Biosafety Level 1 (BSL-1)

BSL-1 applies to working with microorganisms that are generally not disease causing in healthy humans and therefore are of minimal potential hazard to laboratory personnel and the environment (Box 5.2). This safety level is used in municipal water-testing laboratories, high school laboratories, and in some community colleges teaching introductory microbiology classes with organisms that are not considered to be pathogenic and/or hazardous. These laboratories typically include a sink for hand washing, benchtops, sturdy furniture, windows with fly screens if they can be opened, and readily available disinfectants and antiseptics. The laboratory should be easily cleaned, decontaminated, and have procedures posted for the safe disposal of materials being used.

The laboratory at this safety level does not have to be isolated from other parts of the building; however, a door that can be closed while work with agents is in progress is highly desirable. Hazard warning signs (Figure 5.1) should be posted on doors, indicating any hazards that may be present. A sink for hand washing needs to be available because hand washing is one of the simplest yet most important procedures used by laboratory personnel to remove unwanted microbial agents or chemicals used in the laboratory.

Standard microbiological practices at BSL-1 include the use of mechanical pipetting devices and prohibition of eating, drinking, and smoking. People working in the laboratory should be wearing laboratory coats and gloves when working with biological agents. Adequate, efficient methods and procedures for disposal of materials used are also established.

The laboratory supervisor of a BSL-1 laboratory needs to have general training in microbiology or a related science and is responsible for establishing the general laboratory safety procedures, for training laboratory personnel, and for the updating of these procedures.

Biosafety Level 2 (BSL-2)

BSL-2 is similar to BSL-1 regarding the agents that are being handled. However, for BSL-2 work, the facility, containment devices, administrative controls, practices and procedural standards, and guidelines are designed to maximize safe working conditions for laboratories working with agents of moderate risk to personnel and the environment. Agents manipulated at BSL-2 are considered a moderate risk. They often include pathogens to which personnel have previously been exposed and to which they have had an immune response (e.g., childhood diseases) or against which they have received immunization (Box 5.3). Immunization is recommended before working with certain agents, for example, immunization against the hepatitis B virus which is recommended by OSHA for people at high risk of exposure to blood and blood products. In addition to procedures established in the BSL-1 level laboratory, the BSL-2 laboratory requires that:

Biosafety Level 3 (BSL-3)

BSL-3 applies to clinical, diagnostic, teaching, research, or production laboratories using original or exotic agents (Box 5.4). Such agents can potentially cause serious disease or even lethality if exposure occurs. In addition to the laboratory procedures described in BSL-1 and BSL-2, laboratory personnel require specific training in handling pathogenic and potentially lethal agents with on-site supervision by scientists experienced and qualified in working with such agents. Many additional standards are necessary to qualify as a BSL-3 laboratory. Some of the additional procedures include but are not limited to:

The National Institute of Allergy and Infectious Diseases (NIAID) in partnership with the American Society of Microbiology (ASM) conducted a brief survey of academic, biotechnology, and pharmaceutical facilities in the United States to provide the NIAID with information about the location, capacity, and status of existing and operating facilities capable of BSL-3 containment. This information can be viewed at http://www.asm.org/Policy/index.asp?bid=37789.

Biosafety Level 4 (BSL-4)

BSL-4 is required for working with dangerous and exotic agents that present a high risk of aerosol-transmitted laboratory infections and life-threatening disease (Box 5.5). All laboratory staff is required to have specific training in handling extremely hazardous infectious agents. The facility is located either in a separate building or in a controlled area within a building that is completely isolated from all other areas of that building. In addition to all other laboratory procedures described previously, additional protocols and procedures are necessary for BSL-4. Some of these include but are not limited to the following:

It is important to note that the description of the different biosafety levels (BSL-1 to BSL-4) in this chapter is an introduction only and does not reflect complete, more detailed, and specific governmental suggestions, standards, and regulations. Information about BSL-4 laboratories in the United States is summarized in Table 5.2.

Fire Extinguishers

In addition to fire alarms, fire extinguishers are required in all laboratories. They are classified and assigned a letter or symbol for that classification to a particular fire type they are designed to extinguish (Figure 5.3). However, multipurpose extinguishers are often recommended because they are effective against different types of fires. Extinguishers are located at specified places in a laboratory. They should be inspected at least every 12 months and records kept readily visible and easily accessible by appropriately trained persons in a given institution, usually safety officers. Fire extinguishers are classified as:

All laboratory workers should be appropriately trained by safety personnel in the proper use of fire extinguishers. These training procedures should be published, records kept, and the current status of testing of laboratory personnel maintained under the direction of the laboratory supervisor. Some laboratories will also have sand or absorbent material available to extinguish small fires. These materials are generally stored in highly visible dispensers labeled for use according to the type of fire.

Fume Hoods

Fume hoods are the primary control method for exposure to noxious or poisonous vapors in the laboratory environment. They act by drawing ambient air from the laboratory, past the laboratory operator (who is located in front of the hood), and into the hood. The ability of a hood to provide adequate protection depends on a variety of factors including, but not limited to, the control of the velocity of the ambient air at the hood face, air movement and flow patterns in the room, and turbulence within the hood. All laboratory workers with access to a fume hood should be familiar with its particular use, capabilities, and limitations. The various hoods found in different laboratories include the following:

Fume hoods should always be in good condition and capable for routine use, which requires regular inspections and current records. An emergency plan should be posted on or near the hood in case of ventilation malfunction.

Autoclave

An autoclave (see Figure 4.3 in Chapter 4, Microbiological Laboratory Techniques) is a common, routinely used piece of equipment in biological laboratories. It is a device that uses superheated steam under pressure to sterilize equipment and other objects. Sterilization is the elimination of all transmissible agents (see Chapter 4, Microbiological Laboratory Techniques) and autoclaves are widely used for this purpose. However, as an exception, it has been shown that certain archaea (strain 121) and prions have the capacity to survive autoclaving.

Autoclaves, if not operated properly, pose many hazards including physical (heat, steam, and pressure) and biological hazards. Each autoclave has its unique characteristics and therefore operators need to be trained appropriately and regularly performed maintenance recorded to ensure proper functioning. Before using any autoclave, it is necessary to review and understand the manual on instructions for use in order to prevent possible injuries. Autoclave maintenance is an important aspect of proper function and safety and should be performed periodically in accordance with the manufacturer’s recommendations.

Eyewashes and Safety Showers

Eyewashes and safety showers are emergency units present in both public and private industry to protect employees from injury in case of contact with hazardous materials. Employees must be instructed and trained in the proper use of eyewashes and safety showers regardless of their previous experience background. The first seconds after exposure to hazardous materials are critical and decontamination equipment must be in reach for the exposed individual within 10 or fewer seconds after exposure. Therefore, eyewashes and safety showers should be in a clearly marked, accessible location within a laboratory. They provide effective protective treatment in the event of contact from a chemical or biological spill onto the skin or clothing. Many OSHA standards address problems that potentially can occur in the laboratory environment and protocols have been developed for the emergency eyewash and shower equipment (ANSI [American National Standards Institute] Z358.1-2004). The types of equipment addressed by the ANSI standard are listed below and in Figure 5.4.

Refrigerators/Freezers

Laboratory refrigerators, freezers, and ultralow freezers (down to −85° C) need to be carefully selected for specific chemical or biological needs and records of routine, periodical inspections as well as removal of contents noted. The laboratory refrigerator, freezer, cooling unit, or ultralow freezer should be labeled as such with appropriate hazard signs posted on them (Figure 5.5). Containers placed in a given unit should be completely sealed or capped, securely placed, and have readily visible permanent labels.

Disposal of Hazardous Waste

Materials used in any laboratory environment must be disposed of in designated containers. Broken glass is placed in cartons labeled for broken glass and plastics in plasticware containers (Figure 5.6). Biohazardous wastes are disposed of in autoclave bags (Figure 5.7) and used sharps are placed in biohazard sharps containers (Figure 5.8).

Gloves

Hand protection is a necessity in many laboratory settings and the selection of appropriate protection depends on the type of materials to be handled. In the microbiological and healthcare environment, gloves are used to ensure protection from exposure to potentially harmful infectious agents. Traditionally, medical and laboratory gloves are made of latex and powdered with cornstarch; however, because of an increase in occurrence of latex allergies in the general population and among health professionals, nonlatex materials such as vinyl or nitrile rubber are becoming useful alternatives. In addition, powder-free gloves are also available for individuals allergic to powder or in environments where powder is not desirable (e.g., medical clean rooms or electronic clean rooms). Special needs, such as for heavy insulated gloves for protection against burns from ultralow temperatures encountered during transfer or removal of materials from ultralow freezers or while removing hot items from an autoclave, should be identified and personnel trained in their use and limitations.

Respiratory Protection

Various types of respiratory protection are used in laboratory:

Clothing

Laboratory coats should be worn at all times in laboratory areas, but not outside the laboratory because of possible absorption and accumulation of contaminants on the material of the coat. In the presence of infectious material laboratory coats must be worn closed. Shoes need to be worn at all times in the laboratory but should not be sandals or any type of open-toed shoes to avoid exposure to potential spills. In addition, Kevlar-reinforced toe shoes may be required and their use enforced. The selection of the type of protective clothing is the responsibility of the laboratory director in accordance with the procedures manual. For some laboratory procedures additional aprons, either rubber or plastic, might be required. All clothing worn in laboratories handling infectious substances must be disposed of in a safe manner.

Eye Protection

Eye protection is mandatory in many laboratories where a potential for injury exists. The type of eye protection required depends on the hazard present in a given environment. For most situations safety glasses with side shields are adequate, but when there is danger of splashing of any kind goggles are required. The wearing of corrective lenses is no substitute for wearing protective eye equipment such as goggles or transparent face shields as required (Figure 5.9).