Guide to Aspectic Technique

Before attempting ANY procedures that involve bacteria, you MUST learn aspectic technique. Learning just a few precautions will prevent contamination of other experiments and sickness in those who are working with, or around, bacteria.

Even harmless bacteria can cause problems if given the right opportunity. Therefore it is essential that these steps be followed TO THE LETTER. If your students are unable to handle following these safety precautions, you should NOT have them do this experiment!!

So what does aspectic technique do, and why should one bother? First of all, aspectic technique will protect you, your students, and their experiments from harmful bacteria, fungi, and other such microorganisms. Aspectic technique is fairly easy to learn, and should be learned to prevent sickness.


Aspectic Technique:
1. First, your work area should be clean. Bacteria and fungi lurk everywhere, so precautions should be taken against contamination from these unwanted organisms by wiping down tables with a strong disinfectant and washing hands. Bleach and a commercial disinfectant called NABC are good tabletop disinfectants. Dial and other antibacterial handsoaps are good soaps. 60% ethanol also is a good hand or tabletop disinfectant. For good reasons, the alcohol content should NOT go above 70% or below 45 or 50%.

2. A bunsen burner with a blue flame should be used to clean metal inoculating loops. If you are using disposable (plastic) loops, a beaker with some tabletop disinfectant should be handy to put the loops into when finished. The loops should then be autoclaved and thrown away.

3. If using metal loops, the loops should be sterilized by placing them in the hottest part of the flame (this is the middle of the flame, at the tip of the cone. The flame should be blue and should not have any orange sparks) and letting the loop become ORANGE or RED. The loop should be kept there for a moment, making sure it is sterile. When sterilizing loops, it is a bad idea to wave the loop around in the air to cool it. This will defeat the whole purpose of sterilizing it in the first place. Hot loops can still be used when inoculating. Loops should ALWAYS be sterilized before AND after using them! Sterilize before using different bacteria, different plates, or different experiments. You MUST keep loops sterile in order for your experiment to come out right!
Here is a picture of how the loop should be held and sterilized.

4. Using petri plates: If you are inoculating a petri plate with agar, there are certain guidelines you should use to prevent fungi and other bacteria from contaminating the plate. All loops should be sterile. To inoculate: when bacteria from another plate or a broth is being transferred onto another petri dish, carefully lift the lid of the plate, making sure that the lid stays OVER the bottom. The theory behind this is that air falls downward, carrying spores of bacteria and fungi onto the agar of the plate. Agar is the perfect breeding place for almost any type of fungi or bacteria, so preventing the "fall" of these spores onto the agar will prevent contamination. Do not take the lid completely off the plate!! Only a corner really needs to be lifted, and only high enough to slide a loop in without touching the sides! If you were not careful enough to keep the cover over the bottom, you will know when the plates have been incubated. Large, fuzzy spots of fungi will often appear, or strange colonies of bacteria will be mixed in (usually on the sides) with your bacteria.
Here is a picture on how to hold a loop and the lid of the plate. The loop is already in the plate and slightly touching the agar.

5. Using tubes: Usually tubes with bacterial cultures will be stoppered to prevent contamination. Taking the tops off, holding the test tube and the loop, and trying to inoculate a plate is often a awkward job! Flexible fingers are often a must, but holding all these things can easily be learned! First, hold the test tube in your left hand. This is usually the best hand to use, even if you are left-handed. Hold the sterile loop in your right hand like you would a pencil. Now, using your pinky and ring-finger of your right hand, unstopper the test tube. To see how to hold the loop and test tube, click here. Notice how I can hold the cap and the loop at the same time. This should become easy with practice. Your left hand is completely free to hold the tube, replace the tube, and lift the lid of a petri plate! The tube should be held at an angle such that air "falling" will not contaminate the tube, and nothing will spill out! Do NOT hold the test tube upright!! At this point, you should still be holding the stopper and loop with your right hand. With your left, pass the mouth and the upper part of the test tube through the flame once or twice, like this. This should kill anything that MIGHT have gotten into the tube. Do NOT pass the bottom of the test tube through the flame. You will kill the bacteria! Now, insert the loop into the broth (or if using slants, pass the loop over a colony, making sure to catch a small amount of bacteria), being careful NOT to touch the sides of the test tube, and make sure, when using broths, to go to the bottom of the tube. Most bacteria don't like oxygen, so they will be as far from it as possible. Click here to see a picture. The loop should have a small droplet of liquid in the center. Pass the test tube through the flame again. Restopper the test tube, making sure that NO LIQUID is dropped from the loop. Place the test tube in its stand, then use your now free left hand to open the petri dish as described above. You can now inoculate your plate. (*whew*) I know that sounds really hard, but with practice, you will soon be able to do it quickly and efficiently, with minimal or no contamination.

Now you know the basics of aspectic technique! It is best to have someone who knows aspectic technique to SHOW how it is done, but often that is impossible. I have recently added the pictures to aid in the description. Still, it is better to be shown!! Just remember to keep everything sterile, and use common sense! Wash hands and tables BEFORE AND AFTER working with bacteria. It is a good idea not to have books and bookbags lying around the lab. Something could be spilled, and could infect the student. Eyewear not necessary, but they are highly recommended! Autoclave at the proper temperatures and pressures to ensure killing.

And if you are unsure about anything, ask around to see if someone knows anything about your problem. I will admit, I am not a microbiologist (yet), but I know a LOT about it. If you have questions, feel free to email me. I will be happy to answer any question I can!