The following recommended practices and procedures for working safely on microbiology projects in a teaching laboratory environment are based on “Guidelines for Biosafety in Teaching Laboratories,” from the American Society for Microbiology (ASM). The full documents may be viewed at:

http://www.asm.org/images/asm_biosafety_guidelines–FINAL.pdf

http://www.asm.org/images/Education/FINAL_Biosafety_Guidelines_Appendix_Only.pdf

What is Biosafety?

Microbiology is a science that investigates the biology of microscopic organisms. Although individual cells of these organisms may be directly observed with a microscope, and their shapes and activities observed, to investigate other characteristics such as metabolism or genetics, growing cells in populations (called cultures) is the preferred approach. For many types of experiments, all of the cells in the population must be essentially the same; such populations are called pure cultures. A set of techniques, mostly developed in the late 19th century by Robert Koch, Louis Pasteur, and their collaborators, permitted the isolation of bacteria from their natural environments and separation into pure cultures for further study. Probably more than anything else, these are the techniques that define microbiology as a scientific field of study.

It has been estimated that less than 1% of bacteria can be grown in culture in a laboratory. However, many types can be, and those are the ones on which we will focus. Microbiologists culture bacteria by providing them with food, water, and other growth requirements in an environment with a constant and comfortable growth temperature. These requirements vary, depending on the natural growth conditions for the microbial populations under study.

Food is provided in the culture media we use. Media may be in the form of a liquid (called a “broth”) or solid or semi-solid forms, either in tubes or in culture dishes (Petri plates). The choice of media depends on what you want to do or need to know about the bacteria in your cultures.

To ensure that we culture only the specific bacteria we want, and nothing else from the environment, microbiologists use a set of strict aseptic techniques, which protects us from the bacteria in the cultures, and also protects our cultures from contaminants in the environment. These methods will be demonstrated in class. In addition, specific laboratory rules must be followed for containment of microbial cultures in the laboratory, for the safety of all. For this laboratory, these practices are listed below.

Laboratory Safety Practices and Procedures

1. Remember that all bacteria are potential pathogens that may cause harm under unexpected or unusual circumstances. If you as a student have a compromised immune system or a recent extended illness, you should share those personal circumstances with your lab instructor.
2. Know where specific safety equipment is located in the laboratory, such as the fire extinguisher and the eyewash station.
3. Recognize the international symbol for biohazards, and know where and how to dispose of all waste materials, particularly biohazard waste. Note that all biohazard waste must be sterilized by autoclave beforeit can be included in the waste stream.
4. Keep everything other than the cultures and tools you need OFF the lab bench.
5. All of the equipment and supplies used in experiments involving bacterial cultures should be sterilized. This includes the media you use and also the tools used for transferring media or bacteria, such as the inoculating instruments (loops and needles) and pipettes for liquid transfer.
6. Transfer of liquid cultures by pipette should NEVER involve suction provided by your mouth.
7. Disinfect your work area both BEFORE and AFTER working with bacterial cultures.
8. In the event of an accidental spill involving a bacterial culture, completely saturate the spill area with disinfectant, then cover with paper towels and allow the spill to sit for 10 minutes. Then carefully remove the saturated paper towels, dispose of them in the biohazard waste, and clean the area again with disinfectant.
9. Wear gloves when working with cultures, and when your work is completed, dispose of the gloves in the biohazard garbage. Safety glasses or goggles are also recommended.
10. Long hair should be pulled back to keep it away from bacterial cultures and open flame.
11. Make sure that lab benches are completely cleared (everything either thrown away or returned to storage area) before you leave the lab.

Bacteria pose varying degrees of risk both in a controlled laboratory environment and in their natural settings. Therefore, the level of containment necessary for working safely with bacterial cultures also varies according to a system that classifies microbes into one of four biosafety levels (BSL), which provides minimum standards for safe handling of microbes at each level. BSLs are defined and containment practices are detailed by the Centers for Disease Control and Prevention (CDC) for laboratories in the United States. The full document, “Biosafety in Microbiological and Biomedical Laboratories,” can be viewed in its entirety at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.

Prior to the first meeting of the laboratory, navigate to the following web page to review the BSL guidelines as outlined by the CDC, which includes an interactive quiz:

http://www.cdc.gov/training/Quicklearns/biosafety/

Most of the bacterial cultures we will be working with are classified as BSL-1. Below, list three practices that should be used while you are working with BSL-1 bacteria:

1. __________________________________________________________________________

2. __________________________________________________________________________

3. __________________________________________________________________________

In a few of the labs, we will be working with bacteria that are classified as BSL-2. What additional safety practices should you employ when you work with these bacteria?

1. __________________________________________________________________________

2. __________________________________________________________________________

3. __________________________________________________________________________

After your instructor has discussed any additional safety procedures for your lab, complete the biosafety concept assessment on the following page (or one provided by your instructor) and sign the affirmation. Return the completed document to your instructor.

Microbiology Laboratory Biosafety Guidelines

Complete the following questions by writing the letter of the correct answer on the line.

___ 1. Biological agents are assigned to Biosafety Levels (BSLs) based on

a. whether they are bacteria, viruses, or other microorganisms.

b. their susceptibility to laboratory disinfectants.

c. the risk they pose to human health and the environment.

d. the amount of the agent that will be used in the lab.

___ 2. BSL 1 practices, equipment, and facilities are used when working with which type of microorganism?

a. Bacteria that are well characterized and not known to consistently cause disease in healthy adult humans.

b. Bacteria that are considered moderate-risk agents present in the community and associated with human diseases.

c. Indigenous or exotic bacteria with potential for aerosol transmission and associated with human disease.

d. Dangerous/exotic agents with a high risk of life-threatening disease.

___ 3. The primary hazards to people working in a BSL 1 or 2 laboratory relate to

a. accidental skin punctures caused by mishandling of sharp lab tools.

b. mucous membrane exposure due to accidental splashing of cultures.

c. accidental ingestion of infectious material.

d. All of the above are primary hazards in a BSL 1 or 2 lab.

___ 4. Which of the following is required when working with BSL 2 agents?

a. Biohazard warning signage on doors and lab areas where agents are kept or used.

b. Self-closing, double door access to the lab facility.

c. Clothing change when entering or leaving the lab.

d. There are no special precautions needed for a BSL 2 lab.

___ 5. What is the appropriate disposal method for a bacterial culture grown on an agar plate?

a. Throw the culture away in any one of the several garbage containers in the room.

b. Throw the culture away in the garbage container marked with a biohazard sign.

c. Allow the agar to dehydrate in the incubator and then discard in any garbage.

d. Bacteria grown on an agar plate medium should never be disposed of.

Microbiology Laboratory Biosafety Affirmation

By signing your name below, you affirm that:

• Potential hazards in the microbiology laboratory have been identified and explained.

• You understand that the bacterial cultures used in lab require either BSL 1 or BSL 2 containment procedures.

• Safety practices for BSL 1 and 2 have been explained and/or demonstrated to you.

• You fully understand the hazards associated with BSL 1 and 2 bacterial cultures and are willing to assume the risk.

• You are responsible for your own actions while working in a laboratory with potentially hazardous materials.

PRINT your FULL NAME __________________________________________________________

SIGN your name __________________________________________ Date _________________