|Enabling the Safe Use of Biotechnology Principles and Practice (World Bank, 1996)|
|Part two: Practice|
The following examples of guidelines for good laboratory practice and good industrial large-scale practice are taken from the Organization for Economic Co-operation and Development (OECD) study Safety Considerations for Biotechnology.
Good Laboratory Practice
Human error, poor laboratory practice, and misuse of equipment cause the majority of laboratory accidents and related infections. This chapter provides a compendium of techniques designed to correct or minimize the most commonly reported accidents caused by these factors.
Techniques in the Use of Pipettes and Pipetting Aids
· Cotton-wool-plugged pipettes will reduce the possibility of contaminating the pipetting aid.
· Air should never be blown through a liquid containing infectious agents.
· Infectious material should not be mixed by alternate suction and expulsion through a pipette.
· No infectious material should be expelled forcibly from a pipette.
· To avoid the hazards of accidentally dropping infectious cultures from pipettes, a disinfectant soaked cloth should be placed on the working surface and autoclaved after use.
· Mark-to-mark pipettes are preferable to other types because they do not require expulsion of the last drop.
· Fluids should be discharged down the inner wall of the tube or bottle or beneath the surface of the liquid in the container.
· Contaminated pipettes should be completely immersed in a suitable disinfectant before being autoclaved.
· A discard pan for pipettes should be placed within the biological safety cabinet, not outside it.
· A syringe fitted with a sharp hypodermic needle must not be used as a pipetting device. Blunt cannulae should be substituted for needles.
Techniques to Avoid Dispersal of Infectious Material
· A pipetting aid should always be used. Mouth pipetting should be prohibited.
· The circle of a microbiological loop should be completely closed and the arm not more than 6 centimeters long.
· When there is risk of spatter of infected material in a Bunsen flame, a micro-incinerator should be used. Plastic disposal loops are a safe alternative.
· Catalase tests should not be done on slides. Tube methods should be used or cover-glass methods used in an exhaust protective cabinet. Catalase tests may also conveniently be performed by touching a microhematocrit capillary tube loaded with hydrogen peroxide on to the surface of a colony.
· Discarded specimens and cultures should be placed in leak-proof containers for disposal.
· Working areas must be cleaned with a suitable disinfectant when each work period is finished.
· Horizontal outflow cabinets (clean air work stations) are not microbiological safety cabinets and should not be used as such.
Techniques in the Use of Biological Safety Cabinets
· The use and limitations of cabinets must be explained to all potential users.
· The cabinet must never be used unless the fan is switched on and the air flow indicator is in the safe position.
· If it has an operable glass viewing panel, this must be raised when the cabinet is in use.
· Apparatus and materials must be kept to a minimum during operation.
· A Bunsen burner must not be used in the cabinet. The heat produced might distort the air flow and the filters might be burned. A microincinerator is permissible, but disposable plastic loops are preferable.
· All work must be done in the middle to the rear of the cabinet and be visible through the glass window.
· It must be understood that the cabinet will protect neither the hands nor the worker from gross spillage, breakage, or poor technique.
· The cabinet fan should be run for at least fifteen minutes after completion of work in the cabinet.
Techniques to Avoid Ingestion of Infectious Material
· Larger particles and droplets (>5 micrometers) released during microbiological manipulations settle rapidly on the bench surfaces and the hands of the operator. Hands should be washed frequently. Workers should avoid touching their mouth and eyes.
· Food and drink should not be stored or consumed in the laboratory.
· There should be no smoking or gum-chewing in the laboratory.
· Cosmetics should not be applied in the laboratory.
Techniques to Avoid Injection of Infectious Material
· Injection may result from accidents with hypodermic needles, Pasteur pipettes, and broken glass.
· Accidents with hypodermic needles can be reduced only by greater care and making less use of syringes and needles. If syringes must be used for measurement, blunt cannulas should be substituted for needles.
· Accidental inoculation with Pasteur pipettes and broken glass may be avoided only by greater personal care.
Techniques for the Separation of Serum
· Only properly instructed laboratory staff should be employed for this work.
· To prevent splashes and aerosols, good microbiological technique should be observed. Potentially infected fluids, including blood, should be pipetted carefully, not poured. Mouth pipetting must be forbidden.
· Pipettes should be discarded and completely submerged in hypochlorite or some other suitable disinfectant. They must remain in the disinfectant at least overnight before disposal.
· Discarded specimen tubes containing blood clots and the like should be put in suitable leak-proof containers (with the caps replaced) for autoclaving or incineration.
· A solution of sodium hypochlorite should be provided for cleaning splashes and spillage of blood and serum.
Techniques for the Use of the Centrifuge
General. The following precautions should be observed:
· Mechanical safety is a prerequisite in the use of clinical centrifuges.
· Infectious airborne particles may be ejected when centrifuges are used improperly. These particles travel at speeds too high to be captured and retained if a centrifuge is placed in a traditional Class I or Class II safety cabinet.
· The centrifuge should be operated according to the manufacturers' instructions.
· Good centrifuge technique and sealed centrifuge buckets offer adequate protection from microorganisms and agents in Risk Groups 3 and 4.
Centrifugation of risk group 2 microorganisms, agents, and materials. The following precautions should be observed:
· Centrifuge buckets and bunions should be paired by weight and should be properly balanced with tubes in place.
· To avoid dislodging bunions and spilling the contents of the tubes, the motor should be started slowly and speed increased gradually.
· Centrifuges should be placed at such a level that workers of less than average height can see into the bowl to place the bunions correctly on the rotor.
· Centrifuge tubes and specimen containers to be used in the centrifuge should be made of thick-walled glass or plastic and should be inspected for defects before use.
· The interior of centrifuge bowls should be inspected daily for evidence of bad techniques, indicated by staining or soiling at the level of the rotor, and should be cleaned if necessary.
· Angle head should be used for microbiological work except in special high-speed centrifuges. With ordinary angle heads some fluid, even from capped tubes, may be ejected because of the geometry of the machine.
· Except in ultracentrifuges and with small prothrombin tubes, a space of at least 2 centimeters should be left between the level of fluid and the rim of each centrifuge tube. Tubes containing infectious material should be capped.
Centrifugation of risk groups 3 and 4 microorganisms, agents, and materials. The following precautions should be observed, in addition to those above:
· Centrifugation should be done in batches separate from other material.
· Centrifuge tubes or bottles should have screw caps and should be marked in a way agreed locally to indicate that the contents are in Risk Groups 3 and 4.
· Sealed centrifuge buckets (safety cups) should be used.
· The sealed buckets should be loaded, sealed, and opened in a biological safety cabinet.
Techniques for the Use of Homogenizers and Shakers
· Caps and cups or bottles should be sound and free from flaws or distortion. Caps should be well-fitting and gaskets must be in good condition.
· Aerosols containing infectious particles may escape from shakers and homogenizers between the cap and the vessel. A pressure builds up in the vessel during operation. Teflon homogenizers are recommended because glass homogenizers may break releasing infectious material and possibly wounding the operator.
· Machines should be covered when in use by a transparent plastic housing of strong construction. This should be disinfected after use. When possible, these machines, under their plastic covers, should be operated in a biological safety cabinet.
· After shaking or homogenization, all containers should be opened in a biological safety cabinet.
· Sonicators should be used inside biological safety cabinets. Hearing protection should be provided.
Techniques for the Use of Tissue Grinders Such as Griffith's Tubes and TenBroek Grinders
· Grinders should be held in a wad of absorbent material in a gloved hand when tissues are ground.
· They should be operated in a biological safety cabinet.
Techniques for Opening Ampoules that Contain Lyophilized Infectious Materials
Care should be taken when ampoules of freeze-dried materials are opened as the contents are in a vacuum and the sudden inrush of air may disperse the contents into the atmosphere. Ampoules should always be opened in safety cabinets.
The following procedure is recommended for opening ampoules:
· The outside of the ampoules should be decontaminated before use.
· A file mark is made on the tube near the middle of the cotton-wool plug.
· A red-hot glass rod is applied to the file mark to crack the glass.
· The top is removed gently and treated as contaminated material.
· The cotton-wool plug, if still above the contents of the ampoule, is removed with sterile forceps.
· Liquid for resuspension is added slowly to the ampoule to avoid frothing.
Storage of Ampoules that Contain Infectious Material
· Ampoules containing infectious material must never be immersed in the liquid phase of liquid nitrogen because cracked or imperfectly sealed ampoules may break or explode on removal.
· If very low temperatures are required, ampoules may be stored in the vapor phase only (that is, above the level of the liquid nitrogen).Whenever possible, infectious agents should be stored in mechanical deep freeze cabinets or on dry ice rather than in liquid nitrogen.
· The outside of ampoules stored in these ways should be decontaminated when they are removed from storage.
Techniques for Care, Use, and Operation of Refrigerators and Freezers
· Refrigerators, deep freeze, and dry-ice chests should be checked, cleaned out, and defrosted periodically to remove any ampoules or tubes containing hazardous materials that may have broken during storage. Rubber gloves should be worn during cleaning.
· All materials, especially infectious or toxic materials, stored in refrigerators or deep-freeze should be labeled with the scientific name of the material, the date stored, and the name of the individual storing the material.
· Do not store flammable solutions in nonexplosion-proof refrigerators.
Good Industrial Large-Scale Practice
The OECD study on safety considerations for biotechnology worked out the principles for handling organisms with novel traits in industrial use. This report sets out the principles and criteria recommended for the safe use of such organisms in industry and is an appropriate basis for regulating this sector.
An important general point made in the 1986 OECD report is that hazards associated with recombinant DNA (rDNA) organisms can be assessed and managed like those associated with any other organisms. It is expected that the vast majority of rDNA organisms to be used in industrial large-scale production can be handled using good industrial large-scale practice (GILSP).
Irrespective of the intrinsic safety of the organisms concerned, zero risk is not realistic even for GILSP organisms.
Central to the concept of GILSP are:
· The assessment of the recombinant organism according to identified criteria to determine that it is as safe as the low-risk host organism · The identification and adoption of practices ensuring the safety of the operation.
Recombinant DNA organisms that meet the GILSP criteria and are therefore of low risk can thus be handled under conditions already found to be appropriate for the relevant hosts.
GILSP applies to organisms considered to be of low risk and classified in the lowest-risk class. In order to ensure that, for each individual case, a rDNA organism merits the designation of GILSP, the criteria elaborated by the OECD must be taken into consideration in an integrated way. Two clear examples of other classes of organisms that warrant the GILSP designation, provided they are nonpathogenic and without adverse consequences for the environment, are:
· Those constructed entirely from a single prokaryotic host (including its indigenous plasmids and viruses) or from a single eukaryotic host (including its chloroplasts, mitochondria or plasmids but excluding viruses).
· Those consisting entirely of DNA segments from different species that exchange DNA by known physiological processes.
Organisms that do not meet all the criteria for GILSP are not GILSP organisms. However, after the case-by-case evaluation, they may be found to be of low risk. In such circumstances these organisms may be handled using GILSP. Care must be taken when extrapolating GILSP to other organisms to evaluate whether specific practices in addition to GILSP are required to mitigate a specific concern.
Organisms that can be handled on a large scale under conditions of minimal controls and containment procedures will be:
· Those meeting the criteria of GILSP organisms
· Those other classes of organisms described above
· Other organisms not meeting either of these sets of criteria but which have been demonstrated to be of low risk, as described above. When handling GILSP and other low-risk organisms, established principles of good occupational and environmental safety must be followed.
1. OECD, Safety Considerntions for Biotechnology.