Section 1 Biological Safety Chapter 3 Standard Laboratory Practice and Technique

STANDARD LABORATORY PRACTICE AND TECHNIQUE
Biohazard Warning Signage A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. Biosafety Level 1: The sign may include the name of the agent (s) in use, and the name and phone number of the laboratory supervisor or other responsible personnel. Biosafety Level 2: Posted information must include the name of the agent (s), laboratory’s biosafety level, supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. Biosafety Level 3: Posted information must include the name of the agent (s), laboratory’s biosafety level, supervisor’s name (or other responsible personnel), telephone number(s), and required procedures for entering and exiting the laboratory. Personal Protective Equipment Once a biological hazard has been identified, the supervisor and employee must agree on the appropriate personal protective equipment (PPE) to be worn as the primary barrier of protection. PPE may include, but is not limited to face protection, lab coats and gowns, respirators, and shoe-covers/booties. Supervisory personnel are responsible for the initial demonstration and periodic follow-up of proper use. Appropriate PPE should be donned before handling potentially hazardous biological materials and removed immediately and replaced if gross contamination of the equipment occurs. PPE should be removed before exiting the laboratory. Face Protection: When splash or splatter of infectious substances or other biological materials is anticipated, appropriate face protection should be worn if work is performed outside a biological safety cabinet. Such equipment would include but is not limited to goggles, side-shielded safety glasses and chin length face shields. Lab Coats and Gowns: Long sleeved lab coats or gowns should be worn to protect skin and street clothes from contamination. In circumstances when splash or splatter is anticipated, the garment must be resistant to liquid penetration. A cuffed lab coat or gown should be worn when working with potentially infectious materials, and MUST be worn when working with agents requiring Biosafety Level-3 containment. Reusable clothing should be laundered on-site or by a laundering service. Personnel should not launder laboratory clothing at home.

Section 1 Biological Safety Chapter 3 Standard Laboratory Practice and Technique

Gloves: Gloves should always be worn when handling biohazardous materials. Disposable gloves can provide an adequate barrier between the lab employee and most biohazardous materials. Double gloves and/or cut-resistant gloves should be considered when handling sharp items and biohazardous materials. Respirators: When engineering controls (i.e. BSCs) are not available to provide adequate protection against aerosolized agents or when mandated by federal regulations, respirators shall be worn. Duke’s Respiratory Protection Program requires that employees be medically cleared, fit-tested, and trained on proper usage and care before allowed to wear a respirator. Details of the Program can be viewed here. Disposable Shoe-covers/Booties: When significant splash and splatter are anticipated, shoe-covers/booties should be considered. Prior to exiting the laboratory, these must be removed and disposed of properly. Handwashing Hands should be washed as soon as possible when they come in contact with potentially infectious materials. A vigorous handwashing with a mild soap for 20 full seconds is appropriate. Hands should also be washed as soon as feasible after gloves are removed, and before exiting the laboratory. Eating, Drinking, Smoking, Applying Cosmetics and Handling Contact Lenses Eating, drinking, smoking, applying cosmetics and handling contact lenses is prohibited in work areas in which potentially infectious materials are being manipulated. Food and drink must not be stored in refrigerators in which laboratory materials are kept. Housekeeping Good housekeeping in laboratories can reduce the risk of accidents occurring. Work benches should be kept as clutter-free as feasible, and aisles should always be free of trip hazards. Benches should be wiped down with an approved disinfectant at least once a day and immediately after a spill of potentially infectious materials. Pipetting Pipetting infectious agents can lead to personnel exposures by inhalation, contact, or ingestion if not performed properly. The following are a few safety precautions to be followed when pipetting in the laboratory: 1) Never mouth pipette; pipetting aids should always be used, 2) Pipette contents should be allowed to run down the wall of the container, making sure not to release the contents from a height, 3) Place absorbent paper on benchtops to reduce the risk of aerosols being generated by accidental dripping of infectious materials from pipette tips, and 4) Place disposable pipettes into pipette disposal boxes which have been lined with an autoclave bag, and then steam sterilize (autoclave) for 90 minutes at 121 degrees Celsius (see Waste Management Section).

Section 1 Biological Safety Chapter 3 Standard Laboratory Practice and Technique

Sharps The use of needles, glass pipettes, glass slides and cover slips, scalpels and lancets should be eliminated, when possible. Appropriate precautions should be taken to avoid percutaneous injuries. These items should be disposed of immediately after use by placing them in an appropriate puncture-resistant container. Bending, recapping or clipping of needles is prohibited. If recapping is absolutely necessary, a mechanical device or the one handed scoop method must be used. Plasticware should be used whenever possible, such as plastic graduated cylinders, funnels, aspirators, etc. Safety devices (i.e. mylar-coated capillary tubes, Eclipse safety needles) should be used when available. Decontamination The purpose of decontamination is to make a hazardous material safe for further handling. A decontamination procedure can range from sterilization to simple cleaning with soap and water. The following includes a description of the four main categories of physical and chemical means of decontamination. Heat: Wet heat is the most dependable method of sterilization. Steam autoclaving is the most convenient method available to the Duke laboratories for decontaminating biological waste and sterilizing glassware and media. Note: Autoclaves that are used for decontamination of biohazardous wastes should be monitored for the efficacy of treatment. This is accomplished by the use of biological indicators (i.e. spore strips). The generator of the waste (the lab) is responsible for performing and documenting this testing. Liquid Disinfection: Many types of liquid disinfectants are available under a variety of trade names. The most practical use of liquid disinfectants is for surface decontamination. Agents included in the category include, but are not limited to, quaternary ammonium compounds, phenolic compounds, halogens, aldehydes, alcohols and amines. A tuberculocidal disinfectant or diluted household bleach should always be used for decontamination when human materials are handled. NOTE: When household bleach is used for the decontamination of spills, a fresh solution (at least 10% household bleach) must be prepared. Bleach solutions used for routine surface decontamination must be made up at least weekly. Each solution container must be labeled with either a made-on or an expiration date. Vapors and Gases: The use of vapors and gases as decontamination methods usually involve the decontamination of biological safety cabinets, but can also be used for whole building or room decontaminations. Agents used in this category include ethylene oxide, formaldehyde, gas, hydrogen peroxide and peracetic acid. Radiation: Ultraviolet radiation (UV) is sometimes used in biological safety cabinets for inactivating contaminants, but because of the low penetrating power of UV, dusty or soiled areas may limit its usefulness in the laboratory. Because UV can cause serious

burns to eyes and skin, it must not be used when work areas are occupied. Whole room UV is not recommended. Do not rely on just radiation for your disinfection process. Decontaminants and Their Use in Laboratories
Decontaminant Active Temp Contact Vegetative Lipo Tubercle Hydrophilic Bacterial Ingredient/ (°C) time bacteria viruses bacilli viruses spores Concentration (min.) Steam Heat 0.2-3% 0.01-5% 70-85% 4-8% 121 50–90 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + _ + + + + + 6491-60 929 10-30 10-30 10-30 10-30

Section 1 Biological Safety Chapter 3 Standard Laboratory Practice and Technique

Autoclave Incinerator Phenolic compounds Chlorine compounds Alcohol (ethyl or isopropyl) *Formaldehyde Hydrogen peroxide

*Gluteraldeyhyde 2% 6%

10-600 + 10-600 +

+ very positive response + less positive response — negative response *irritating characteristics of agent precludes use for routine spill cleanup