Chapter 5: Laboratory Biosafety Practices

The foundations of protective practices in a laboratory lie in an individual’s laboratory experience, technical knowledge, personal work habits, and attitude toward laboratory safety. Unlike administrative controls, which are behaviors dictated by regulation or laboratory policy, the term “protective behavior” is used to define an innate part of each individual worker’s personal approach to the laboratory environment. As such, “protective behaviors” form the first and most important line of defense against injury or exposure in the biomedical workplace.

Laboratory Practices

Basic Laboratory Practices

Prudent practices and good techniques are of primary importance in laboratory safety. Both are based on sound technical knowledge, experience, common sense, and an attitude of courtesy and consideration for others.

Techniques and practices are spelled out in detail as “Standard Microbiological Practices” in the CDC/NIH’s Biosafety in Microbiological and Biomedical Laboratories, 6th edition and the NIH’s Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, as well as in the National Research Council’s Biosafety in the Laboratory – Prudent Practices for the Handling and Disposal of Infectious Materials (National Academy Press, Washington, D.C., 1989). Many laboratory safety text and reference books also contain good information.

At a minimum, the seven basic rules of biosafety, based on the National Research Council’s Prudent Practices document, should be the basis of any personal laboratory work ethic. They are noted in Table 1.

Table 1: Biosafety practices and blocked routes exposure

Laboratory Practice and Technique

The most important element of containment is the strict adherence to standard microbiological practices and techniques.

Persons working with infectious agents or infected materials must be aware of potential hazards and be trained and proficient in the practices and techniques required for handling such material safely. The PI is responsible for ensuring that laboratory personnel are properly trained; the PI may delegate the provision of training to the laboratory supervisor or a designee, but the responsibility remains with the PI.

Each laboratory should adopt this Biosafety Manual and develop written procedures if there are lab-specific hazards not addressed in the Manual or those needing specific additional details that will or may be encountered. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with the handling of infectious agents must direct laboratory activities.

When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The PI is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure. The BSO or EHS may be consulted for more information.

Laboratory personnel safety practices and techniques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices.
Each laboratory will designate a person as the Laboratory Safety Coordinator (LSC).

Laboratory Housekeeping and Personal Hygiene

Personal safety is greatly enhanced by keeping work space areas neat, clean, and orderly. Injuries and exposures are more likely to occur in poorly maintained, disorderly areas.
If work space is shared, the importance of maintaining a neat, clean area increases significantly. Persons that share work spaces must understand their responsibilities to keep the work area and equipment clean and properly maintained. Coworkers must rely on one another to maximize efficiency and safety. Personal materials should be properly labeled, waste discarded, and the shared space disinfected or cleaned prior to leaving it for the next user.
The following guidelines should be observed in the laboratory:

• Routine housekeeping ensures work areas are free of sources of contamination and hazards;
• Establish and follow housekeeping procedures in order to maintain a clutter free and well organized work environment. Laboratory personnel are responsible for the routine cleaning of laboratory benches, equipment, and areas that are used during research work;
• Access to exits, sinks, eyewashes, emergency showers, and fire extinguishers must not be blocked;
• The workplace should be free of physical hazards;
• Follow electrical safety. Use of extension cords are temporary measures and placement must avoid heavily traffic areas. Equipment should be properly grounded. Overloaded electrical circuits and the creation of electrical hazards in wet areas are to be avoided;
• Work benches should be free of infrequently used chemicals, glassware, and equipment;
• Do not store unnecessary items on floors, under benches, or in corners;
• All compressed gas cylinders should be properly secured.

Personal hygiene, including proper handwashing techniques, is also a means by which to enhance personal protection in the laboratory. Scrubbing immediately after de-gloving ensures that contamination of the hand by glove micropuncture or prior exposure is cleaned.

The laboratory is also an inappropriate place to perform personal cosmetic tasks, such as applying makeup, cleaning or trimming fingernails, or brushing hair including wearing or removal of contact lenses. These activities provide new opportunities for exposure and contribute to retrograde contamination of the laboratory environment.

Universal Precautions

Prudent practices often overlap with a set of practices known as “universal precautions.” The overarching universal precaution espoused by the Bloodborne Pathogens (BBP) Standard (see Appendix K for a list and more detailed discussion of universal precautions) should be adopted by all laboratory personnel.

Universal precautions require that all human blood and tissues be handled as though they are infectious. Adopting and applying universal precautions to all laboratory reagents clearly creates a heightened awareness of potential risk and adds another level of caution to activities involving reagents.

Administrative Controls

Administrative controls are policies and procedures designed to assist with the safe handling of potentially hazardous biological materials. They include training, medical surveillance, vaccinations, access control, etc.

Biological Hazard Information

Laboratory workers must be knowledgeable about the hazards associated with the biological agents present in the laboratory and have hazard information available to them. The following are sources of hazard information for biological agents.

Microbial Agents The CDC/NIH’s Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th edition has descriptions of biosafety levels and recommended biosafety practices for specific biological agents. The Canadian Laboratory Centre for Disease Control maintains Material Safety Data Sheets for microbial agents.

Toxins

Isolated biological toxins are chemical hazards, although many such toxins produce adverse effects at doses significantly below that of “traditional” laboratory chemicals. Laboratory use of isolated toxins falls under the BU Lab Safety Manual and Chemical Hygiene Plans, and Safety Data Sheets (SDSs) must be maintained and available. SDSs for a specific toxin should be obtained from the vendor upon receipt of the toxin. Toxicology textbooks, such as Casarett & Doull’s Toxicology, are also good sources of hazard information for toxins. Biological toxins that are listed under the select agent category and are over the permitted quantities are regulated by CDC/APHIS. The IBC also requires laboratories to register these toxins with their office regardless of the amount.

Each laboratory should adopt this Biosafety Manual, the CDC/NIH publications Biosafety in Microbiological and Biomedical Laboratories, 6th edition, and Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. However, because these cover relatively general topics, individual laboratories should develop written procedures if there are lab-specific hazards not addressed in the Manual or CDC/NIH documents that cover the biosafety concerns and laboratory procedures for that particular laboratory.

For example, laboratory-specific SOPs should address safe manipulation of specific organisms, specific exposure control methods, and specific decontamination and waste-handling requirements. Appendix U provides a recommended standard format for SOPs, and laboratories are encouraged to use this format to effectively convey the biosafety information (including use of pictures and illustrations). The laboratory-specific SOPs do not need to duplicate the more general SOPs contained in this manual or the CDC/NIH documents, but should serve as supplements.

Security and Inventory of Biological Agents

Each PI must develop site-specific criteria that safeguard all biological materials, regardless of their risk group, from unauthorized removal. It is the PI’s responsibility to ensure that his or her laboratory implements sufficient security measures and procedures to prevent unauthorized access to biological agents.

Prevention of Aerosols and Droplets

Handling of liquids or dry powders may generate aerosols or droplets. In practice, high-energy procedures, such as centrifuging, vortexing and mixing, tend to produce respirable aerosols that stay airborne for extended periods and are small enough to be inhaled,while low-energy procedures, including opening containers and streaking plates, produce droplets that settle quickly on surfaces, skin, and mucous membranes.

Utilization of Biological Safety Cabinets

In general, the following guidelines are recommended when using biological safety cabinets (BSCs):

• The BSC should be certified when it is initially installed and recertified after it is moved and installed into its new place, and annually thereafter (for information on cabinet certification, call (617) 358-7840 at BUMC and (617) 353-4094 at CRC.
• The magnahelic gauge should be checked each time before the BSC is used. This gauge will normally run at a relatively fixed value. When it deviates significantly, the cabinet should not be used until the cause of the deviation has been identified and fixed.
• Personnel should know how the BSC works.
• Personnel should be familiar with the safe and effective use of any UV lamps inside the BSC (if equipped) and use appropriate precautions to avoid any UV exposure.
  Note: EHS has discouraged the use of UV lamps because they have a short half-life and are difficult to maintain.
• The BSC’s protective airflow pattern should not be disrupted. The user should avoid rapid arm movement, and heavy foot-traffic in front of the BSC while in use. Also avoid opening and closing nearby laboratory doors, as those activity may disrupt the airflow pattern and reduce the cabinet’s effectiveness.
• Preplanning of the work and organization of necessary materials needed minimize the need to exit and reenter the BSC work space.
• Avoid the accumulation of materials inside the BSC and keep only the materials needed to conduct the work to reduce the disruption of airflow.
• The BSC should be left running whenever the cabinet is in use.
• Use of only appropriate disinfectants to avoid damaging the cabinet’s interior.
• Work surface should disinfected with an appropriate chemical disinfectant. If 10% bleach solution is used, it must be followed by 70% ethanol to prevent surface damage from the bleach solution. Each item needed for the planned procedures should be wiped off with 70% ethanol and placed in the cabinet.
• After the work volume is set up, the BSC should run for at least 5 minutes to allow for stabilization of air flow before starting any procedure.
• If an equipment such as a centrifuge or blender could create air turbulence in the BSC, it should be placed in the back one-third of the cabinet. All other work should be stopped while this equipment is operating.
• Open flames should be avoided because they create air flow turbulence that may compromise sterility. In addition, the heat buildup may damage the HEPA filters. If a flame is necessary, a burner with a pilot light should be used. Electric devices, such as loop sterilizers, are often satisfactory alternatives to open flames.
• A pan with disinfectant and/or a sharps container should be placed inside the BSC for pipette/sharps disposal. Vertical pipette discard canisters on the floor outside the cabinet should be avoided.
• Contaminated and clean items should be segregated, and personnel should work from “clean to dirty.” The biohazardous waste collection bag should be in a rigid container. Do not block air flow into the front and rear exhaust grilles.
• Move arms slowly when removing items from or introducing items into the cabinet work volume.
• Protect the facility vacuum system from biohazards by using dual aspirator flasks in series (A and B) and placing an in-line hydrophobic HEPA filter (C) between the vacuum trap and the source valve (D) in the cabinet:
  
  Note: EHS requires that the flasks are placed in a secondary container, such as a Nalgene tub.
• All spills in the cabinet should be cleaned immediately. Upon clean up, the BSC should be allowed to filter and purge its air and work should not resume for 10 minutes.
• When work is complete, all materials should be removed from the BSC and all interior surfaces should be wiped with an appropriate disinfectant.
• Gloves must be removed before exiting the BSC, and after touching or handling contaminated materials. Discuss the appropriateness of other alternatives, such as use of double gloves, with the Biosafety Officer before deploying as part of the protocol.
• Laboratory coats must be removed and hands thoroughly washed before leaving the laboratory.

Utilization of Pipettes

Pipettes are used for volumetric measurements and the transfer of fluids that may contain infectious, toxic, corrosive, or radioactive agents. Laboratory-associated infections have occurred from oral aspiration of infectious materials, mouth transfer via a contaminated finger, touching face (eyes, nose, etc.) and inhalation of aerosols. Exposure to aerosols may occur when liquid from a pipette is dropped onto the work surface; when cultures are mixed by pipetting; or when the last drop of an inoculum is blown out.

The following outlines safe pipetting techniques to minimize the potential for exposure to hazardous materials:

• Do not mouth pipette. Always use a pipetting aid.
• If working with biohazardous or toxic fluid, confine pipetting operations to a biological safety cabinet.
• Always use cotton-plugged pipettes when pipetting biohazardous or toxic materials, even when safety pipetting aids are used.
• Do not prepare biohazardous materials by bubbling expiratory air through a liquid with a pipette.
• Do not forcibly expel biohazardous material out of a pipette.
• Never mix biohazardous or toxic material by suction and expulsion through a pipette.
• When pipetting, avoid accidental release of infectious droplets.
• Use “to deliver” pipettes rather than “to contain” pipettes, which require “blowout.” Be careful not to dislodge the residual liquid.
• Do not discharge material from a pipette at a height. Whenever possible, allow the discharge to run down the container wall.
• Place contaminated reusable pipettes horizontally in a pan containing enough liquid disinfectant to completely cover them. Autoclave the pan and pipettes as a unit before processing them for reuse.
• Discard contaminated, broken, or intact Pasteur pipettes and broken glass in a sharps container. Dispose of the container properly when it is, at most, three-fourths full.
• Pans or sharps containers for contaminated pipettes should be placed inside the BSC, if possible.
• Proper procedures for disposal of plastic pipettes are presented in Chapter 9: Biohazardous and Medical Waste Disposal.

Utilization of Centrifugation

Hazards associated with centrifuging include mechanical failure and the creation of aerosols. To minimize the risk of mechanical failure, centrifuges must be maintained and used according to the manufacturer’s instructions. Users should be properly trained and operating instructions that include safety precautions should be prominently posted on or near the unit.

Aerosols are created by activities such as filling centrifuge tubes, removing plugs or caps from tubes after centrifugation, removing supernatant, and re-suspending sediment pellets. A significant aerosol hazard can be created if a tube breaks during centrifugation.

To minimize the generation of aerosols when centrifuging biohazardous material, the following procedures are recommended:

• Use sealed tubes and safety buckets that seal with O-rings. Before use, inspect tubes, O-rings, and buckets for cracks, chips, erosions, bits of broken glass, etc. Do not use aluminum foil to cap centrifuge tubes because it may detach or rupture during centrifugation.
• Only use appropriate centrifuge tubes. Open and fill the centrifuge tubes, rotors, and accessories in a biological safety cabinet. Avoid overfilling centrifuge tubes to prevent closures from becoming wet. After tubes are filled and sealed, wipe them down with disinfectant.
• In the event of a tube breakage during centrifugation or an abnormal stoppage that could result into a spill in the centrifuge bucket, the bucket should be brought into the BSC and opened for inspection of potential breakage or spills. The bucket containing the spilled sample should be decontaminated inside the BSC and debris removed prior to reuse.
• Always follow the manufacturer’s instructions when operating the centrifuge and balance the buckets, tubes, and rotors properly before centrifugation.
• Avoid decanting or pouring off supernatant; unless the supernatant must be retained, use a vacuum aspirator with appropriate in-line reservoirs and filters.
• Work in a BSC when re-suspending pelleted material. Use a swirling rotary motion rather than shaking. If shaking is necessary, wait a few minutes to permit the aerosol to settle before opening the tube.
• Small, low-speed centrifuges may be placed in the BSC during use to reduce the aerosol escape. High-speed centrifuges pose additional hazards. Precautions should be taken to filter the exhaust air from vacuum lines, to avoid metal fatigue resulting in disintegration of rotors, and to use proper cleaning techniques and centrifuge components. Manufacturers’ recommendations must be meticulously followed to avoid metal fatigue, distortion, and corrosion.
• Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous materials. Celluloid centrifuge tubes are highly flammable and prone to shrinkage with age. They distort on boiling and can be highly explosive in an autoclave. If celluloid tubes must be used, an appropriate chemical disinfectant must be used.

Utilization of Cryostats

Use of cryostats is very common in many research laboratories. These devices may pose potential hazards associated with sharp cutting edges and cold environments and should be handled with extra care.

The following guidelines should be followed when using cryostats:

• Frozen sections of unfixed human tissue or animal tissue infected with an etiologic agent pose a risk because freezing tissue does not necessarily inactivate infectious agents. Use of freezing propellants under pressure is not recommended with frozen sections because they may cause spattering of droplets of potentially infectious material.
• Appropriate gloves should be worn during preparation of frozen sections.
• When working with human or infected animal tissue, consider the contents of the cryostat to be contaminated and decontaminate it frequently with 70% alcohol.
• Ribbons and trimmings from the frozen tissue samples can accumulate in the cryostat during its use. The machine should be cleaned up and decontaminated routinely.
• Defrost and decontaminate the cryostat with a tuberculocidal hospital disinfectant once a week and immediately after use with tissue known to contain bloodborne pathogens, M. tuberculosis, or other infectious agents.
• Use disposable knife blades. Handle microtome knives or blades with extreme care. Cut-resistant gloves with stainless steel mesh or similar gloves should be worn when changing knife blades.
• Solutions used for staining potentially infected frozen sections should be considered contaminated.
• Always use the safely lock of the equipment before mounting or unmounting tissues or replacing the blade.

Utilization of Inoculating Loops

Flaming inoculating loops can result in spatter and the release of aerosols and droplets. Use of an electric microincinerator, or disposable loops are the preferred alternative, to minimizing this issue.

Use of Absorbent Materials

Work surfaces should be covered with absorbent paper or “diaper” sheets to absorb splashes and drips and to minimize the spread of contamination. The absorbent paper should be changed at the end of the laboratory procedure as part of the final cleanup, or at least daily during use.

Utilization of Miscellaneous Aerosol-Producing Devices and Activities

Use of any of the devices listed below results in considerable aerosol production. Blending, cell-disrupting, and grinding equipment should be used in a BSC when working with biohazardous materials.

Blenders

Safety blenders, are designed to prevent leakage from the bottom of the blender jar. They provide a cooling jacket to avoid biological inactivation and can withstand sterilization by autoclaving.

• Safety blender rotors used for infectious materials should be leak-proof. They should initially be tested with sterile saline or dye solution prior to use with any biohazardous material.
• The use of glass blender jars is not recommended because of the potential for breakage. If they must be used, glass jars should be coated with a polypropylene to prevent spraying of glass and contents in the event the blender jar breaks. The blender must be operated within a secondary containment basin.
• A towel moistened with disinfectant should be placed over the top of the blender during use.
• When opening blenders, be cognizant of potential contamination hazards in the form of droplets that might become airborne or fall on the surfaces; liquid residue on the cap; and possible expansion of the volume due to aeration.
• Before opening the blender jar, allow the unit to rest for at least one minute to allow the aerosol to settle.
• Placing the blender in a BSC will provide protection against airborne hazards and placement of a tray lined with absorbent pads would assist with contamination control.
• The device should be decontaminated promptly after use.

Lyophilizers

Depending on type and model, potential aerosol production may occur when material is loaded into or removed from the lyophilizer unit.

• If possible, sample material should be loaded in a BSC.
• The vacuum pump exhaust should be equipped with a filter to filter out any hazardous agents or, alternatively, the pump can be vented into a BSC.
• After lyophilization is complete, all surfaces of the unit that have been exposed to the agent should be disinfected.
• If the lyophilizer is equipped with a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination.
• Handling of cultures should be minimized and vapor traps should be used wherever possible.

Sonicators

Sonication is the use of sound-wave energy for dispersion, disruption, or inactivation of biological materials, such as viruses. Sonicators generate sound waves at very high frequencies (~20,000 + Hz range), which is outside normal hearing range. The following are hazards associated with sonicators:

• Noise: Although the 20,000-Hz frequency is outside normal hearing range, there are other sources of noise, such as vibration from any loose equipment or other items on the bench or the liquid itself. If the noise levels are high, hearing protection devices should be worn.
• Aerosols: Aerosols present a more serious potential hazard and must be taken into consideration. Precautions listed for blenders and lyophilizers should be observed.

Ampoules

Glass ampoules have been widely used in packaging cultured materials. Opening ampoules containing liquid or lyophilized cultured material should be performed in a BSC to control any aerosol produced. Sealed-glass ampoules used to store biohazardous material in liquid nitrogen have exploded, causing eye injuries from glass microparticles in the snap of the ampoule. The use of polypropylene tubes (cryovials) eliminates this hazard. These tubes are available dust-free or pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat-sealable polypropylene tubes are also available.

• Individuals should be trained on the safe handle and opening of glass ampoules.
• Gloves and protective eyewear must be worn when opening ampoules or cryovials.
• To open a sealed-glass ampoule, nick the neck of the ampoule with a file, wrap it in disinfectant-soaked disposable towel, hold the ampoule upright, and snap it open at the nick.
• Reconstitute the contents of the ampoule by adding liquid slowly to avoid aerosolization of the dried material.
• Mix the contents without bubbling and withdraw it into a fresh container. Discard the disposable towel and the ampoule’s top and bottom as medical waste.

Loop Sterilizers and Bunsen Burners

Sterilization of inoculating loops or needles in an open flame generates small-particle aerosols that may contain viable microorganisms.

• Alternatively, disposable plastic loops and needles may be used for culture work where electric incinerators or gas flames are not available.
• Continuous flame gas burners should not be used in a BSC. These burners can produce turbulence that disturbs the cabinet’s protective airflow patterns. Additionally, the heat produced by the continuous flame may damage the HEPA filter. If a gas burner must be used, one with a pilot light should be selected. Electric sterilizers should also be considered.

Personal Protective Equipment (PPE)

Personal protective equipment (PPE) must be provided without cost to personnel. Although not a substitute for the use of BSCs and good laboratory practices, PPE is considered a primary barrier to infectious agents and proper use will reduce the likelihood of infection. PPE is the least-desirable exposure control method because its failure results in direct exposure to the agent.
PPE is most effective when used to supplement primary control methods such as biological safety cabinets, safety centrifuge cups, and other containment devices. Appropriate clothing may also protect the experiment from contamination
The following PPE requirements apply to all individuals visiting research and teaching laboratories:

• Full-length pants, or equivalent must be worn at all times;
• Closed toe footwear, that covers the top of the foot, must be worn at all times; and
• Long sleeves or laboratory coat covering both arms.

All other individuals entering laboratory space must follow the PPE requirements described above for the visitor, as well as the following:

• Laboratory coats must be worn over personal clothing at all times;
• Long hair and facial hair must be secured or tied back;
• Flame resistant laboratory coats must be worn when working with pyrophoric chemicals, water reactive chemicals, and high volumes of flammable chemicals;
• Disposable gloves that are protective against the hazardous or potentially hazardous materials being used must be worn. Gloves must be replaced when soiled, contaminated or damaged; and
• Eye protection must be available and used when danger to splashing of hazardous or potentially hazardous materials could occur.

Respirators

Respirators are selected based on the hazard involved and the protection factor required. Certain laboratory and clinical situations require respiratory protection to prevent inhalation of infectious agents. Regulations, as well as good safety practice, require that personnel be medically evaluated, specifically trained, and fit tested prior to wearing respiratory protective equipment.

Contact EHS if respiratory protective equipment is required or if there are questions about the respiratory protection program.

Further guidance on the use of PPE can be found in the Personal Protection Equipment in Laboratories Policy and Chemical Hygiene Plan.

Storage and Labeling of Biological Agents

Biological agents must be stored using leak proof and sealed containers. Containers must be clearly labeled with the identity of the agent and should include the universal biohazard symbol (see below) as physical space on the container permits. At a minimum, secondary (or outside) containers must include the universal biohazard symbol (identity of contents is also desirable).

Freezers, refrigerators, and other storage areas must also be labeled with the biohazard symbol; exceptions to this policy will be considered on an individual basis by the IBC. Waste and contaminated equipment or other objects to be decontaminated must also be labeled with the biohazard symbol.

Universal Biohazard Symbol

The OSHA Bloodborne Pathogen Standard specifically requires that containers of human blood or other potentially infectious material (OPIM), contaminated waste, and refrigerators, freezers, and other storage containers used to store or transport blood or OPIM be labeled with the universal biohazard symbol (fluorescent orange or orange-red):

Biohazard Labels and Signs

Each laboratory must have a sign at the entrance that provides safety information to visitors and service personnel. Room signs must contain designations for all laboratory hazards in use within the laboratory (carcinogens, acutely toxic agents, reproductive hazards, biohazards, radioactive materials, lasers, and magnetic fields). EHS will prepare the signs for each door in accordance with the requirements of NFPA 704 and BSL3.

Biohazard signs will be posted at the following:

• Entrances to laboratories and animal rooms that use agents classified as BSL2, BSL3 or BSL4.
• Entrances to animal rooms used for housing animals infected with ABSL2, ABSL3, or ABSL4 agents.

For examples of BU door signage, see Appendix S: Laboratory Door Signage.

Certain other areas and pieces of equipment within a laboratory may also require signs. Refrigerators, freezers, cabinets, and other storage facilities require the biohazard symbol whenever they are used to store infectious agents of Risk Group 2 or higher; human blood or blood products; unfixed tissues; cell or organ cultures; body fluids; or excreta. Large pieces of equipment for handling such materials (e.g., centrifuges, biological safety cabinets) must be similarly labeled.