LABORATORY BIOSAFETY

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Potential Laboratory Hazards

Chemicals Includes different classes of hazardous chemicals

Biohazards Cells, animals, biological / patient samples, viruses,

bacteria

Allergens Chemical, animal, latex

Radioactive Material

Physical / Equipment Hazards

Electrical, sharps, hi/low temperature and pressure

Mixed hazards & Multiple simultaneous hazards

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Definitions

Biohazard: Any agent of biological origin that has the capacity to produce harmful effects in humans.

Biosafety: Safety against biohazards

A laboratory acquired infection is defined as one that resulted from laboratory work, whether it occurred in a laboratory worker or in another person who happened to be exposed as a result of research or clinical work with infectious agents.

Antiseptic :– A substance that inhibits the growth and development of micro-organisms without necessarily killing them. Antiseptics are usually applied to body surfaces.

Disinfectant :– A chemical or mixture of chemicals used to kill microorganisms, but not necessarily spores. Disinfectants are usually applied to inanimate surfaces or objects.

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Definitions…cont Biocide – A general term for any agent that kills

organisms.

Sporocide – A chemical or mixture of chemicals used to kill microorganisms and spores.

Decontamination – Any process for removing and/or killing microorganisms. The same term is also used for removing or neutralizing hazardous chemicals and radioactive materials.

Disinfection – A physical or chemical means of killing microorganisms, but not necessarily spores.

Sterilization – A process that kills and/or removes all classes of microorganisms and spores.

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What is a biohazard Any agent of biological origin that has the capacity to

produce harmful effects in humans. Examples of biohazards:

Micro-organsims such as bacteria, viruses, fungi and parasites and their toxins.

Blood and body fluids as well as tissues from humans and animals.

Transformed cell lines and certain types of nucleic acids.

Every infectious microbial agent which has been studied in the laboratory has caused infection in lab personnel

Fewer than 20% of all LAIs wereassociated with a known accident. 80% go unknown or unrecognized

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Organisms proved to have been transmitted in the lab

Q fever Brucellosis Typhoid fever Hepatitis Tularemia Tuberculosis Dermatomycosis Venezuelan equine

encephalitis Typhus Psittacosis Coccidioidomycosis. Leptospirosis

C. difficile E. coli O157:H7 N. meningitidis Salmonella Shigella HIV HBV HCV

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Route Lab Practices and/or Accidents

Protection

Inhalation Procedure s that produce aerosols vapours, dust, mists, gases and biological agents

fume hood, masks, respirator or BSC as appropriate

Ingestion Mouth pipettingSplashes into mouthContaminated hands /utensilsEating, drinking, smoking etcLeaking contaminated items

NO eating or drinking or makeup application in the lab. NO mouth pipetting

Inoculation Needle stick injuryCuts from sharpsAnimal and insect bites

limit use of sharps and if possible handle indirectly with forceps. Use sharps container

Other - contact

Spills and splashesContact with contaminated itemsTransfer by hand –to-face actions

Standard Precautions and GMP

Epidemiology of laboratory acquired infections

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CLASSIFICATION OF INFECTIVE MICROORGANISMS

WHO recommends that the Health Authorities of every country make lists of organisms in the different risk groups, relevant to the local circumstances , so that appropriate precautions may be applied.

Risk group 1 :

Organisms in this group represents low risk to the lab worker & to members of the community. They are unlikely to cause human disease. (e.g. saprophytic bacteria, animal/plant organisms)

Risk group 2 : Organisms in this group offer a moderate risk to the lab

worker limited risk to members of the community.

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CLASSIFICATION OF INFECTIVE MICROORGANISMS…cont

Risk group 2 ….cont They can cause human disease but preventive measures

& treatment /vaccines etc are available risk of spread in the community is not great E.g : staph, strep., enterobacteriaceae, clostridia, vibrio,

polio, coxsackie & hepatitis viruses.

Risk group 3 : Presents high risk to Laboratory worker Low risk to community Vaccines & treatment available for most pathogens Eg ; Brucella, Mycobacterium tb, S. typhi, Pasteurella,

Francisella, many arboviruses, Ricketttsiae , Chlamydia, HIV.

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CLASSIFICATION OF INFECTIVE MICROORGANISMS…cont

Risk group 4 : High risk to Lab worker & community. Cause serious disease & spreads easily & rapidly

in the community No vaccines or chemotherapy agents available. E.g : Hemorrhagic fever viruses including

Marburg, Lassa, Ebola, & some encephalitis & arboviruses.

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CLASSIFICATION OF LABORATORIES

Basic Laboratory (Bio-safety level 1) Standard Good Laboratory practices (GLP)

required Simplest kind – BSCs not required.

Basic Laboratory (Bio-safety level 2) Suitable for Risk group 2 GLP as outlined with all the standard

precautions Training Lab personnel in handling infectious

organisms & suspensions. BSCs ( Class 1 or Class 2) in addition to all other

equipment including autoclaves Employee health policy

(GLP includes aseptic tech, personal hygiene area clean-liness, following standard precautions and PPE, etc)

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CLASSIFICATION OF LABORATORIES…cont

Containment Laboratory (Biosafety Level 3)

Suitable for Risk group 3 Controlled access by authorized staff only Special Lab design & engineering & HVAC with

careful control of air movement. Wearing of special protective clothing &

accessories. Laminar flows, Biological safety cabinets - class

2 Effective Employee Health policy with baseline

sera stored for comparison with acute sera.12vivek bhat tmc

CLASSIFICATION OF LABORATORIES…cont

Containment Laboratory (Biosafety Level 4) Suitable for Risk group 4 Includes all facilities of Level 3 plus

Separate buildings/units with strict controlled access through air locks & exit decontamination showers & pressure gradient rooms.

Special training for staff. Requirement of BSC Class 3 Effective Employee Health Policy with baseline

sera stored for comparison with acute sera. Most are Research/ Defense Laboratories

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BiologicalSafety

Cabinet

SterilGARD® III Advance, The Baker Company14

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Use of Biological safety cabinets

The use and limitations of biological safety cabinets should be explained to all potential users. Written protocols or safety or operations manuals should be issued to staff.

In particular, it must be made clear that the cabinet will not protect the operator from spillage, breakage or poor technique.

Do not use the cabinet unless it is working properly.

Do not open the glass viewing panel when the cabinet is in use.

Apparatus and materials in the cabinet must be kept to a minimum. Air circulation at the rear plenum must not be blocked.

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Use of Biological safety cabinets ….cont

Bunsen burners must not be used in the cabinet. The heat produced will distort the airflow and may damage the filters. An electric micro-incinerator is permissible but sterile disposable transfer loops are better.

All work must be carried out in the middle or rear part of the working surface and be visible through the viewing panel.

Traffic behind the operator should be minimized. Air grills must not be blocked with notes, pipettes or other

materials, as this will disrupt the airflow causing potential contamination of the material and exposure of the operator.

Wipe the surface of the BSC using an appropriate disinfectant after work is completed and at the end of the day.

The cabinet fan should be run for at least 5 min before beginning work and after completion of work in the cabinet.

Do not place paperwork inside BSCs.

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Safe laboratory environment

Use of Biohazard and safety symbols

Protective clothing : Overalls, lab coats to be worn over normal clothing

to protect from splashes, droplets containing micro-organisms/chemicals.

Waterproof aprons may be worn when indicated, poly-cottons & flammable – resistant fabric is ideal.

Laundering regularly, soiled clothing is placed in a special bag & disinfected with 1% v/v Na Hypo-chlorite & then sent to laundry. (or sent and processed as per CSSD protocol)

Gloves must be worn when handling specimens of blood or body fluids or cultures & while washing lab ware.

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How do you know when to use personal protective equipment? Wear gloves when

you anticipate being in contact with blood or body fluids.

Wear protective eyewear, masks, and gowns in addition to gloves when you think splashing of blood or body fluids could occur.

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Wear gloves when there is reasonable likelihood of hand contact with blood or other potentially infectious materials, mucous membranes or non-intact skin; when performing vascular access procedures; and when handling contaminated items or surfaces. Change disposable gloves when they become contaminated, torn or punctured. Wash hands before donning AND after removing gloves.

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Eye Protection

Z87 Type Goggles

O29

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Vinyl synthetic examination gloves: tasks that involve minimal stress on the glove. low risk of exposure to blood and other potentially infectious

materials. Consideration needs to be given to manipulation and other

stresses placed on the glove material.

Suggested use: For changing bed linens. for briefly suctioning endo-tracheal secretions; for emptying emesis basins; For discontinuing an IV line; and for handling and preparing food.

Not recommended: When there is moderate to high risk of exposure to blood or

body fluids; for preparing, handling or administering chemotherapeutic agents.

for handling chemicals or other caustic agents; for performing environmental services or housekeeping duties; If sensitivity or clinical reactivity to vinyl compounds.

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Natural rubber latex examination gloves the gold standard in barrier protection. Natural rubber provides dexterity, tactile sensitivity,

flexibility and durability. preferred for procedures and tasks considered moderate to

high risk for exposure to blood and other potentially infectious materials and when a non-sterile hand covering is indicated.

Latex exam gloves may need to be changed every 15 to 30 minutes depending on the task or procedure, the amount of blood and fluid exposure and the contact with needles and other sharp instruments.

Suggested use: For direct patient care involving exposure to blood or other potentially infectious materials and for contact with blood and body fluid specimens or items

contaminated with blood or body fluids.

Not recommended: For individuals with a known or suspected allergy or clinical reactivity to natural rubber latex

protein and for prolonged contact with high-level disinfectants, such as

gluteraldehyde.

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Nitrile examination gloves: Preferred choice for those individuals who have a

sensitivity or clinical reactivity to latex AND may be at moderate to high risk of exposure to blood and other potentially infectious materials.

Nitrile gloves typically have better chemical resistance than natural rubber latex, especially to hydrocarbon-based products (e.g., products containing mineral oil, petrolatum or lanolin).

Suggested use: For personnel who are allergic or sensitive to latex and who perform tasks or procedures. involving prolonged exposure to blood, body fluids,

chemo-therapeutic agents, cleaning solutions and other chemicals.

Not recommended: For individuals who have Sensitivity or clinical reactivity to nitrile compounds. 32vivek bhat tmc

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Safe laboratory environment….cont

l) Personal health & safety measures ….cont

Covering any cuts, sores, wounds etc with water proof dressing

Do Not wear lab coats (and gloves) in the following areas: All offices, bathrooms, elevators, public hallway Coffee/ lunch rooms, departmental libraries Student carrel area outside of the lab Other non-lab areas of the building.

No licking gum labels, putting pen/pencil in mouth/hair.

Remove your gloves before using instruments, telephone, and leaving the laboratory

Always wash your hands before you leave and especially before eating.

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Laboratory hygiene Don’t apply cosmetics in

lab

Avoid wearing jewellary. (pendants, necklaces, bracelets

Don’t touch your face, mouth or eyes without washing hands

Not eating, drinking, chewing gum, smoking, applying cosmetics or sitting on lab benches.

No storing food/drink in lab refrigerators. 36vivek bhat tmc

Hand Hygiene Techniques1. Alcohol hand rub: 20 to 30 sec. (60 - 95% ethanol or

isopropyl alcohol)

2. Rapid decontamination of hand: no visible soiling 15 sec. (chlorhexidine 1% in alcohol)

3. Hand wash procedure (before Aseptic procedures): 1 minute (chlorhexidine - 2% handwash)

4. Surgical wash: 3-5 minutes (chlorhexidine - 4% handwash)

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Areas Most Frequently Missed

HAHS © 1999Distribution of areas missed during hand washing 38

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Do staff wash their hands enough ? No

• Wards 16 – 48%• NICU 29%• ICU 30 – 81%

Why

Don’t Staff Wash their Hands

(Compliance estimated at less than 50%)

• Skin irritation• Inaccessible hand washing facilities• Wearing gloves• Too busy• Lack of appropriate staff• Being a physician

(“Improving Compliance with Hand Hygiene in Hospitals” Didier Pittet. Infection Control and Hospital Epidemiology. Vol. 21 No. 6 Page 381) 41

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SPECIFIC MICROBIAL HAZARDS

Aerosolization: An aerosol ( 1—3 µ) is a suspension of

fine solid particles or liquid droplets in a gas

Small droplets evaporate & leave behind droplet nuclei consisting of bacteria or viruses which are too light to settle & they move around by air currents & ventilating systems.

Particles >10 µm diameter are filtered in nose, smaller ones especially < 10 µm are inhaled right into the lungs where they initiate infection

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MICROBIAL HAZARDS….cont

Aerosolization results from Pouring supernatant fluids (from a height into a

discarding container). Vigorous tapping of the tube to re-suspend a

sediment. Opening cultures & rapid snap-closing of

specimens or cultures. Heating a contaminated wire loop in an open

burner flame. Using a long springy loop. Rapid rinsing of Pasteur pipettes. Open centrifugation, opening lid before rotation

stops, opening centrifuge immediately following the breakage of tube before aerosols settle.

Mouth pipetting & blowing out fluids (especially last drop)

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PipettesRaymond A. Lamb, LLC

VWR International

Beckman Coulter, Inc.

Aerosol ProducersVortex

Tubes

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WORKING SAFELYPrevent aerosolization Pouring infectious material safely

Fluids should be poured carefully down the side of a funnel emptying in disinfectant in a jar.

Opening cultures & ampoules safely Liquid films containing microorganisms form

between the rims & bottoms of Petri dishes & rims & stoppers of bottles & tubes. they must be separated carefully preferably in a safety cabinet.

Inoculating loops & safe looping out Loops must be fully closed - of smaller diameter -

length of wire must be short (< 6mm) Use hooded burners or safety cabinets Cool the loops before use.

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WORKING SAFELY….contShaking & homogenizing safely

Capping the culture containers tightly while shaking, vortex mixer may be used.

Avoiding infection from spillages & breakages Specimens, culture tubes & bottles should always be

placed in racks so that they cannot fall over & spill their contents.

Spilled material & broken culture vessels should be covered with a cloth soaked in disinfectant, left for 30 min & then cleaned up using a metal dust pan & stiff card board

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WORKING SAFELY….contSafe pipetting & dispensing Mouth pipetting has been the cause of many

infections in lab personnel, some of which have been fatal.

Serious chemical injuries may occur with chemicals.

WHO recommends that mouth pipetting be banned.

Low cost pipette fillers are available commercially.

Air should never be blown through a liquid containing infectious agents.

Infectious materials should not be mixed by alternate suction and expulsion

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How do Needlestick Injuries Occur?Disposing of needlesOverfilling containerEmptying sharps container rather than disposing once filledAdministering injectionsDrawing bloodRecapping needles.Handling trash and dirty linens. Improper disposal in regular garbage

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Sharp injury -POA Wash the area thoroughly with 2% chlorhexidine antiseptic

soap solution and water. Skin sites and wounds that have been in contact with blood/body

fluids shall also be immediately washed as above. Do wash mucosal surfaces like mouth or conjunctiva with warm

water or saline.

Do not scrub the affected area. Do not suck the area. Do not apply any caustic agents (hypochlorite etc); Do not inject antiseptic/disinfectant into the wound. Do not apply tight bandages.

Baseline titres for HIV, HBsAg, HCV, Anti- HBs done depending on vaccination history.

ART, HB- Ig, HBV vaccine etc provided on case to case basis.

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Exposure to Human Blood or Body FluidsIf you are exposed to human blood or body fluids through acut, stab or splash:– First Aid - wash immediately with soap and water (5 min)– Eyes flush with water for 10 min– Cuts, stab scrub area to bleed– Remove gloves or clothing if requiredReport accident and seek medical aid and follow-up

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Spills outside of a containment device

The spill is not inside of a Biological Safety Cabinet (BSC), Centrifuge, Refrigerator, Incubator, Freezer, Lab instrument etc.

1. Close off spill area to traffic, and notify coworkers.

2. If the spill may involve an aerosol, (e.g. event involving dropping material onto floor, high mechanical force, a forceful expulsion of liquid) leave the room for 30 minutes to allow aerosols to settle.

3. Remove contaminated lab coat or clothing and wash exposed skin.

4. Put on clean gloves and lab coat.

5. Prepare enough volume of a 1:10 dilution (to give 5g/l available chlorine) of chlorine bleach or other approved disinfectant to saturate the contaminated area. If dilution is not possible, undiluted household bleach can be used. However eye protection must be worn. 66vivek bhat tmc

Spills outside of a containment device ……cont

1. Contain the spill with paper towels or other absorbent material

2. Flood the spill area with disinfectant. Leave on for 10 - 30 min.

3. Push the absorbent material at the edge of the spill into the spill's center. Add more paper towels as needed. If glass is present, do not use bare hands! Use tongs (large pieces) forceps (small pieces) followed by a dustpan to remove pieces.

4. Discard the paper towels into a regulated medical waste container. If contact with bleach occurs with skin, mucous membranes or eyes, flush area with copious amounts of water.

5. Discard gloves into regulated medical waste container. Wash hands thoroughly. Autoclave an overtly contaminated lab coat.

6. Report incident to supervisor.

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A general all-purpose laboratory disinfectant should have a concentration of 1 g/l available chlorine. A stronger solution, containing 5 g/l available chlorine, is recommended for dealing with bio-hazardous spillage and in the presence of large amounts of organic matter. Sodium hypochlorite solutions, as domestic bleach, contain 50 g/l available chlorine (5% w/v) and should therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l and 5 g/l, respectively. (tap water for dil- freshly prep soln). 1: 5 dilution ( 1% hypochlorite) may also be used for dirty conditions ( as we do in TMC)Industrial solutions of bleach have a sodium hypochlorite concentration of nearly 120 g/l and must be diluted accordingly to obtain the levels indicated above.

General lab disinfectant

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Gluteralde--hyde

2.0 – 3.2%

High/CS + + + + + + -- + + +

Hydrogen peroxide

3.0 – 25%

High/CS + + + + + ± ± -- + +

Chlorine 1000ppm Cl-

High + + + + + ± + + + +

Isopropyl alcohol

60 – 95%

Int + + + ± + -- ± -- + +

Phenolic compounds

0.5-5.0%

Int + + + ± + -- -- + + +

Iodophors 50ppm free I

Int + + + + ± -- ± + + +

Q.A.Cs 0.4 – 1.6%

Low ± + ± -- -- -- -- + + +

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CBWTF- landfill afterstabilization Or Recycling

Mercury Spillcleanup

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Spills in a centrifuge. Biohazardous spills in centrifuges can be quite difficult to

disinfect. Some but not all centrifuges have closed rotors, buckets or other carriers with leak proof lids, designed to contain spills and allow efficient, safe emptying and decontamination. However, not all centrifuges are equipped with these containment devices.

If unusual sounds from a centrifuge suggest that breakage and a spill has occurred, or, if breakage and a spill is discovered after the machine has stopped, wait at least 30 minutes after centrifuge has stopped before opening. This will allow hazardous aerosols to settle in the centrifuge.

Don lab coat, gloves, and face shield prior to opening centrifuge and then open carefully to assess the situation. Use of a respirator is recommended and double gloving is advisable if glass tubes were used and broken.

Attempt to determine if the spill is contained in a closed cup, bucket or tray carrier, or within a closed rotor.

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Centrifuge spills ….cont Remove rotors and buckets to nearest biological safety cabinet for clean-up.

Carefully retrieve unbroken tubes, wipe outside with disinfectant, The broken glass tube (s) must be removed with a forceps and the pieces can then be disposed of in a sharps container.

After proper decontamination with disinfectant , carriers, rotors etc. can be washed with a mild detergent according to the manufacturer’s instructions.

Thoroughly wipe the inside of the centrifuge chamber with disinfectant saturated paper towels. Allow for adequate contact time before wiping up excess liquid

chlorhexidine + cetrimide (Savlon) or 70% alcohol may be used if bleach is corrosive

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WASTE MANAGEMENT

The Science: The only documented risk of transmission of infections from

waste to healthcare workers is through sharps There is however a potential for transmission of several

microbial infections due to dumping of untreated wastes by healthcare facilities.

Mixing of a small quantity of infectious waste with municipal garbage converts the entire waste to “ infectious”

Segregation of wastes at source followed by appropriate treatment is the key to the success of a waste management

strategy

The Reality:IgnoranceCommercialization of scienceApathy

The concerns:OccupationalPublic healthEnvironmental

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Hospital waste

Hazardous Non-hazardous

Noninfectious Infectious

Kitchen Recyclables

Non-sharps

Patient contaminated waste

Anatomical

Equipment

Specimens

Laboratory waste

Non-plastics

Sharps: needles, scalpel blades, scalp veins, glass contaminated with blood

Radioactive

Cytotoxic drugs

Toxic Chemicals

Plastics

PVC, PE PET, PS contaminated cotton waste, gauze, linen

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Category of waste

Waste category Treatment & Disposal Color coding

1 Human anatomical waste ( human organs, tissue , body parts)

Incineration/deep burial

yellow

2 Animal waste ( animal parts, organs , body parts, bleeding parts, fluid, blood, animals used in research, waste from veterinary hospitals, colleges, animal houses)

Incineration/deep burial

yellow

3 Microbiology and Biotechnology waste Waste from laboratory cultures, stocks, or specimens with microorganisms, live or attenuated vaccines, human & animal cell culture used in research, infectious agents from research and industrial laboratories, wastes from production of biologicals, toxins, dishes and devices used for transfer of cultures)

Autoclaving/ microwaving incineration.

Yellow/red

4 Waste sharps: (needles, syringes, scalpels, blades, glass, etc. that may cause puncture and cuts. This includes both used and unused sharps)

Disinfection/chemical treatment/autoclaving /microwaving and mutilation/ shredding

Blue/white puncture proof container

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5 Discarded medicines and cytotoxic drugs: wastes comprising of outdated contaminated and discarded medicines

Incineration/destruction and disposal in secured landfill

Black/yellow

6 Solid waste: (Items contaminated with blood and body fluids incluidng cotton dressings, soiled plaster casts, lines beddings, other materials contaminated with blood

Incineration/autoclaving/microwaving

Yellow/red

7 Solid Waste (wastes generated from disposable items other than the waste sharps such as tubings, catheters, IV sets etc)

Disinfection by chemical treatment/autoclaving/microwaving and mutilation shredding

Red/ blue or white bag

8 Liquid Waste (waste generated from laboratory and washing, cleaning, housekeeping activities

Disinfection by chemical treatment and discharge into drains

--

9 Incineration ash: (ash from incineration of any biomedical waste)

Disposal in municipal landfill

Black

10 Chemical Waste (chemicals used in production of biologicals, chemicals used in disinfection, as insecticides etc)

Chemical treatment and discharge into drains for liquids and secured landfill for solids

Black

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Thank you

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