Appendix: Sterile Technique, etc.

Nov. 14, 1990

C. Helms

Introduction

The most basic technical skill learned in the molecular biology lab is good sterile technique. Contamination, whether it is from a chemical or a biological source, can ruin an experiment by rendering the results invalid. Sterile technique begins with the scientist becoming aware of the potential sources of contamination and using the proper technique to reduce the likelihood of contamination during experimental procedures.

Fingers and the open air are the most likely sources of biological contamination of sterile surfaces. Work as quickly as is comfortable to minimize exposure of materials to the air. Because they are nutrient rich, the different growth media are more likely to produce noticable biological contamination (by turning color, developing clumps, or becoming turbid), but most other liquids used in the lab also can support the growth of organisms. Refer to the solution sections of the lab manual procedures to learn the best method to sterilizing each solution (autoclave or filter sterilization).

Many solutions are prepared from sterile components, and care must be taken to avoid contaminating them during preparation. A solution which is contaminated with another solution may not be easily detected (most often they are discovered through trouble-shooting efforts after failed experiments). To prevent the inadvertant cross-contamination of solutions, learn the techniques described below and keep your mind on your work.

Suggested times in the autoclave for sterilization are: 1) a minimum of 20 minutes for dry items wrapped in foil, 2) a minimum of 30 minutes for loosely capped containers with less than 1 liter liquid volume, and 3) 45 minutes to 1 hour for volumes of 1 to 2 liters. Check the autoclave during the run; the chamber pressure should not fall below 18 lbs. Note that autoclave tape is used only as a temperature indicator and develops black stripes when the autoclave chamber reaches 121 degrees C during the run, i. e., the material is not necessarily sterile if the tape is striped.

Note: Tissue culture (TC) work has even more rigid sterile technique guidelines than the molecular lab. TC technicians receive more specialized instruction about sterile technique in the TC lab from Rose Veile. You should know that TC work is carried out using a stock of supplies (pipets, tubes, flasks, etc.) kept separate from the molecular lab supplies; do not use these supplies in the molecular labs.

Routine practices for the molecular biology lab

1. Periodically wash the benchtop and shelves with 10% bleach, followed by 70% ethanol. Before beginning work each day, wipe down the lab bench using 70% ethanol, or cover with clean bench paper. Wash hands frequently, and wear gloves when working with toxic materials.
2. When pouring media plates, keep the lids on the plates until just before pouring. Lift the lid a short distance up with one hand while pouring the medium with the other, and keep the media bottle at a slant to reduce dust contamination. When the source container is empty, promptly flush with hot water.
3. When plating out phage, bacteria, or yeast, keep the plate uncovered for as short a time as possible. Hold the lid with the inside surface face down to minimize dust contamination. Avoid breathing on open plates.
4. To sterilize a glass rod used in spreading cells on a media plate, dip the rod in 95% ethanol, then pass through a flame. Touch the hot surface to a cold sterile surface (to cool it before touching cells). To sterilize a metal needle or loop, hold it in a bunsen burner flame for a short time (till red hot). Cool it in ethanol, then pass it through the flame to burn off the ethanol. Touch the surface to another sterile surface before touching the cells.
5. When picking plaques or colonies, orient the plate with media side up so the plaque/colony is easily visible, place the index finger over the colony to be picked, and lift the plate up with the other fingers on that hand. With the other hand holding the sterile toothpick/needle, pick the plaque/colony and return the plate immediately to its lid. Be careful to not cross contaminate: use a new sterile toothpick or loop for each sample. The same applies to streaking out colonies.
6. Use a bunsen burner to briefly flame the openings of sterile containers before and after removing contents. When pipetting solutions, use one or two of the smaller fingers on one hand to hold the lid of the container (inside surface face down) and use the thumb and index finger of the same hand to hold the container while the other hand manipulates the pipet. Also briefly flame the tips of pipets before using.
7. Label all solutions prepared with the components and concentrations.
8. Use sterile plastics when preparing human DNA and solutions for PCR. Glass tubes and containers are often not cleaned sufficiently to remove all traces of DNA and can be the source of plasmid DNA contamination.
9. The lab has sterile containers and stock solutions kept in common areas (e. g., TE, 2X LB, 1M MgCl2). Bottles of these solutions should not be returned to the stock shelves once they have been opened; keep the bottle at your bench or dispose.
10. If fingers or the nonsterile surface of a tool ever touches the inside surface or contents of a sterile container, consider the container contaminated and dispose, or resterilize immediately.
11. When removing a volume from a stock bottle, tilt the bottle slightly to reduce the possibility of dust entering the mouth of the bottle. When pipetting, avoid touching the inside edges of the container. Use a sterile pipet/pipetman tip, and return the cap to the sterile container as quickly as possible.
12. We use a lot of supplies that are sterile and individually wrapped. When using a bulk item, e.g., a package of pipets, open the package and leave it lying horizontal until it is empty. Pull out the pipets by inserting as little of your fingers as possible, and as the pipet is withdrawn, hold it free of any other surface to prevent its contamination. Use blowout pipets containing cotton plugs.
13. Remember to use a new sterile pipet for each sterile solution. Never use material from a pipet that has flowed back to the mouth of the pipet, even if the pipet is plugged with cotton. Pipets used with biohazard materials should be disposed of in biohazard bags or biohazard pipet containers prior to autoclaving.
14. Swab down the outside of pipetmen from time to time with 70% ethanol. Use the appropriate pipetman and sterile tip for the volume being pipetted -- i. e., don't use a P200 to pipet 225 ul. Never tilt a loaded pipet up so that the liquid flows back to the pipetman barrel. Periodically remove and clean the pipetman barrel.
15. Each lab has reserved pipetmen for use with human DNA stock tubes. Do not use them for any other purpose than to remove human DNA from the stocks, and never use any other pipetmen for the removal of human stocks. If a pipetman contaminated with clone DNA or nucleases introduces the contamination to a stock tube, many experiments will be ruined. Our human DNA stocks are precious resources and hard to replace; it can be a long time for contamination to be discovered and traced to a stock, so be especially careful with them. The same precautions apply to the enzyme stocks.
16. Remember that experiments using PCR can be easily ruined if the template DNA is contaminated. Extreme care must be taken in preparing solutions and template used in PCR. Each lab should have pipetmen designated for PCR use. Clean the barrels of the pipetmen frequently. Use only sterile plastics that have never been used before.

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