Laboratory Biosafety and Good Laboratory Practices

Laboratory Biosafety and Good Laboratory Practices

Intended Learning Outcomes

• Execute laboratory safety practices and SOPs when doing laboratory exercises.
• Enumerate different protocols established at the international and national levels to ensure biosafety in institutions involved in biotechnology research and development.

Introduction

• Good microbiological techniques (GMT) were insufficient in the early days of rDNA technology development.
• June 1976: After the Asilomar Conference, the NIH published guidelines for research involving recombinant nucleic acid molecules, ensuring safety in biotechnology.

WHO Laboratory Biosafety Manual

• 1983: UN WHO published the Laboratory Biosafety Manual (aka WHO Manual).
• Established basic concepts and practices for the safe handling of pathogenic microorganisms.
• Encouraged countries to develop national codes of practice for implementation.

Importance of Adhering to International Standards

• To protect plant, animal, or human life.
• To protect the health of citizens.
• To facilitate trade in biotech products.

Risk Categories of Microorganisms

• NIH guidelines and WHO Manual recognize 4 categories based on the risk of infection to:
  • Laboratory workers
  • Community in the event of an escape from the lab

How to Assign a Microorganism to a Risk Category

• Pathogenicity of the organism.
• Host range and mode of transmission of the organism.
• Local availability of effective measures to prevent a disease outbreak.
• Local availability of effective treatment.

Risk Groups (RG) of Microorganisms

• RG 1:
  • Unlikely to cause human or animal diseases.
  • Pose little or no risk to individuals and the community.
  • Generally regarded as safe (GRAS).
• RG 2:
  • Pathogenic, but unlikely to pose a serious hazard to lab workers, livestock, the community, or the environment.
  • Effective treatment and preventive measures are available to limit the spread of infection.
  • Moderate risk to individuals and low risk to the community.
• RG 3:
  • Pathogenic and can cause serious human or animal diseases but are not contagious.
  • Effective treatment and preventive measures to limit the spread of infection are available.
  • High risk to individuals, but low risk to the community.
• RG 4:
  • Causes serious diseases in humans and animals and is readily transmitted (direct or indirect).
  • Effective treatment and preventive measures are unavailable.
  • High risk to both individuals and the community.

Biosafety Levels (BLs)

• Four BLs for handling organisms corresponding to the 4 RGs.
• Relies on:
  • Standard practices of GMT
  • Physical barriers: procedures, equipment, and lab installations based on the estimated biohazard.
• BLs for specific research work depend on:
  • The assessed RG of the organisms handled.
  • Professional judgment of risk associated with the activity.

Relation of RGs to BLs, Practices, and Equipment

Physical Containment

• Strict adherence to good microbial practices.
• All personnel working with recombinant or synthetic nucleic acids should be trained in GMT.
• Four levels of physical containment: BL1 to BL4.
• Facilities range from low to high potential hazard.
• Involve: Standard microbiological practices; Special practices; Containment equipment; and Laboratory facilities.

Biosafety Level Specifications

Biosafety Level 1

• Standard microbiological practices:
  • Access to the laboratory is limited or restricted at the discretion of the Principal Investigator (PI).
  • Work surfaces are decontaminated once a day; all liquid and solid wastes are decontaminated before disposal.
  • Mouth pipetting is prohibited.
  • Eating, drinking, smoking, or storing food in refrigerators is prohibited.
  • Procedures are performed carefully to prevent the formation of aerosols.
  • Good hygiene, including washing hands and wearing protective clothes, is encouraged.
• Special practices:
  • Contaminated materials to be decontaminated at a site away from the laboratory are transported in durable, leak-proof containers with closed lids.
  • An insect and rodent control program is required.
• Containment equipment:
  • Generally, not required for BL1.
• Laboratory facilities:
  • The laboratory should be designed to be easily cleaned.
  • Benchtops should be resistant to water, acid/alkali/organic solvents, and should have sinks for hand-washing.

Biosafety Level 2

• Standard microbiological practices:
  • As described for BL1.
  • Experiments of lesser biohazard can be conducted concurrently in demarcated areas of the laboratory.
• Special practices:
  • As described for BL1.
  • PI limits access to the laboratory and establishes policies and procedures whereby persons entering the laboratory are aware of the hazard and meet any specific entry requirements (such as immunization).
  • A hazard-warning sign with the universal biosafety symbol, details of the agent used, contact information of the PI, and any special requirements for entry are to be posted on the access door.
  • Protective clothing used exclusively in the laboratory is required; gloves are to be used to prevent skin contamination with experimental organisms.
  • Only needle-locking hypodermic syringes are used, placed in puncture-proof containers after use, and decontaminated before disposal.
  • A biosafety manual is prepared and adopted for the safety of personnel.
  • Baseline serum samples of all laboratory and at-risk personnel should be collected and stored in accordance with institutional policy.
• Containment equipment:
  • Biological safety cabinets (class I or II) or other appropriate personal protective devices are used.
• Laboratory facilities:
  • As described for BL1.
  • An autoclave is required for decontamination.

Hazard vs Risk

• HAZARD: something that has the potential to harm you
• RISK: The likelihood of a hazard causing harm

Risk Assessment

• Hazard identification:
  • Is there a problem?
  • What is it: pathogen or associated food?
• Hazard characterization:
  • What happens when the pathogen is ingested?
  • How much of it causes illness?
• Exposure assessment:
  • What is the probability of eating contaminated food?
  • How many pathogens are likely to be in the food when you eat it?
• Risk characterization:
  • What are the nature and likelihood of the health risk?
  • Who is likely to become ill? How many?
  • What are the sources of variability and uncertainty in the information used?

Biosafety Level 3

• Standard microbiological practices:
  • As described for BL2.
  • Persons below 16 years of age are not permitted entry.
• Special practices:
  • As described for BL2.
  • Laboratory doors are kept closed when experiments are in progress.
  • Laboratory clothing that protects street clothes is to be worn in the laboratory, removed when exiting the laboratory, and decontaminated prior to laundry or disposal.
  • Molded surgical masks or respirators are worn in rooms containing experimental animals.
  • If animals are housed with a conventional caging system, personnel must wear protective devices that include wrap-around gowns, head covers, gloves, shoe covers, and respirators; personnel shall shower on exit from areas where these devices are required.
  • Alternatively, laboratory animals shall be housed in partial-containment caging systems; no animals other than the experimental animals are allowed.
  • Vacuum lines are protected with high-efficiency particulate air (HEPA) filters and liquid disinfectant traps.
  • Spills and accidents that result in potential exposure to modified organisms are immediately reported to the Biological Safety Officer, Institutional Biosafety Committee (IBSC), and the NIH Office of Science Policy.
  • Written records are to be maintained on appropriate medical evaluation, surveillance, and treatment provided.
• Containment equipment:
  • Biological safety cabinets (class I, II, or III) or other appropriate personal protective devices (such as special protective clothing, masks, gloves, respirators, centrifuge safety cups, sealed centrifuge rotors, containment cages for animals) are used.
• Laboratory facilities:
  • The laboratory is to be separated from open areas within the building and accessed through two sets of doors.
  • Physical separation of a high-containment laboratory from other laboratories or activities may be provided by a double-door clothes change room with showers, an airlock, or other double-door access features.
  • Interior surfaces of walls, floors, and ceilings are water-resistant for easy cleaning and should be capable of being sealed for decontaminating the area.
  • Access doors are self-closing.
  • The HEPA-filtered exhaust air from Class I or II biological cabinets is discharged directly to the outside or through the building exhaust system.

Biosafety Level 4

• Standard microbiological practices:
  • As described for BL3.
• Special practices:
  • As described for BL3.
  • Access to the facility is limited by means of secure locked doors; accessibility is restricted to authorized personnel and is supervised and managed by the PI, Biological Safety Officer, or person responsible for the physical security of the facility. A log of entry and exit of personnel is maintained. All personnel are advised of potential biohazards and are to comply with instructions on entry and exit procedures. Protocols for emergency situations are established.
  • Biological material to be removed in an intact state is to be sealed in a primary nonbreakable container, enclosed and sealed in a secondary nonbreakable container, and removed from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for the purpose.
  • Any other material to be removed from the facility is to be autoclaved or decontaminated before exiting the maximum containment laboratory.
  • Personnel enter and exit the facility only through clothing change and shower rooms; shower every time they exit the facility.
  • Street clothing is removed and kept in an outer changing room. Complete laboratory clothing (may be disposable) is provided and to be used by all personnel entering the facility. When exiting, the laboratory clothing is removed in an inner changing room before proceeding to the shower area. The clothing is decontaminated prior to laundering or disposal.
  • Supplies and materials are brought into the facility through a double-door autoclave, fumigation chamber, or airlock.
• Containment equipment:
  • All procedures within the maximum containment facility are conducted in the Class III biological safety cabinet, or in a Class I or II biological safety cabinet used in conjunction with one-piece positive pressure personnel suits ventilated by a life-support system.
• Laboratory facilities:
  • The maximum containment facility is to be housed in a separate building or a clearly demarcated and isolated zone within a building. Access to the facility requires outer and inner change rooms separated by showers for entry and exit of personnel, and a double-door autoclave, fumigation chamber, or airlock for the passage of materials, supplies, and equipment.
  • Internal surfaces of walls, floors, and ceilings of the facility should be water, acid, and alkali resistant; the facility should be sealable for fumigation, and animal and insect-proof. Drains in the floor contain traps filled with suitable chemical disinfectant and are connected directly to the liquid-waste decontamination system. Sewer and other ventilation lines contain HEPA filters.
  • Benchtops have seamless surfaces impervious to acids, alkalis, organic solvents, and moderate heat; construction of the facility should have adequate space for accessibility for cleaning.
  • Access doors are self-closing and locking.
  • An individual supply and exhaust air ventilation system that maintains pressure differentials and directional airflow ensures that airflow inwards from areas outside the facility toward areas of highest potential risk within the facility. The supply and exhaust airflow is monitored by manometers to assure inward (or zero) airflow at all times.
  • Exhaust air from the facility is filtered through HEPA filters before discharge to the outside.
  • A specially designed suit area may be provided in the facility entry into which is through an airlock fitted with airtight doors. The air pressure within the suit area is maintained greater than that of adjacent areas. Personnel who enter this area shall wear a one-piece positive pressure suit ventilated by a life-support system. A chemical shower is provided to decontaminate the surface of the suit before the worker exits the area.

Biological Containment

• The growth and dissemination of organisms are naturally limited due to:
  • The infectivity or host specificity of a vector or virus
  • Its spread and survival in the environment
• Example of biological containment strategies:
  • Survival of the vector in its host outside the lab
  • Transmission of the vector from the propagation host to other nonlaboratory hosts
• Purpose of physical and biological containment for research:
  • To prevent unintentional transmission or release of recombinant or synthetic nucleic acid molecules.

Good Lab Practices (GLP)

• GLP embodies a set of principles that provide a framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived (Dolan, 2007).
• Primary purpose of GLP:
  • Ensure uniformity, consistency, and reliability of safety tests (nonclinical) for pharmaceuticals, agrochemicals, aroma and color food/feed additives, cosmetics, detergents, novel foods, nutritional supplements for livestock, and other chemicals.
• GLP is mandatory to evaluate safety or toxicity of products intended to undergo clinical trials.
• Compliance of GLP requires:
  • Tests conducted by qualified personnel.
  • Each study have a Study Director.
  • The lab study and data are audited by a Quality Assurance Unit.
  • All lab activities performed were according to SOPs.
  • All test articles and reagents must be ID, characterized, and labeled.
  • Equipment must be maintained and calibrated.

Standard Operating Procedure (SOP) for General Safety in the Lab

• Wear a lab gown while in the lab.
• Clean your lab area & material/equipment with disinfectant before and after use.
• If you do not understand something, ask.
• Any spillage must be immediately addressed.
• Carefully read the activity and systematically plan the workflow before conducting.

SOP for Biosafety Cabinet Class II

• Decontaminate the BSC surface and the materials to be placed inside (70% EtOH).
• Turn on the blower 10 mins before beginning work; turn off UV lamps when the room is occupied.
• Heat sources are strictly prohibited inside the BSCs (will disrupt the laminar flow of air).
• Keep the work area free of unnecessary equipment or supplies.

Biosafety Cabinet

• A partially enclosed workspace that has built-in protection for the worker, environment, and the material inside.
• Outside air is filtered (HEPA - High-efficiency particulate absorber).

SOP for Laminar Air Flow

• Close the sash and keep the UV on for 20 mins.
• Turn on the blower 10 mins before beginning work.
• Surface sterilize (70% IPA v/v) all materials to be placed inside.
• Keep the work area free of unnecessary equipment or supplies.

Laminar Floodhood (Tissue Culture Hood)

• Enclosed bench designed to prevent contamination of biological samples.

SOP for Waste Disposal

• Decontaminate, and waste should not be stored for more than 1 week.
• Used sharps should be placed in a safety box (close when 3/4 full).

Waste Disposal Containers

• Red Container: Sharps (Blades, Razors, Needles, Staples, Ampules, Glass, Trocars, Others).
• Red Liner/Container: Biohazard (Blood Items, Infectious Waste, Cultures, Medication Vials, Pads, Discarded PPE, Gloves, Gowns).
• Black Container: RCRA Hazard (RCRA Hazardous Meds, RCRA Partial Doses, Hazardous Bulk Meds, P, U, and D-listed chemicals, Medication aerosols and inhalers).
• Yellow Container: Trace/Residual Chemo (IV Tubes, Empty IV bags, Tubes, Stacks & Syringes, Packaging).
• Blue Container: Pharmaceutical (Expired pills, Unused drugs and vaccines, Antibiotics).
• Shielded Containers (w/Radioactive Logo): Cesium, Cobalt, Iodine, Ionizing Radiation, Plutonium, Radium, Uranium.

SOP for Compound Microscope

• Clean the lenses with cotton soaked in xylene (before and after use).
• Observe with both eyes when using a microscope.
• Handle all the parts with care.
• Turn off and cover the microscope when not in use.

SOP for Autoclave Machine

• Sterilization time and temperature are dependent on the composition and nature of items (15 mins at 121°C at 15 lbs pressure min).
• Small items should be bagged or wrapped with a label.
• Sterile packs stored on open shelves are safe to use for up to 1 month (dry and intact).

Autoclave Machine

• Sterilization refers to the complete killing of all living organisms (wet/dry heat, chemicals, and radiation).

SOP for PCR Machine

• Protect the outer sleeves from damage or sharp objects.
• Only the power cord of the machine supplied with the workstation should be used to connect.

PCR Machine (Thermocycler)

• Used to amplify segments of DNA.
• Has a thermal block with holes where tubes can be inserted.

Personal Protective Equipment (PPE)

• To protect against potential chemical exposures.
• It is a supplement and not a substitute for feasible engineering and administrative controls.
• Will vary with the nature of contaminant, route(s) of exposure, and contaminant concentration.

Personal Protective Equipment - Respirators

• May be required when engineering controls are not sufficient to reduce the concentration of air contaminants below an acceptable level.
• Types ranging from filtering face pieces (dust masks) to full face covering, depends on the concentration and form of contaminants (dusts, mists, fumes).

Examples of Respirators

• Filtering half facepiece.
• Surgical N95.
• Elastomeric half facepiece.

Examples of Masks and Facial Coverings

• Face mask, surgical (FDA).
• Comfort mask.
• Facial covering.
• Face mask, non-surgical (FDA).
• Procedure mask.

Personal Protective Equipment - Eye and Face Protection

• Protective eyewear/face shield combination may be needed to prevent projectiles, airborne particles, thermal burns, chemical splashes, vapors, mists, dusts, or radiant energy from damaging the eyes and face.

Personal Protective Equipment - Hand Protection

• Gloves provide protection for the hands from many types of hazards (chemical absorption).
  • Butyl rubber gloves - chemicals except for aliphatic and aromatic hydrocarbons and halogenated solvents
  • Natural (latex) rubber gloves - thin, elastic low barrier protection against acids, alkaline, salts, ketones

Glove Material and Intended Use

Personal Protective Equipment - Continued

• Neoprene gloves - similar in texture and appearance to latex gloves but more resistant. Low barrier protection against hydraulic fluids, gasoline, alcohols, organic acids, and alkalis.
• Nitrile gloves - similar in texture and appearance to latex or neoprene gloves. Made of copolymer and provide protection from chlorinated solvents such as trichloroethylene and perchloroethylene.

Personal Protective Equipment - Body Protection

• Protective body apparel against potential contact through spills or splashes.
• In high-risk splash hazards, the body apparel should fully cover the torso, arms, and legs.
• Low hazard situations - lab coats made of cotton or flame-retardant material.

Examples of Body Protection

• Coveralls.
• Scrubs.
• Reflective Safety Vest.
• Cold Weather Coveralls.
• Lab Coat.
• Chemical Splash Apron.

Fire Emergency - Fire Extinguisher Types

• CLASS A: trash, wood, paper involve ordinary combustible materials (quenched by water or multipurpose (ABC) dry chemicals agent)
• CLASS B: Liquid flammable liquid (quenched by dry chemicals and CO2)
• CLASS C: Electrical equipment (dry chemicals or CO2)
• CLASS D: Combustible metals like magnesium, titanium, zirconium, and lithium (quenched by sodium carbonate, graphite, bicarbonate, NaCl, and salt-based chemicals)
• CLASS K fire: Cooking oil

Fire Classes and Materials

Fire Extinguisher Types and Uses