All persons using laboratories at Texas A&M-Kingsville are bound to the safety procedures stated in the University’s Chemical Hygiene Plan. This appendix deals with specific procedures pertaining to microbiology laboratories and is designed as a supplement to the Chemical Hygiene Plan. Refer to various sections within the Chemical Hygiene Plan to obtain information on safety procedures not covered in this appendix.

1.0 GENERAL SAFETY PROCEDURES

The following procedures have been established to minimize the health and safety risks while conducting procedures in a microbiology laboratory. It is the responsibility of the laboratory supervisor to demonstrate these and other appropriate aseptic procedures to ensure that all students are proficient at making proper aseptic transfers.

Every microorganism used in the lab should be treated as a pathogen. Even though microbial lab organisms may be altered forms that are normally not dangerous, they can mutate into pathogenic forms, and on occasion you may work with organisms that are not lab strains.

It is imperative to learn the names of instruments and other items used in microbiology, and know the proper use of each. Transferring microbial cultures from one container to another is a common practice. This requires proper "aseptic techniques" which will minimize the risk of contact with the microorganisms and reduce the risk of contaminating cultures. Proper aseptic techniques include, but are not limited to, the following:

1. Flame inoculating needles and loops properly, both before and after contact with microorganisms.
   
2. Keep lids on Petri dishes and caps on culture tubes when not in use. Remove lids and caps only for making transfers, and replace immediatly to prevent contamination.
   
3. When making transfers to and from glass culture tubes, flame the tops of the tubes after removing the cap, and reflame the tops before replacing the cap to prevent contamination.
   
4. When making transfers, keep your face away from the microbial cultures. This minimizes the risk of your own microbes contaminating the cultures, and reduces the risk of inhalation of aerosols from the cultures.
   
5. Use rubber bulbs or mechanical pumps to pipette fluids. Never pipette materials by mouth.
   
6. Never place contaminated materials (pipettes, swabs, inoculating needles, etc.) on the workbenches.
   
7. Microscopes are important instruments in microbiology. Individuals should become very familiar in the proper usage of this instrument, especially with the use of the various objectives (lenses). Immersion oil should only be used with specially designed oil-immersion objectives. Oil will damage the "dry" objectives. Do not use the coarse focus mechanism when observing slides with longer-length objectives. Clean the objectives before and after use to keep them free of oil and dirt.
   
8. Place used items in the appropriate disposal containers for sterilization. These containers should be clearly labeled with the type of waste they contain. Do not mix different waste types.
   
9. Do not take equipment, media, or bacterial cultures out of the laboratory.
   
10. Report all spills and accidents, no matter how small, to the laboratory supervisor. Spills shall be covered with paper towels and drenched with disinfectant. Leave the disinfectant in contact with the spill for at least 15 minutes.
    
11. Wash hands before and after working in the laboratory, using antimicrobial soap.
    
12. Notify your instructor if you become ill during lab. If you become ill outside of class, notify your instructor before you start lab work. The instructor may decide to curtail your lab activities for that period if your condition may be worsened, or if your condition poses a danger to other students. Also, if you are pregnant or become so during the term, it is your responsibility to notify the instructor so that any necessary precautions can be taken.

2.0 SPECIFIC LAB PROCEDURES FOR LAB TECHNICIANS

The following are minimum guidelines for microbiological laboratory technicians and should be adhered to at all times.

1. Always maintain fresh stock cultures. New cultures should be inoculated into fresh media every two months. If this is not practical, existing stock cultures should be reinoculated into fresh media every two months. Screening for mutants is desirable (using replica-plating, isolation culturing, or similar techniques).
   
2. Prepared media should be used promptly. Freshly prepared media is best, but if the lab uses pre-made media from a supply company, be sure to use the media before the expiration date. Expired media should be autoclaved and disposed.
   
3. Rotation of disinfectants is recommended on a weekly basis. A rotation of sodium hypochlorite solution (bleach), isopropyl alcohol (70%), and quaternary ammonium cleaner ("Quat") usually works well.
   
4. Students should be supplied with adequate waste containers. Suggested labels for these containers are as follows:

Disposable items (swabs, plastics, etc.) for autoclaving
Reusable items (glassware) for autoclaving
Reusable pipettes (a container partially filled with liquid detergent should 
    be available for these)
Noncontaminated waste

APPENDIX - B - prepared by:
Thomas Neil McCrary
Texas A & M University-Kingsville
College of Agriculture
February 27, 1995

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