Microbiology Laboratory

LABORATORY PROTOCOL – DO’S AND DON’T’S OF MICROBIOLOGY LABORATORY

Written by MicroDok

ATTIRE / LAB COAT / APRON
A lab coat or apron must be worn at all times in the microbiology laboratory. It will protect your clothing from accidental contamination and stains in the lab. When leaving the laboratory, remove the coat or apron. In addition, long hair must be secured in a ponytail to prevent injury from Bunsen burners and contamination of culture material.

HAND WASHING
Before you start working in the lab, wash your hands with a liquid detergent and dry them
with paper toweling. At the end of the period, before leaving the laboratory, wash them again. This practice is good for your personal hygiene and health; and it will also help to contain the spread of germs from your hands.

TABLE TOP DISINFECTION
The first chore of the day when you enter the microbiology laboratory will be to sponge down your desktop or bench top with a disinfectant. This process removes any dust that may be present and minimizes the chances of bacterial contamination of cultures that you are about to handle. Your instructor will indicate where the bottles of disinfectant and sponges are located. At the end of the period before leaving the laboratory, perform the same procedure to protect students that may occupy your desk in the next class.

BUNSEN BURNER USAGE
When using a Bunsen burner to flame loops, needles, and test tubes, follow the procedures outlined in the SOP (standard operating procedure) of your Lab. Inoculating loops and needles should be heated until they are red-hot. Before they are introduced into cultures, they must be allowed to cool down sufficiently to prevent killing organisms that are to be transferred. If your burner has a pilot on it and you plan to use the burner only intermittently, use it. If your burner lacks a pilot, turn off the burner when it is not being used. Excessive unnecessary use of Bunsen burners in a small laboratory can actually raise the temperature of the room. More important is the fact that unattended burner flames are a constant hazard to hair, clothing, and skin. The proper handling of test tubes, while transferring bacteria from one tube to another, requires a certain amount of skill. Test-tube caps must never be placed down on the desktop or bench top while you are making inoculations. Techniques that enable you to make transfers properly must be mastered.

PIPETTING
Transferring solutions or cultures by pipette must always be performed with a mechanical suction device. Under no circumstances is pipetting by mouth allowed in the microbiology laboratory. Disposable hand pipettes and mechanical pipetting by the use of suction tubes should always be used in any pipetting techniques in the microbiology laboratory. This is important because it will help reduce the risk of personal contamination and disease or pathogen spread.

DISPOSAL OF CULTURES AND BROKEN GLASS
The following rules apply to culture and broken glass disposal:
1. Petri dishes must be placed in a plastic bag to be autoclaved.
2. Unneeded test-tube cultures must be placed in a wire basket to be autoclaved.
3. Used pipettes must be placed in a plastic bag for autoclaving.
4. Broken glass should be swept up into a dustpan and placed in a container reserved for broken glass. Don’t try to pick up the glass fragments with your fingers.
5. Contaminated material must never be placed in a wastebasket.

ACCIDENTAL SPILLS
All accidental spills, whether chemical or biological, must be reported immediately to your instructor. Although the majority of microorganisms used in the microbiology laboratory (especially at the undergraduate level) are non-pathogens, some pathogens will be encountered still. It is for this reason that we must treat all accidental biological spills as if pathogens were involved. Chemical spills are just as important to report because some agents used in the microbiology laboratory may be carcinogenic; others are poisonous; and some can cause dermal damage such as blistering and depigmentation.

DECONTAMINATION PROCEDURE
Once your instructor is notified of an accidental spill, the following steps will take place:
1. Any clothing that is contaminated should be placed in an autoclavable plastic bag and autoclaved.
2. Paper towels, soaked in a suitable germicide, such as 5% bleach, are placed over the spill.
3. Additional germicide should be poured around the edges of the spill to prevent further
aerosolization.
4. After approximately 20 minutes, the paper towels should be scraped up off the floor with an autoclavable squeegee into an autoclavable dust pan.
5. The contents of the dust pan are transferred to an autoclavable plastic bag, which may itself be placed in a stainless steel bucket or pan for transport to an autoclave.
6. All materials, including the squeegee and dustpan, are autoclaved.

ADDITIONAL IMPORTANT REGULATIONS
Here are a few additional laboratory rules:
1. Don’t remove cultures, reagents, or other materials from the laboratory unless you have been granted specific permission.
2. Don’t smoke or eat food in the laboratory.
3. Make it a habit to keep your hands away from your mouth at all times. Obviously, labels are never moistened with the tongue; use tap water or self-adhesive labels instead.
4. Always clean up after yourself. Gram-stained slides that have no further use to you should be washed and dried and returned to a slide box. Coverslips should be cleaned, dried, and returned to their casing. This also applies to glass slides. Staining trays should be rinsed out and returned to their storage place.
5. Return all bulk reagent bottles to places of storage.
6. Return inoculating loops and needles to your storage container. Be sure that they are not upside down.
7. If you have borrowed something from someone, return it after use.
8. Do not leave any items on your desk at the end of the period.
9. Do not disturb another class at any time. Wait until the class is dismissed.
10. Treat all instruments, especially microscopes, with extreme care. If you don’t understand how a piece of equipment functions, ask your instructor.
11. Work cooperatively with other students in group assigned experiments, but do your own analyses of experimental results.
12. Do not operate any equipment without prior approval, knowledge and guidance of the Lab instructor.

Source:
Benson: Microbiological Applications Lab Manual. Eight Edition. The McGraw-Hill Companies

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