Appendix L - BIOLOGICAL HAZARDS AND PATHOGENS

Introduction

Much of the microbiological work in university laboratories is not concerned with known pathogenic (i.e. disease causing) organisms. However, certain safety measures are essential because (1) the dividing line between pathogens and non-pathogens is not clear cut; (2) the possibility of adventitious growth of pathogens exists and cross-contamination of cultures may occur. The teaching of the principles and techniques of handling microorganisms safely is an essential component of training workers using microbiological techniques. Before beginning work involving pathogens, contact the Chemical Hygiene Officer for additional information.

General Precautions
Before beginning work, ensure that supplies of an appropriate disinfectant are available. Carry out all work in a thoughtful and meticulous way. Treat all microorganisms as if they are pathogenic. Swab benches and worktops with a suitable disinfectant before leaving the laboratory.
Do not leave contaminated material unattended unless it is sealed and clearly labeled. If contaminated material is stored in a refrigerator or cold room, stand it in a tray large enough to contain the contents should the container break. Seal contaminated materials in a suitable container before sterilization or incineration.
To minimize contamination through spillage, stand vessels containing infectious materials in trays and cover the working surface with disposable absorbent material, which can be incinerated after use.
Keep work spaces as uncluttered as possible.
Do not eat, drink, apply cosmetics or smoke in laboratories, animal houses or any other potentially contaminated place, and avoid putting any objects, such as a pen or pencil, into your mouth. Gummed labels must not be licked: use self-adhesive labels or a glass pencil. Never pipette anything by mouth, but use an automatic pipette. If pipettes are to be used with infected material, plug them with non-absorbent cotton wool. If only small drops of culture are required, use a plugged Pasteur pipette with a rubber teat.
Wear appropriate protective clothing and use automatic devices when appropriate. Seal any abrasions of the skin before entering a contaminated zone to prevent contamination by infectious materials. Remove contaminated clothing before leaving the contaminated area and then wash your hands. Arrangements should be made for the sterilization of contaminated clothing. Do not wear contaminated clothing in any place where eating, drinking or smoking is permitted. Wash protective gloves after handling contaminated apparatus or materials, and also before removing them, and then wash hands thoroughly. Clean under and around fingernails but do not abrade the skin by vigorous scrubbing. All accidents and spillages must be recorded even when no personal injury was involved.
Before use, flame the apparatus or glassware after removing the plug or cap. A hooded Bunsen burner or a micro-incinerator is the safest way of sterilizing wire loops. Materials which spit from overcharged loops may not have been sterilized. Always support culture tubes, bottles, etc. in a rack, preferably one made from plastic coated wire: do no prop them up or lay them down on the bench.
Sterilize all contaminated apparatus and glassware before sending it for cleaning. Immerse used pipettes, tips downwards, in a jar of suitable disinfectant and compatible detergent. Leave the pipettes long enough to be sterilized. Replenish the disinfectant regularly.
Smears of infected material and cultures on microscope slides may still be infectious after fixation and staining so they should be sterilized in a similar manner.

Aerosols

Aerosols are a major route of infection. Finely disseminated droplets are formed whenever the surface film of a liquid is broken, or when dry material is being crushed, ground, shaken or vibrated.
Once disseminated they cause extensive contamination of the laboratory and may persist in the air for some time.
Accidental breakage of containers is an obvious source of aerosol formation. The precautions necessary to minimize aerosols formation include the following:

Handle reagents gently at all times.
Never blow material from pipettes.
Never bubble gases through liquid cultures, unless there is a filtered exhaust for the exit gas.
Do no wet the cap or plug when shaking vessels containing liquid cultures.
Use disposable loop or loops made of platinum or soft wire.
When transferring infectious material into or out of a rubber stoppered bottle, wrap a disinfectant soaked swab around the hypodermic needle and the stopper of a bottle before removing the needle. Do this with negative pressure in the bottle. Do not leave the bottle under greater than atmospheric pressure after transferring fluid through the stopper.
Expel any air and excess fluid from a syringe into a disinfectant soaked swab.
Remove the needle from a syringe after finishing and place in "sharp" disposal box; also scalpel blades.
Place all instruments in trays of disinfectant after use.
Carry out all procedures likely to cause aerosols such as agitating, blending, grinding, post-mortem dissection and the inoculation of animals in a microbiological safety cabinet.

D. Accidents
When culture vessels are broken, the debris should be immediately covered with a disposable towel or cloth soaked in a suitable disinfectant (5% solution sodium hypocholorite – bleach). It should be possible to do this without the operator bringing his or her face into the aerosol cloud. This should then be left for at least 10 minutes to allow the disinfectant to work.
The debris end of the cloth should then be put into a discard bin or an autoclavable dustpan and hand brush.
Sweeping brooms should not be used. The area can then be swabbed with disinfectant using disposable cloths or paper towels. Heavy duty gloves should be worn and care taken to avoid wounding from glass splinters.
Immunization and regular booster injections should be offered to professors and laboratory technicians as a supplemental safety precaution. As a minimum, those who conduct experiments involving potential contact with blood or body fluid should have a current hepatitis immunization, and those who have frequent contact with soil should have a current tetanus immunization.