a lit Bunsen burner.

  The danger isn't the disinfectant on its own; it's a possibly flammable spray meeting a naked flame.

• Work close to the lit Bunsen: the air it heats flows upwards and stops microorganisms settling.
• Flame the loop until it glows red to sterilise it, then let it cool. A hot loop would kill the bacteria in your sample.
• Pass the neck of the bottle through the flame after opening it and again before replacing the lid, to stop bacteria escaping.
• Have a goFlaming the neck of the bottle of bacteria: is it keeping unwanted microorganisms OUT of the culture, or the cultured bacteria IN?

  It stops the cultured bacteria escaping from the bottle and contaminating the air.

  Not every flame protects the culture. This one protects the air around you from the bacteria you are growing.

• Lift the plate's lid only to about 45 degrees and replace it quickly, so microorganisms from the air don't settle on the agar.
• Attach the lid with two pieces of tape, not all the way round, because the bacteria being cultured need oxygen for aerobic respiration.
• Label the underside of the plate. In school, incubate it upside down at a maximum of 25 °C to prevent the growth of pathogens.
• Have a goThe incubator in your school lab is set to 30 °C. Is that okay for culturing bacteria in school?

  No. In school the maximum is 25 °C, to prevent the growth of pathogens.

  25 °C is a ceiling, not a target. A warmer plate may grow more, but in school that is the wrong direction.

• If a patient's bacteria are tested against antibiotic discs, contamination could obscure the clear zones and lead to the wrong antibiotic.

The big picture

Aseptic technique is the set of practical methods that stop unwanted microorganisms contaminating you, your work area or your samples when you culture bacteria. Every step answers one question: how could an unwanted microorganism get into the culture, or a cultured one get out? Know the reason and the method stops being a list to memorise.

⚠ Watch out

Treating the steps as rituals, and especially thinking the safest plate is the one sealed all the way round. It isn't: a full seal would stop oxygen entering, and the bacteria being cultured need oxygen for aerobic respiration.

Memory hook

OUT, IN, SAFE. Every step keeps unwanted microorganisms OUT of the culture, keeps your cultured bacteria IN, or keeps people SAFE.

Check yourself

Cover the page and talk through the method from sterilising the equipment to incubating the plate. Give one reason for each step. Which reason did you have to think hardest about?