Laboratory Biosafety and Good Laboratory Practices Notes

Laboratory Biosafety and Good Laboratory Practices

Intended Learning Outcomes

Execute laboratory safety practices and SOPs.
Enumerate protocols to ensure biosafety at international and national levels.

Introduction

Early rDNA technology development relied on GMT (good microbiological techniques).
June 1976: Asilomar Conference focused on ensuring safety in biotechnology.
NIH Guidelines for Research Involving Recombinant Nucleic Acid Molecules were established.

WHO Manual

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

Importance of International Standards

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

Risk Categories of Microorganisms

Based on NIH guidelines and WHO Manual.
Four categories based on infection risk to laboratory workers and the community.

Assigning Microorganisms to Risk Categories

Pathogenicity of the organism.
Host range and mode of transmission.
Local availability of effective preventive measures.
Local availability of effective treatment.

Risk Groups (RG) of Microorganisms

RG 1: Unlikely to cause human or animal diseases; little to no risk.
RG 2: Pathogenic but unlikely to pose serious hazard; effective treatments available; moderate risk to individual, low risk to community.
RG 3: Pathogenic, can cause serious diseases, but not contagious or have effective treatment; high risk to individual, low risk to community.
RG 4: Causes serious diseases, readily transmitted, no effective treatment; high risk to individuals and community.

Biosafety Levels (BLs)

Four BLs corresponding to the four RGs.
Relies on: Standard practices of GMT; Physical barriers.

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

Different biosafety levels require different laboratory practices and safety equipment.

Physical Containment

Strict adherence to good microbial practices.
All personnel working with recombinant or synthetic nucleic acids should be trained in GMT.

Levels of Physical Containment

BL1 to BL4 representing facilities of increasing potential hazard.
Standard microbiological practices; Special practices; Containment equipment; Laboratory facilities.

Biosafety Level Requirements

Isolation of laboratory, room sealability, ventilation, and air filtration vary by level.
Autoclave availability and biological safety cabinet use increase with BL.

Biosafety Level 1

Limited access, daily decontamination, no mouth pipetting, and basic hygiene.
Contaminated materials transported in leak-proof containers.
Insect and rodent control required; easy-to-clean facilities with sinks.

Biosafety Level 2

As per BL1, plus limited access, hazard warning signs, and protective clothing.
Needle-locking syringes, biosafety manual, and baseline serum samples.
Biological safety cabinets (class I or II) required; autoclave needed for decontamination.

Biosafety signs

Signs must include the universal biosafety symbol, agent details, PI contact information, and entry requirements.

Hazards vs Risks

HAZARD: potential to cause harm.
RISK: likelihood of a hazard causing harm.

Hazard identification

Identify pathogens or associated foods.

Hazard Characterization

Determine what happens when a pathogen is ingested and how much causes illness.

Exposure Assessment

Assess the probability of eating contaminated food and the number of pathogens likely in the food.

Risk Characterization

Understand the nature and likelihood of health risks, who is likely to become ill, and sources of variability and uncertainty.

Biosafety Level 3

As per BL2, plus no entry for those under 16, and closed lab doors.
Protective lab clothing, masks for animal rooms, and HEPA filters for vacuum lines.
Spills reported to Biosafety Officer and NIH; Maintain written records for medical evaluation.
Separated laboratory with double doors and water-resistant surfaces; HEPA-filtered exhaust air.

Biosafety Level 4

As per BL3, plus restricted access with locked doors and entry/exit log.
Materials sealed in nonbreakable containers, and autoclaved before removal.
Personnel enter/exit through clothing change and shower rooms; supplies enter via autoclave or airlock.
All procedures in Class III BSC or Class I/II BSC with positive pressure suits; separate building or isolated zone.

Biological Containment

Limits growth and dissemination of organisms naturally.
Strategies include limiting vector survival and transmission.

Purpose of Containment

Prevent unintentional transmission or release of recombinant or synthetic nucleic acid molecules.

Good Lab Practices (GLP)

Framework for planning, performing, monitoring, recording, reporting, and archiving laboratory studies.

Primary Purpose of GLP

Ensure uniformity, consistency, and reliability of safety tests (nonclinical).
Mandatory for evaluating safety/toxicity of products for clinical trials.

Compliance of GLP

Tests by qualified personnel, a Study Director, and auditing by a Quality Assurance Unit.
All activities follow SOPs, and test articles/reagents are identified, characterized, and labeled.
Equipment maintained and calibrated.

Standard Operating Procedures (SOPs)

Wear lab gown, clean area before/after use, ask questions if unsure, and address spills immediately.

SOP for Biosafety Cabinet Class II

Decontaminate surface with 70% EtOH, turn on blower before work, and turn off UV lamps when the room is occupied.
No heat sources inside; keep work area clear.

Biosafety Cabinet

Enclosed workspace with built-in protection using HEPA filtration.

SOP for Laminar Air Flow

Close sash, turn on UV for 20 mins, then turn on blower before work.
Surface sterilize all materials with 70% IPA; keep work area clear.

SOP for WASTE DISPOSAL

Red container = sharps
Red liner container = biohazard
Yellow container = trace/residual chemo
Black container = RCRA Hazard
Blue container = pharmaceutical
Shielded container = Radioactive

Compound Microscope Usage

Clean lenses with xylene, observe with both eyes, handle parts carefully, and cover when not in use.

SOP for Autoclave Machine

Sterilize at 121°C at 15 lbs pressure for 15 mins minimum.
Label small items and store sterile packs safely (1 month if dry and intact).

Autoclave

Sterilization: Complete killing of all living organisms (wet/dry heat, chemicals, radiation).

SOP for PCR Machine

Protect sleeves from damage; use the supplied power cord.

PCR Machine

Thermocycler used to amplify segments of DNA.

Personal Protective Equipment (PPE)

Supplement to engineering and administrative controls.
Varies with contaminant nature, exposure routes, and concentration.

Respirators

Required when engineering controls are insufficient.
Types range from filtering face pieces to full face coverings depending on contaminants.

Eye and Face Protection

Needed to prevent projectiles, particles, burns, splashes, vapors, mists, dusts, or radiant energy damage.

Hand Protection

Gloves protect from chemical absorption.
Butyl rubber: chemicals except hydrocarbons.
Latex: low barrier protection against acids, alkaline, salts, ketones.
Neoprene: resistant to hydraulic fluids, gasoline, alcohols, organic acids, and alkalis.
Nitrile: protects from chlorinated solvents.

Body Protection

Protective apparel against spills or splashes.
Lab coats (cotton or flame-retardant) for low hazard; full coverage for high-risk splashes.

Fire emergency

CLASS A: trash, wood, paper (water or ABC dry chemicals).
CLASS B: flammable liquid (dry chemical and $CO_2$ ).
CLASS C: electrical equipment (dry chemicals or $CO_2$ ).
CLASS D: combustible metals (sodium carbonate, graphite, NaCl).
CLASS K: Cooking oil