Laboratory Methods & Laboratory Safety

Standard Precautions

assume all specimens are infectious

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Hierarchy of controls

1. Standard Precautions

2. Hierarchy of controls

3. Risk assessment before procedure

4. Reporting and documenting incidents

4 Principles of Laboratory Safety

BSL-1

non-pathogenic organisms, basic PPE

BSL-2

moderate risk agents; use biosafety cabinet for aerosols

BSL-3

airborne pathogens; directional airflow, restricted access

BSL-4

high-risk; full containment suits, specialized facilities

1. Gown/Lab coat

2. Mask or respirator

3. Eye protection/ face shield

4. Gloves

Donning (Putting On) PPE – Correct Sequence

1. Gloves

2. Gown/Lab coat

3. Hand hygiene

4. Eye protection/ face shield

5. Mask or respirator

6. Final hand hygeine

Doffing (Removing) PPE – Correct Sequence

Latex gloves

Good elasticity, tactile sensitivity; avoid if allergic.

Nitrile gloves

Suitable for low-risk, short-duration tasks; less protective.

Vinyl gloves

Suitable for low-risk, short-duration tasks; less protective

Extended cuff gloves

Use when working with large volumes of liquids or high-contamination risk.

handling blood from hepatitis or HIV-positive patients

High risk scenarios requiring double-glove

Surgical Masks

Protect against droplets (≥5 μm).

Surgical Masks

Used in BSL-1 or BSL-2 labs for standard microbial work.

Surgical Masks

Protect the specimen from contamination by the wearer’s respiratory secretions

Respirators (e.g., N95, P100, PAPRs)

Protect against airborne particles (<5 μm) and aerosols.

Respirators (e.g., N95, P100, PAPRs)

Required in BSL-3 or higher, or when working with:

– Mycobacterium tuberculosis

– SARS-CoV-2 in aerosol-generating procedures

– Any unknown respiratory pathogen with airborne transmission potential

Respirators (e.g., N95, P100, PAPRs)

Must be fit-tested and seal-checked before use.

Non-pathogenic, well-characterized

AGENT OF BSL-1

Minimal

Risk Level of BSL-1

Standard Laboratory

Facility requirement for BSL-1

Standard microbiological practices, PPE (gloves, lab coats)

Practices and Safety Equipment for BSL-1

Pathogenic

AGENT OF BSL-2

Moderate

Risk Level of BSL-2

Limited access, biohazard signs

Facility requirement for BSL-2

BSCs for aerosols, enhanced PPE, sharps precautions

Practices and Safety Equipment for BSL-2

Indigenous or exotic

AGENT OF BSL-3

Serious

Risk Level of BSL-3

Controlled access, negative air pressure

Facility requirement for BSL-3

BSCs for all activities, respirators, controlled access

Practices and Safety Equipment for BSL-3

Dangerous and exotic

AGENT OF BSL-4

High

Risk Level of BSL-4

Isolated facility, full-suit protection

Facility requirement for BSL-4

Full-body air-supplied suits, extensive decontamination

Practices and Safety Equipment for BSL-4

Green

Color indicator for BSL-1

Yellow

Color indicator for BSL-2

Orange

Color indicator for BSL-3

Red

Color indicator for BSL-4

Bloodborne Pathogens Standard

Protects workers from health hazards presented by bloodborne pathogens. Requires exposure

control plans and proper PPE usage.

General Duty Clause

Requires employers to provide a safe workplace by removing recognized hazards, including biological hazards, that can cause serious harm or death.

Biosafety in Microbiological and Biomedical Laboratories (BMBL)

A comprehensive manual detailing biosafety levels, practices, equipment, and facility requirements. Widely adopted as the primary guide for biosafety in labs.

NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

Guidelines for safe handling of genetically engineered organisms and viruses, to prevent misuse and ensure safety in genetic research.

Resource Conservation and Recovery Act (RCRA)

Governs the disposal of biohazardous and other hazardous wastes, ensuring environmentally safe management and disposal practices

OSHA

Body of Bloodborne Pathogens Standard

OSHA

Body of General Duty Clause

CDC & NIH

Body of Biosafety in Microbiological and Biomedical Laboratories (BMBL)

NIH

Body of NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

EPA

Body of Resource Conservation and Recovery Act (RCRA)

Biological Safety Cabinets (BSCs)

Enclosures that prevent airborne contaminants from escaping into the laboratory environment. They are essential for work with toxic vapors or particulate matter that might be infectious.

HVAC Systems with HEPA Filters

High-efficiency particulate air (HEPA) filters in ventilation systems ensure that any air exiting the lab is free of infectious agents.

Autoclaves

Used for sterilizing equipment and deactivating biohazards before disposal.

Pass-Through Boxes

Sealed containers that allow materials to be passed into and out of containment areas without direct exposure.

Training Programs

Regular training on the proper handling of biohazards and response procedures for potential exposure or spills.

Access Restrictions

Limiting laboratory access to trained and authorized personnel only

Standard Operating Procedures (SOPs)

Detailed procedures for safely working with biohazards, which include specific instructions on using, storing, and disposing of biological agents safely.

Emergency Response Plans

Protocols that outline steps to be taken in the event of an accidental spill or exposure, including communication strategies and remediation measures.

Gloves

Protect hands from exposure to hazardous materials and prevent the transfer of contaminants.

Gowns or Lab Coats

Provide body protection from spills and splashes of infectious or toxic substances.

Respirators or Masks

Essential for preventing inhalation of infectious aerosols, particularly in BSL-3 and BSL-4 settings

Face Shields or Goggles

Protect the eyes and face from splashes or sprays of hazardous substances.

Transport & Accessioning

Use leak-proof, labeled secondary containers with biohazard bag

refrigeration or ambient as required

Temperature control for transport & accession

Legal/forensic samples

Proper chain of custody is needed for what sample?

verify labels, assign lab accession number, log details

Proper Accessioning

20 sec, soap & water or alcohol-based rub

Proper hand hygiene

Surface disinfection

contact time, dilution for bleach

evacuate, disinfect, material-specific protocol

Proper handling spills

never recap needles & use puncture-resistant containers

Sharps safety?

Internal QC

controls, calibration, maintenance schedules

External QA

proficiency testing, inter-lab comparisons

Documentation

QC logs, corrective actions, preventive maintenance

Centrifuges

balance loads, use sealed rotors for infectious material

Autoclaves

verify cycle parameters with biological indicators

Pipettes

regular calibration and correct technique

Microscopes

cover when not in use; service schedules

spills, exposures, fires, needlestick injuries

Incidents that require immediate action

first aid, reporting, testing, prophylaxis

What are the post-exposure protocols?

Perform root cause analysis

How do you maintain incident log?

Handle, Use, Waste, Label

Microscopy & Staining— Safe Practices

Wear, Keep, Ask, Practice

Practical Tips for Students