Biosafety in the laboratories

• Biosafety is the prevention of large-scale loss of biological integrity, focusing both on ecology and human health.

• It is a system for the safe handling of toxic and dangerous biological and chemical substances

• In Medicine-• It refers to the levels of lab containment

protocols, measured as Bio Safety Level (BSL) 1, 2, 3, 4 in rising order of danger

• Safe handling, storage and disposal of-Specimens-Chemicals-Instruments-Radio active components

• Fire safety• Electrical safety

• Disaster management in areas risk prone for natural calamities like Earthquakes, Hurricanes or snowstorms

From-

•Bacteria

•Viruses

•Fungi

•Parasites

•Prions

•Recombinant DNA

Source-•Various specimens•Human blood

•Unfixed tissue

•Human cell lines

To-

•Lab personnel

•Community

Infections of special concern•Tuberculosis•Hepatitis B•HIV•Enteric infections

Routes of infections•Inoculation•Ingestion•Inhalation

• Fundamental objective biosafety program- the containment of potentially harmful biological agents.

• “Containment”- safe methods, facilities and equipment for managing infectious materials in the laboratory environment where they are being handled or maintained.

• The purpose of containment- reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents.

• The use of vaccines may provide an increased level of personal protection.

• Laboratories should recognize hazards of processing infectious agents

• Guidelines should be developed to protect workers in microbiological and medical labs through engineering controls, management policies and standard work practices.

• It is issued under the Environmental (Protection) Act of 1986

• Rules for manufacture, use/import/export and storage of hazardous microorganisms/genetically engineered organisms or Cells, 1989, Dec 5

• There is a new law—the Biotechnology Authority of India (BRAI)—under consideration of the Central government.

1. Consider all the specimens potentially infectious for HIV and other blood borne infections

2. All specimens should be placed in a leak-proof impervious container for transport

3. Use gloves while handling all samples, especially when there is contact with body fluids, non-intact skin or mucous membrane.

4. If there is likelihood of spattering, use face mask with glasses and gowns. Wrap around gowns should be preferred. These should not be used outside the lab.

5. Cover cuts or abrasions present over skin with waterproof bandage.

6. Decontaminate the laboratory work surfaces immediately in case of spillage of blood or any other body fluids7. Follow ‘no needle recapping’ strategy8. All sharps should be collected and disposed away properly.9. Never pipette by mouth. Use mechanical pipetting devices.10. There should always be a system working efficiently for management of hospital generated waste.11. It is advisable for the laboratory personnel to be vaccinated against Hepatitis-B12. Facilities should be available easily for post exposure prophylaxis in case of exposure to HIV & HBV.

• NOT permitted in laboratories:Eating DrinkingStoring food and drinkSmokingHandling contact lensesPipetting by mouth

Do’s•controlled access to the laboratory•Frequent hand wash•Mechanical pipetting•Appropriate waste management & Sterilization and disinfection measures•Training to the workers

Don’ts•Eating, drinking, smoking, handling contact lenses, •storing food•Mouth pipetting

• - Primary barriers

• - Secondary barriers

• Primary barriers: Physical barriers or personal protective equipments for lab worker

•Gloves, masks, Goggles, aprons, suits, special breathing apparatuses

• structural aspects of the laboratory that make working environment safer against infection• Sinks for hand washing

• Special containment areas

• Special air ventilation patterns

• Sterilization equipments

• Tuberculosis• Rubella• Hepatitis B• Typhoid• Cholera• Anthrax• Rabies• Poliomyelitis• Diphtheria

• Precautions to be taken by people researching or trying to identify organisms

• Labs must adhere to these specific safety regulations

• A biosafety level is the level of the bio containment precautions, required to undertake while handling dangerous biological agents in an enclosed facility

• These are the guidelines developed to protect workers in microbiological and medical labs through a combination of safeguards which include-engineering controls-management policies-standard work practices.

• These BSL help you to understand why every lab cannot perform tests for every organism

• Labs are divided into 4 biosafety levels; in which protective practices increase with each level

•Biosafety Level 1 labs - work with least dangerous agents, require fewest precautions•Biosafety Level 4 labs - have strictest methods because dealing with agents that are most dangerous to human health

BSL Laboratory type Laboratory practices Safety equipment

1 Basic teaching, research Good microbiological techniques

None

Open bench work

2 Primary health

services; diagnostic

services, research

Good microbiological techniques,

protective clothing,

biohazard sign

Open bench PLUS

biological safety cabinet for potential aerosols

3 Special diagnostic

services, research

As BSL 2 PLUS

special clothing,

controlled access,

directional airflow

Biological safety cabinet and/or other primary devices for all activities

4 Dangerous

pathogen units

As BSL 3 PLUS

airlock entry, shower exit,

Class III biological safety cabinet, positive pressure suits,

BSL The Micro organisms Remarks

1 canine hepatitis virus, non-pathogenic Escherichia coli, other non-infectious bacteria, B.subtilis

Minimal protection required

2 C. difficile, most Chlamydiae, hepatitis A, B, and C virus, HIV, orthomyxoviruses (other than smallpox), influenza A, Lyme disease, Salmonella, mumps, measles, scrapie, MRSA, and VRSA, B. anthracis

Can cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting

3 Yersinia pestis, Francisella tularensis, Leishmania donovani, Mycobacterium tuberculosis, Chlamydia psittaci, West Nile virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, SARS coronavirus, Coxiella burnetii, Rift Valley fever virus, Rickettsia rickettsii, several species of Brucella, rabies virus, and yellow fever virus

Can cause serious or potentially lethal disease after inhalation in humans but for which treatments DOES exist

4 Bolivian and Argentine hemorrhagic fevers, Marburg virus, Ebola virus, Lassa virus, Crimean-Congo hemorrhagic fever, and various other hemorrhagic diseases

Dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, these cause severe to fatal disease in humans for which vaccines or other treatments are NOT available

• BSL1 - microorganisms that don’t consistently cause disease in healthy adults

•E. coli , polyoma virus

•Basic laboratory

•Standard Microbiological Practices

• Standard practices required: • frequent hand washing• door that can be kept closed when working;• limits on access to the lab space when working; • no smoking, eating, drinking, storage of food in

laboratory;• care to minimize splashes and actions that may create

aerosols (tiny droplets);• decontamination of work surfaces after every use after

any spills;

• Standard practices (continued): • decontamination of laboratory wastes;• use of mechanical pipettes only (no mouth pipetting);• "sharps" precautions, including special containers for

disposing of needles and other sharp objects;• maintenance of insect/rodent control program;• use of personal protective equipment (lab coats, latex gloves,

eye protection or face shields)• Open bench top sink for hand washing

• Agents associated with human disease•Generally required for any human-derived blood, bodily fluids, tissues in which infectious agent may be unknown

•Agents include measles virus, Salmonella species, pathogenic Toxoplasma, Clostridium botulinum, hepatitis B virus

• Primary hazards: • accidental needle sticks• exposure to eyes and nose (mucous membranes)• ingestion of infectious materials

• Agents do not cause lethal infections, are not transmissible via airborne route• (do not cause infection if tiny droplets become airborne and

are inhaled, which might occur if the material were spattered)

• Agents are pathogens for which immunization or antibiotic treatment is available

• Extreme care should be taken with contaminated needles and sharp lab instruments

•  Standard practices include BSL-1 plus: • policies to restrict access to lab;

•biohazard warning signs posted outside lab;• surveillance of laboratory personnel with appropriate

immunizations offered;• biosafety manual with definitions of needed waste

decontamination or medical surveillance policies; • supervisory staff who have experience of working with

infectious agents and specific training for laboratory personnel in handling these agents

• Primary barriers: biosafety cabinets or other approved containment devices• Personal protective equipment: lab

coats, gloves, face protection as needed • Protective clothing removed when

personnel leave laboratory area• Cabinets thoroughly decontaminated

daily and monitored for radiation for personal protection• Secondary barriers: BSL-1 barriers plus

autoclave for glassware

• Also called as biological safety cabinet or microbiological safety cabinet

• It is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level

• BSCs first became commercially available in 1950

CATEGORYCATEGORY TYPE OF WASTETYPE OF WASTE TREATMENT & TREATMENT & DISPOSALDISPOSAL

Category 1Category 1 Human anatomical wastesHuman anatomical wastes Incineration/ deep burialIncineration/ deep burial

Category 2Category 2 Animal wastesAnimal wastes Incineration/ deep burialIncineration/ deep burial

Category 3Category 3 Microbiology & biotechnology Microbiology & biotechnology waste, waste, Liquid wastes, waste Liquid wastes, waste

from Laboratory, blood from Laboratory, blood banks, hospitals, house etc.banks, hospitals, house etc.

Local autoclaving/ Local autoclaving/ microwaving/ microwaving/ incineration/ incineration/

Disinfection by Disinfection by chemicalschemicals

Category 4Category 4 Waste sharps like needles, Waste sharps like needles, syringes, scalpels, blades, glass syringes, scalpels, blades, glass

etcetc

Disinfection Disinfection (Chemical/autoclaving/ (Chemical/autoclaving/

micro waving & micro waving & mutilation/shredding)mutilation/shredding)

Category 5Category 5 Discarded Medicines & cytotoxic Discarded Medicines & cytotoxic drugsdrugs

Incineration/ destruction Incineration/ destruction & disposal in land fills& disposal in land fills

CATEGORYCATEGORY TYPE OF WASTETYPE OF WASTE TREATMENT & TREATMENT & DISPOSALDISPOSAL

Category 6Category 6 Soiled wastesSoiled wastesItems contaminated with Items contaminated with

blood, body fluids including blood, body fluids including cotton, dressings etccotton, dressings etc

Incineration, Incineration, autoclaving, autoclaving, microwavingmicrowaving

Category 7Category 7 Solid wastes like catheters, IV Solid wastes like catheters, IV sets etc.sets etc.

Disinfection by Disinfection by chemical chemical

treatment/autoclavingtreatment/autoclaving/micro waving and /micro waving and

mutilation & mutilation & shreddingshredding

Category 8Category 8 Chemical wastesChemical wastes Chemical treatment & Chemical treatment & discharge into drains discharge into drains for liquid and secured for liquid and secured

land fills for solids.land fills for solids.

• And these 8 categories are again sorted according to special colour coded receptacles for waste of those categories.

• The waste is collected in these colour coded containers and processed further accordingly.

• The following table depicts this categorization.

Colour CodingColour Coding Type of Type of container to be container to be

usedused

Waste Waste Category Category NumberNumber

TreatmentTreatment

YellowYellow Non Chlorinated Non Chlorinated plastic bagsplastic bags

Category Category 1,2,5,61,2,5,6

IncinerationIncineration

RedRed Non Chlorinated Non Chlorinated plastic plastic

bags/puncture bags/puncture proof container proof container

for sharpsfor sharps

Category 3,4,7Category 3,4,7 Disinfection, Disinfection, autoclave, autoclave, microwave, microwave, mutilation & mutilation & shredding, shredding, landfillinglandfilling

BlueBlue Non Chlorinated Non Chlorinated plastic bags plastic bags containercontainer

Category 8Category 8

BlackBlack Non Chlorinated Non Chlorinated plastic bagsplastic bags

Municipal Municipal WasteWaste

• BSL3 - microorganisms that cause serious disease, transmitted by inhalation

•M. tuberculosis, yellow fever virus, hantavirus, Y. pestis (plague)

• Containment lab: double door entry; directional airflow; all work in biosafety cabinet

• Care of patients with tuberculosis starts with a quality assured diagnosis, obtained by growing and identifying Mycobacterium tuberculosis from clinical specimens and conducting DST of the organism to confirm or exclude resistance. Uptake of TB diagnostic technologies requires appropriate laboratory infrastructure and adequate policy reform at country level to enable their effective use in TB screening and diagnostic algorithms

• Laboratory infrastructure, appropriate biosafety measures and maintenance Equipment validation and maintenance Specimen transport and referral mechanisms Management of laboratory commodities and supplies Laboratory information and data management systems Laboratory quality management system are a priority.

• With growing incidences of MDR-TB and XMDR-TB it is highly essential all Microbiology laboratories must install Grade 3 Biosafety cabinets to prevent exposure to Infection. If necessary precaution's are not taken a fraction of Medical and Technical personal will be infected with grave consequences.

• Pathogenic agents are grouped in Risk group 3 which can cause serious diseases

• Effective treatment and preventive measures are usually available

• Little person-to-person spread

• Standard practices include BSL-2 plus: • strictly controlled access to the lab;• specific training for lab personnel in handling potentially lethal agents;•decontaminating all waste;•changing contaminated protective lab clothing, decontaminating lab clothing before laundering;• institutional policies regarding specimen collection and storage from workers to prevent exposure

• Primary barriers:• Similar to BSL-2 personal protective equipment• Respiratory equipmentSecondary barriers:• All BSL-2 barriers• Corridors separated from direct access to lab• Access through self-closing double doors• Air handling systems to ensure negative air flow (air flows into the lab)• Air pumped into lab not re-circulated in building

• BSL4 - microorganisms that cause lethal disease, with no known treatment or vaccineEbola virus, Marburg

virus Maximum

containment lab; positive pressure ventilated suits (moon suits)

• Pathogenic agents are grouped in Risk group 4 which is lethal

• Readily transmittable-direct or indirect

• Effective treatment and preventive measures are not usually available

• Dangerous and exotic agents with high risk of life-threatening disease and are aerosol-transmittion• Related agents with unknown risk of

transmission• Agents (all viruses) include Marburg virus, Ebola virus, viruses that cause Congo-Crimean hemorrhagic fever, Lassa fever

• Primary hazards: • respiratory exposure to infectious aerosols•mucous membrane exposure to infectious droplets• accidental sticks with needles or other sharp objects contaminated with infectious material

• For example• In late 1960s, 25 laboratory-acquired Marburg infections, including 5 deaths• Affected workers studying infected monkeys from Uganda

• Personnel must receive specialized training in handling extremely dangerous infectious agents, containment equipment and functions

• Access to lab is restricted: immunocompromised persons are never allowed to enter the lab

•  Standard practices include BSL-3 plus: •More strictly controlled access to the laboratory;• Changing clothing before entering and exiting lab (showering upon exiting recommended);• Decontaminating all material exiting facility

• Primary barriers:• Biosafety cabinets used at other biosafety levels• Full-body, air-supplied, positive pressure personnel suit

• Secondary barriers:• All physical barriers at BSL-3• isolated zone or a separate building;• dedicated supply and exhaust, vacuum, decontamination systems; • a recommended absence of windows (or sealed and resistant to breakage)

• BSL-1: high schools, community colleges, municipal drinking water treatment facilities

• BSL-2: local health departments, universities, state laboratories, private laboratories (hospitals, health care systems), industrial laboratories (clinical diagnostic companies)

• BSL-3: state health departments, universities, private companies, industry, federal government (NIH, CDC)

• BSL-4: only 15 facilities in the US• 9 federal (CDC, NIH), 4 university (Georgia State University,

University of Texas Medical Branch), 1 state, 1 private• Renovations underway at several labs, new facilities proposed

at additional sites

• All India Institute of Medical Sciences, New Delhi

• Microbial Containment Complex, Pune

• Centre for Cellular and Molecular Biology, Hyderabad

• High Security Animal Disease Laboratory (HSADL), Bhopal

• Four standard biosafety levels are also described for activities involving infectious disease work with commonly used experimental animals.

• Acharya, D.B. and Singh, The book of hospital waste management 2000• World Health Organization. Laboratory biosafety manual. 3rd ed.

Geneva; 2004• http://www.who.int/injection_safety/toolbox/docs/

AM_HCW_Safety.pdf• WHO guidelines on laboratory training for Field Epidemiologists• "Biohazard Symbol History".the original on July 16, 2011.• Biosafety in Microbiological and Biomedical Laboratories, 5th Edition,

U.S. Department of Health and Human Services, Public Health ServiceCenters for Disease Control and Prevention National Institutes of Health, HHS Publication No. (CDC) 21-1112Revised December 2009

• UNC School of Public Health Laboratory Safety Levels• U.S. Centers for Disease Control and Prevention; U.S. National Institutes

of Health (2000). Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets. U.S. Centers for Disease Control and Prevention.