Module 5. Techniques for microbiological analyses

Lesson 22


22.1 Introduction

Biosafety concepts in quality control laboratories ensure that adequate safety conditions are implemented to avoid potential hazards associated with the handling of milk and milk products. The biosafety guidelines define the essential competencies needed by laboratory personnel to work safely in a laboratory. Competencies are measurable and include not only knowledge, skills, and abilities but also judgment and self-criticism.

22.2 Biohazardous Agent

A biohazard can be defined as any organism, or material produced by such an organism, that is known or suspected to cause human or animal disease. Exposure to bio-hazardous agents may occur via puncture, wounds or as a result of absorption through the respiratory tract, digestive system, skin and mucous membranes. Such exposures may result while handling samples, microbiological cultures and radioactive substances to be used for detection of various contaminants. Exposure to bio-hazardous agents is intended to be prevented or limited by establishing and following the appropriate bio-safety level practices and conditions.

22.3 Biosafety Level

The application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or bio-hazards. Biosafety defines the containment conditions under which infectious agents can be safely manipulated. The objective of containment is to confine bio-hazards and to reduce the potential exposure of the laboratory worker, persons outside of the laboratory, and the environment to potentially infectious agents.The term ‘bio-safety’ is used in describing measures used to provide a barrier between the infectious/ pathogenic organisms being handled and the worker (and ultimately, the community at large). The bio-safety levels in microbiological laboratories can be designated as bio-safety level (BSL) 1, 2, 3 and 4. The levels are classified into four groups depending on the safety requirements, the highest safety standards (Level-4) are reserved for the most hazardous pathogens (Risk Group 4), and the least stringent (Level-1) for those which have minimal impact on health (Risk Group 1). Containment is achieved through the use of appropriate safety equipment, facility design and lab procedures and practices. Careful consideration must be given to both facility design and work practices to ensure protection of laboratory personnel, their colleagues and the community as a whole.

Table 22.1 Risks and characteristics associated with pathogens from risk groups 1 to 4, and recommended containment level and class of biological safety cabinet

t 22.1

22.3.1 Biological safety cabinets

Biological safety cabinets reduce the risk of airborne infection by reducing the escape of aerosolized infectious agents into the laboratory environment. In addition to protecting workers, some biological safety cabinets protect the work inside the cabinet from airborne contamination (product protection). Biological safety cabinets minimize contact between the operator and pathogens through the use of directional airflow, high efficiency particulate air (HEPA) filtration of supply and/ or exhaust air, and, in some cases, a physical barrier such as a plastic or glass shield (Fig. 22.1). Filters are an essential component of the biological safety cabinet, and have particle removal efficiencies of 99.97% or better for 0.3 micron diameter particles. This size particle is used as the basis for filter definition because it is considered the most difficult to remove. Thus, a filter that can trap 0.3 micron diameter particles can easily eliminate other sizes. HEPA filters consist of continuous sheets of glass fiber paper pleated over rigid corrugated separators and mounted in a wooden or metal frame.


Fig. 22.1 HEPA filters

22.3.2 Classes of biological safety cabinet

Horizontal and vertical clean benches are not biological safety cabinets. Air filtered through HEPA filters is directed over the work surface and then discharged directly into the room. Thus, these units provide product protection, but do not protect the operator from exposure to the materials being handled; they must not be used for work with potentially infectious or toxic materials. There are three basic types of biological safety cabinet, each providing different levels of containment: Class I

(i) open-fronted (ii) protects operator and environment (iii) for work with low and moderate risk agents (Risk Groups 2 and 3) where product protection is not critical

General Principle of Operation

An inward flow of room air through the work opening, away from the operator, prevents the escape of airborne pathogens into the laboratory. Negative cabinet pressure is created by a blower that exhausts the air, either into the room or to the outside, through a HEPA filter. It is this HEPA filtration of exhaust air that provides environmental protection (Fig. 22.2). A disadvantage of this type of cabinet is that the product is exposed to contaminants that are pulled in from the room environment. In addition, internal air turbulence may result in cross-contamination within the cabinet.


Fig. 22.2 Biosafety level 1 work with open bench tops and standard microbiological practices Class II

(i) open-fronted (ii) protects operator, product and environment from particulate contamination (iii) for work with low to moderate risk agents (Risk Groups 2 and 3).

General Principle of Operation

Escape of pathogens into the worker's environment is prevented by an inward flow of room air which enters the front opening without crossing the work area and by HEPA filtration of exhaust air (this provides environmental protection), while downward flow of HEPA filtered air through the work area removes work zone contaminants and protects the product (Fig. 22.3). The amounts of room air drawn into the intake grille and the amount of air exhausted through the exhaust filter are equal. This balance is critical: positive pressure will allow the outflow of pathogens, while negative pressure will result in inflow of room contaminants. The different types of Class II cabinets (e.g. Type A, Type B or 100% exhaust) vary in: airflow velocities, amount of cabinet air recirculated (from 0 to 70%), amount of cabinet air exhausted (from 30 to 100%), destination of exhaust air (back to lab or outside), exhaust ducting (building system versus dedicated ducts). It should be kept in mind that toxic or radiolabelled chemicals must not be handled in cabinets that recirculate air within the cabinet or exhaust into the laboratory.


Fig. 22.3 Biosafety cabinet level-II and its working Class III

(i) Totally enclosed, gas tight, with glove ports for manipulation of pathogens (ii) provides the greatest level of operator and product protection (iii) for work with high risk pathogens (Risk Group 4).

General Principle of Operation

These cabinets form a physical barrier between the operator and microbiological agent. Internal negative pressure confines any leaks to the inside of the cabinet. Supply and exhaust air is HEPA filtered; a dedicated exhaust fan, separate from that of the facility ventilation system, discharges directly to the outdoors. There is no recirculation of air within the cabinet. A Class III cabinet system must be designed to allow for safe introduction, handling and removal of all materials throughout the procedure. Equipment such as the incubator, refrigerator, centrifuge, autoclave and chemical dunk tank are connected to the cabinet system.

22.4 Working Safely in a Biological Safety Cabinet

Biological safety cabinets must be combined with good work practices for optimum safety and contamination control. Recommended practices when using a biological safety cabinet include – movement of arms into and out of the cabinet can disrupt airflow, adversely affecting cabinet performance. Whenever possible, place all materials needed for a procedure inside the cabinet before starting. Avoid bringing non-essential equipment and supplies into the cabinet. Place supplies, equipment and absorbent towels so that air intake or exhaust grilles are not obstructed. Keep opening and closing of lab doors and other personnel activity to a minimum. If a burner is deemed to be indispensable (remember that burners contribute to the heat load, generate convection currents that interfere with airflow and may damage the filters), use one that has a pilot flame. Attach a HEPA filter cartridge between the vacuum trap and the source valve. Work at least 4-6 inches inside the cabinet window. Carry out work on an absorbent pad to contain small spills. Clean up spills as soon as they occur; remove and disinfect the grille if contaminated. Designate separate areas within the cabinet for contaminated and clean materials; place contaminated material at the rear of the work area.

22.4.1 Biosafety level-1

Biosafety Level 1 is used when working with agents (Risk Group 1) that pose no risk to healthy adults (Fig. 22.2). The laboratory may be near a public area but doors should be kept closed. Work may be carried out on an open bench top. Lab surfaces (walls, ceilings, furniture and floors) should be cleanable. Open windows should have insect screens. Eyewash stations and hand washing facilities should be available. Street clothes and lab coats should not be kept together. Disinfection should be carried out as required, using effective concentrations and contact times; solutions should be replaced regularly.

22.4.2 Biosafety Level-2

Biosafety Level 2 is appropriate for work with Risk Group 2 agents (Fig. 22.3). The following precautions, in addition to those for containment Level 1, are recommended. Standard microbiological practices
  1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
  2. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
  4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
  5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented.
  6. Perform all procedures to minimize the creation of splashes and/or aerosols.
  7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
  8. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method.
  9. A sign incorporating the universal bio-hazard symbol must be posted at the entrance to the laboratory when infectious agents are present.

Fig. 22.4 Biohazard sign

10. An effective integrated pest management program is required.
11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Special practices

None required. Safety equipment (primary barriers and personal protective equipment)
  1. Special containment devices or equipment, such as Biosafety Cabinet (BSCs) Level –II are required.
  2. Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
  3. Wear protective eye wear when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials. Persons who wear contact lenses in laboratories should also wear eye protection.
  4. Gloves must be worn to protect hands from exposure to hazardous materials.

Fig. 22.5 Personal protective equipment Laboratory facilities (secondary barriers)
  1. Laboratories should have doors for access control.
  2. Laboratories must have a sink for hand washing.
  3. The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate and epoxy floorings are recommended.

Fig. 22.6 Epoxy floorings


Fig. 22.7 Doors for access control

22.4.3 Biosafety level -3

Biosafety Level - 3 is recommended for work with Risk Group 3 agents. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents, and must be supervised by scientists competent in handling infectious agents and associated procedures. All procedures involving the manipulation of infectious materials must be conducted within BSCs or other physical containment devices. Measures should include the recommendations outlined for levels 1 and 2, plus the following standard and special safety practices, equipment, and facility requirements apply to BSL-3. Standard microbiological practices

As per BSL-1 ( Special practices

In addition to special practices of BSL-2, all procedures involving the manipulation of infectious materials must be conducted within a BSC, or other physical containment devices. No work with open vessels is conducted on the bench. When a procedure cannot be performed within a BSC, a combination of personal protective equipment and other containment devices, such as a centrifuge safety cup or sealed rotor must be used. Safety equipment (primary barriers and personal protective equipment)
  1. All procedures involving the manipulation of infectious materials must be conducted within a BSC (preferably Class II or Class III), or other physical containment devices.
  2. Workers in the laboratory where protective laboratory clothing with a solid-front, such as tie-back or wrap-around gowns, scrub suits, or coveralls. Protective clothing is not worn outside of the laboratory. Reusable clothing is decontaminated before being laundered. Clothing is changed when contaminated.
  3. Eye and face protection (goggles, mask, face shield or other splash guard) is used for anticipated splashes or sprays of infectious or other hazardous materials. Eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact lenses in laboratories must also wear eye protection.
  4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves must not be worn outside the laboratory. In addition, BSL-3 laboratory workers:
  • Changes gloves when contaminated, glove integrity is compromised, or when otherwise necessary. Wear two pairs of gloves when appropriate.
  • Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
  • Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
  • Eye, face, and respiratory protection must be used in rooms containing infected animals. Laboratory facilities (secondary barriers)
  1. Laboratory doors must be self-closing and have locks in accordance with the institutional policies. The laboratory must be separated from areas that are open to unrestricted traffic flow within the building. Laboratory access is restricted. Access to the laboratory is through two self-closing doors. A clothing change room (ante-room) may be included in the passageway between the two self-closing doors.
  2. Laboratories must have a sink for hand washing. The sink must be hands-free or automatically operated. It should be located near the exit door. If the laboratory is segregated into different laboratories, a sink must also be available for hand washing in each zone. Additional sinks may be required as determined by the risk assessment.
  3. The laboratory must be designed so that it can be easily cleaned and decontaminated. Carpets and rugs are not permitted. Seams, floors, walls, and ceiling surfaces should be sealed. Spaces around doors and ventilation openings should be capable of being sealed to facilitate space decontamination.
  4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment must be accessible for cleaning.
  5. All windows in the laboratory must be sealed.
  6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, heavily travelled laboratory areas, and other possible airflow disruptions.
  7. Vacuum lines must be protected with HEPA filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
  8. An eyewash station must be readily available in the laboratory.
  9. A ducted air ventilation system is required. This system must provide sustained directional airflow by drawing air into the laboratory from ‘clean’ areas toward ‘potentially contaminated’ areas. The laboratory shall be designed such that under failure conditions the airflow will not be reversed.
  10. HEPA filter housings should have gas-tight isolation dampers, decontamination ports, and/ or bag-in/ bag-out (with appropriate decontamination procedures) capability. The HEPA filter housing should allow for leak testing of each filter and assembly. The filters and the housing should be certified at least annually.
  11. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations. A method for decontaminating all laboratory wastes should be available in the facility, preferably within the laboratory (e.g. autoclave, chemical disinfection, or other validated decontamination method).
  12. Equipment that may produce infectious aerosols must be contained in primary barrier devices that exhaust air through HEPA filtration or other equivalent technology before being discharged into the laboratory. These HEPA filters should be tested and/ or replaced at least annually.
  13. Facility design consideration should be given to means of decontaminating large pieces of equipment before removal from the laboratory.
  14. Enhanced environmental and personal protection may be required by the agent summary statement, risk assessment, or applicable local, state, or federal regulations. These laboratory enhancements may include, for example, one or more of the following: an anteroom for clean storage of equipment and supplies with dress-in, shower-out capabilities; gas tight dampers to facilitate laboratory isolation; final HEPA filtration of the laboratory exhaust air; laboratory effluent decontamination; and advanced access control devices, such as biometrics.
  15. The BSL-3 facility design, operational parameters, and procedures must be verified and documented prior to operation. Facilities must be re-verified and documented at least annually.
22.4.4 Biosafety level-4

Level 4 containment is used for work with Risk Group 4 agents. Additional recommendations include: physical isolation of the laboratory, with an airlock for access, entry restricted to authorized personnel and recorded in a log book: no one should work alone, use of class III biological safety cabinets and/ or positive-pressure protective suits, additional safety measures for ventilation, waste treatment, and gas and water services.
Last modified: Wednesday, 7 November 2012, 4:17 AM