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A START-UP MANUAL FOR UNDERGRADUATE RESEARCH STUDENTS IN MICROBIOLOGY
Active Learning from the Very Beginning
Keri Law, Rachel Lamb, and Min-Ken Liao

ASEPTIC TECHNIQUE: WARDING OFF CONTAMINATION

The aseptic, or sterile technique, is a way of working in the lab that decreases the chances of contamination. Since bacteria are microscopic, you will NOT be able to see contamination happening. The aseptic technique MUST be used in the lab. Read and USE the following rules.

1. Have a clear working area as traffic free as possible. The more traffic in your working environment, the more dust and spores and bacteria which will be circulating in the air.

2. Wipe down the lab area BEFORE and AFTER use. Use 70% ethanol to wipe down your area.

3. Autoclave all pipettes, flasks, media, etc.

4. Use the Bunsen burner or alcohol burner when transferring bacteria.

5. DO NOT touch the mouth of a test tube or eppendorf tube with your finger.

6. DO NOT cough or sneeze near the lab bench.

7. Cover the lid of a petri dish if you are about to sneeze!

8. DO NOT place your glass pipette on the bench before use.

9. Open sterile cotton swab packages from the STICK end, NOT the cotton end. By touching the cotton, the swab is no longer sterile.

10. DO NOT leave bottles, plates, etc. open for any period of time: the air is full of microorganisms and they will fall into your media.

The aseptic technique is more of a concept than a technique. Remember, microbes are EVERYWHERE ? on your fingers, in your breath, in the air, on ALL of your lab utensils. USE THE ASEPTIC TECHNIQUE!!!! It is better to start slowly and follow ALL the rules than to have contamination. Soon this will all become second nature.

COMPLETE THE FOLLOWING EXERCISES:

Take the lid off of a new plate and place it on the lab bench.

Place the bottom of the plate (the part WITH the nutrient agar) INSIDE the lid. (This is similar to placing a shoe box in the lid.) The nutrient agar should be exposed to the air.

Does this seem like a GOOD aseptic technique?

Can you see how this could cause contamination?

How clean is our lab?

1. Dampen a cotton swab with distilled water.

2. Swab a small area of your lab. The lab bench, areas around the facets and legs of chairs are all good areas.

3. Using the same cotton swab, swab a sterile nutrient agar plate.

4. Label the plate correctly.

5. Incubate the plate for 24 hours at 30 degrees Celsius.

Even after cleaning the lab, it will NOT be sterile. You need to be sure that flamed loops, pipette tips and other sterile lab utensils are not carelessly placed on the lab bench.

How clean is the air?

1. Remove the lid of a nutrient agar plate.

2. Place the open plate on the lab bench for 30 minutes.

3. Label the plate.

4. Incubate the plate for 24 hours at 30 degrees Celsius.

5. Count the colonies.

The air, which you breathe, is not void of contaminating bacteria. Be sure when you are working the lab to NOT leave sterile containers open to the air.

How clean are YOU?

1. Pat your fingers all over a sterile nutrient agar plate. Note: This will be the LAST time you will EVER do this!

2. Label the plate.

3. Incubate the plate for 24 hours at 37 degrees Celsius.

4. Count the colonies.

Your fingers are NOT clean. The surface of your skin is home to thousands of bacteria. No, don't go try and wash them off. These bacteria are essential for your health. However, they will easily contaminate media, plates and glassware in the lab. Therefore you need to be careful not to touch pipette tips, the insides of plate lids, or any other surface, which are sterile.