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A problem that may occur in the laboratory is an overt biological spill. A spill that takes place in the open laboratory may create a serious problem. Every effort should be taken to avoid such occurrences. A spill poses less of a problem if it happens in a biological safety cabinet, provided splattering to the outside of the cabinet does not occur. Direct application of concentrated liquid disinfectant and a thorough wipe down of the internal surfaces of such cabinetry will usually be effective for decontaminating the work zone, but gaseous sterilants will be required to disinfect the interior sections of the cabinet. Each researcher must realize that in the event of an overt accident, research materials such as tissue cultures, media, and animals within such cabinets may well be lost to the experiment. 

A. Spill in the Open Laboratory 

Advance preparation for spill management is essential. A "spill kit" including leak proof containers, forceps, paper towels, sponges, disinfectant, respirators, and rubber gloves should be readily available. If potentially hazardous biological material is spilled in the laboratory, the first step is to avoid inhaling any airborne material by holding the breath and leaving the laboratory. Warn others in the area and go directly to the wash or change room area. If clothing is known or suspected to be contaminated, remove the clothing with care, folding the contaminated area inward. Discard the clothing into a bag or place the clothing directly in an autoclave. Wash all potentially contaminated body areas as well as the arms, face, and hands. Shower if facilities are available. Reentry into the laboratory should be delayed for a period of thirty minutes to allow the dissipation of the aerosol generated by the spill. Protective clothing should be worn when entering the laboratory to clean the spill area. Rubber gloves, autoclavable footwear, an outer garment, and a respirator should be worn. If the spill was on the floor, do not use a surgical gown that may trail on the floor when bending down. Take the " spill kit" into the laboratory room, place a discard container near the spill, transfer large fragments of material into it and replace the cover. Using a hypochlorite containing solution (1000 ppm available chlorine), iodopher solution containing 1600 ppm iodine or other appropriate disinfectant, carefully pour the disinfectant around and into the visible spill. Avoid splashing. Allow 15 minutes contact time. Use paper or cloth towels to wipe up the disinfectant and spill, working toward the center of the spill. Discard towels into a discard container as they are used. Wipe the outside of the discard containers, especially the bottom, with a towel soaked in a disinfectant. Place the discard container and other materials in an autoclave and sterilize.

Remove shoes, outer clothing, respirator and gloves and sterilize by autoclaving or exposure to ethylene oxide. Wash hands, arms and face, or if possible, shower.
 

B. Spill in a Biological Safety Cabinet

A spill that is confined to the interior of the biological safety cabinet should present little or no hazard to personnel in the area. However, chemical disinfection procedures should be initiated at once while the cabinet ventilation system continues to operate to prevent escape of contaminants from the cabinet. Spray or wipe walls, work surfaces and equipment with a disinfectant. A disinfectant with a detergent has the advantage of detergent activity which will help clean the surfaces by removing both dirt and microorganisms. A suitable disinfectant is a 3% solution of an iodopher such as Wescondyne or a 1:100 dilution of house-hold bleach (e.g. Chlorox) with 0.7% nonionic detergent. The operator should wear gloves during this procedure. Use sufficient disinfectant solution to ensure that the drain pans and catch basins below the work surface contain the disinfectant. Lift the front exhaust grill and tray and wipe all surfaces. Wipe the catch basin and drain the disinfectant into a container. The disinfectant, gloves, wiping cloth and sponges should be discarded into an autoclave pan and autoclaved. The above procedure will not disinfect the filters, blower, air ducts or other interior parts of the cabinet. If the entire interior of the cabinet needs to be sterilized, contact EH&S.
 

C. Biological Spill Response Guidelines

These guidelines are intended to assist the principal investigator, laboratory supervisor, and other responsible individuals who may be involved in the cleanup of biological spills. This guide outlines the basic procedures for dealing with some of the biological spills that may be encountered in a research laboratory. All lab personnel should refer to the specific spill response procedures before initiating their experiments.
 

Biosafety Level 1 (BL1) Spill

• Notify others in the area, to prevent contamination of additional personnel and environment. 
• When BL1 spills occur outside the lab (e.g. hallways, common rooms & corridors) report these BL-1 spills to: (1) Lab Director (2) Biosafety Officer (620-2019) 
• Remove any contaminated clothing and wash exposed skin with soap and water.

Clean-up of BL1 Spill

• Wearing gloves and lab coat, cover spill with paper towels, pour disinfectant around the spill allowing it to mix with spilled material. Allow suitable contact time, at least 15 min.
• Pick up any pieces of broken glass with forceps and place in a sharps container. 
• Discard all disposable materials used to clean up the spill into a biohazard bag. 
• Wash hands with soap and water

Biosafety Level 2 (BL2) Spill

• Notify others in the laboratory regarding the spill 
• Close door, and post with a warning sign. 
• Remove contaminated clothing, turning exposed areas inward, and place in a biohazard bag. 
• Wash all exposed skin with soap and water. 
• Inform Lab director, University Police Department (911), and Biosafety Officer (620-2019)

Clean-up of BL2 Spill

• Allow aerosols to disperse and or settle for at least 30 minutes before reentering the laboratory (if spill outside cabinet). Assemble clean-up materials (disinfectant, paper towels, biohazard bags, and forceps). 
• Put on protective clothing (lab coat, facemasks/face protection, utility gloves, and booties if necessary). 
• Cover the area with disinfectant-soaked towels, and then carefully pour disinfectant around the spill. Avoid enlarging the contaminated area. Use more concentrated disinfectant as it is diluted by the spill. Allow at least a 20 minute contact time.
• Pick up any sharp objects with forceps and discard in a sharps container. 
• Soak up the disinfectant and spill using mechanical means, such as an autoclavable broom and dustpan, since there may be sharps under the paper towels, and place the materials into a sharps container.
• Smaller pieces of glass may be collected with cotton or paper towels held with forceps. If no sharps were involved in the spill discard the materials into an autoclave bag.
• Wipe surrounding areas (where the spill may have splashed) with disinfectant.
• Spray the area with 10% household bleach solution and allow to air-dry (or wipe down with disinfectant-soaked towels after a 20-minute contact time).
• Place all contaminated paper towels and any contaminated protective clothing into a biohazard bag and autoclave.
• Wash hands and exposed skin areas with soap and water. 

D. Engineering Controls

 Engineering controls are tools or equipment that, when used properly, provide significant protection to the operator as well as other laboratory occupants. Examples include biological safety cabinets, autoclaves, and sharps containers. As is true of most tools, there are correct (proper) and improper ways to use engineering controls. Given that there are considerable adverse consequences if used improperly, correct usage of engineering controls is critical. When used properly, they have proven to be, in most circumstances, the most effective and practical way to achieve a safety goal. 

E. Biological Safety Cabinets (BSCs)

BSCs are designed to provide personnel, environmental, and product protection when appropriate use/procedures are followed. They are among the most effective and commonly used primary containment devices in laboratories working with infectious agents. Three types of BSCs (Class I, II, and III) have been developed to meet various research and clinical needs. BSCs use HEPA (high efficiency particulate air) filters in their exhaust and/or supply systems. Biological safety cabinets must not be confused with other laminar flow devices or “clean benches.” Horizontal flow cabinets that direct air toward the operator should never be used for handling infectious, toxic or sensitizing materials. Class I and II biosafety cabinets, when used in conjunction with good microbiological techniques, provide an effective partial containment system for safe manipulation of moderate and high-risk microorganisms, (i.e. BL 2 and 3 agents).

Class I Biological Safety Cabinet

is a ventilated cabinet for personnel protection with an unrecirculated inward airflow away from the operator. This unit is fitted with a HEPA filter to protect the environment from discharged agents. A Class I BSC is suitable for work involving low to moderate risk agents, where there is a need for containment, but not for product protection. 

Class II Biological Safety Cabinet

is a ventilated cabinet for personnel, product and environmental protection, which provides inward airflow and HEPA-filtered supply and exhaust air. There are three basic types of Class II BSCs: Type A, Type B, and 100% exhaust. Type B cabinets are further sub-typed into types B1, B2, and B3. The major differences between the three types is the percent of air that is exhausted or recirculated, and the manner in which exhaust air is removed from the work area.

Class III Biological Safety Cabinet or glove box

is a totally enclosed ventilated cabinet, which provides the highest attainable level of protection to personnel, environment and product. The supply air is HEPA-filtered and exhaust air has two HEPA-filters in series. Work is performed in the cabinet through glove ports with Oring for attaching arm-length gloves to cabinet.

It is important to note that laminar flow clean benches must not be utilized for work with biohazardous or chemically hazardous agents. 

Table 1 – Selection of a Biological Safety Cabinet through Risk Assessment. 

Clean benches provide product protection by ensuring that the product is exposed only to HEPA-filtered air. They do not provide protection to personnel or the ambient environment. The correct location, installation, and certification of a biological safety cabinet are critical to its performance in containing infectious aerosols. All BSCs used for must be inspected and certified annually. Inspection and re-certification is mandatory if the cabinet is relocated, experiences major repairs or after a filter change etc. 

 F. Safe and Effective Use of Biosafety Cabinets 

• All personnel must b trained prior to using the BSC.
• Equipment and/or supplies should not be stored inside the cabinet; unnecessary objects can disrupt airflow in the cabinet.
• Do not use the top of the cabinet for storage. The HEPA filter can be damaged, disrupting the balance of airflow.
• Do not place any objects that will block either the front air intake grille, or block the rear exhaust grille.
• Close the sash when the cabinet is not in use. Keeping the sash closed between uses will assist in minimizing cabinet contamination. Always leave the BSC running.
• Do not eat, drink, chew gum, or store food near the cabinet.
• Avoid sudden movement in or out of the cabinet, as well as areas directly adjacent to the cabinet. Sudden movements can disrupt the airflow and compromise safety. Move arms slowly when removing or introducing new items into the BSC.
• Segregate contaminated and clean items. Work from “clean to dirty” • Protect the building vacuum system from biohazards by placing a cartridge filter between the vacuum trap and the source valve in the cabinet.
• Always decontaminate the interior surfaces prior to working in the cabinet. Clean up spills in the cabinet immediately.
• When work is finished, remove all materials and wipe all interior surfaces with 70% alcohol or any other disinfectant suitable for the agent(s) in use.
• Always extinguish and remove the Bunsen burner from the hood prior to decontamination with alcohol.
• Remove lab coat, gloves and other personal protective equipment (PPE) and wash hands thoroughly before leaving the laboratory.

Table 2 – Summary of Recommended Biosafety Levels for Infectious Agents