1. Laboratory Biosafety Practices
A. I. Bhat
Indian Institute of Spices Research
Calicut , Kerala

2. Why Biosafety Practices?
Protection:
workers
“products”
co-workers
lab support personnel
environment

3. What are the general hazards in a laboratory?
Fire
Breakage of glassware
Sharps
Spillages
Pressure equipment & gas cylinders
Extremes of heat & cold
Chemical hazards
Biological hazards
Radiation
And many more!

4. worldwide literature search from 1979-2004
1,141 overt infections, 24 deaths
Most common causative agents of overt infection include:
1. M. tuberculosis HBV
2. Arboviruses Shigella spp.
3. C. burnetti Salmonella spp
4. Hantavirus HCV
5. Brucella spp N. meningitidis
Many asymptomatic infections
Many newer agents, e.g. SARS-CoV, Cryptosporidium, etc.
No specific exposure event in most cases
Summarized by Harding and Byer, Biological Safety, Principles and Practice, 4th Ed – Excellent Review
No doubt,still an underestimate; also no incidence data
Many other agents implicated…well described in Harding and Byer’s chapter
__________________________________________________________WSLH

5. What were the predominant means of exposure?
As mentioned before no specific exposure event identified in most cases
Those identified included:
Inhalation of aerosols generated by work practices or procedures or spills
Percutaneous inoculation
Contamination of mucous membranes
Ingestion
Percutaneous exposures include needle sticks, cuts with contaminated items, animal bites
For many of these agents, exposure to aerosol (either by breakdown in work practices or procedure (sniffing, not using BSCs and covered centrifuges, vortexing hot looping, tests that generate aerosols, procedures that generate aerosols, accidents eg spillage, or carelessness)is greatest concern…and we’ll revisit this
Note the organisms on these lists classically associated with aerosol transmission
One can easily identify the relevant means of exposure given the pathogen described
While risk of ingestion lessened by ceasing mouth pipetting, hand to mouth contamination still prevelant
__________________________________________________________WSLH

6. Key elements of containment include: Laboratory practice and technique
Objective of biosafety is containment of potentially harmful biological agents
Purpose of containment is to reduce/eliminate exposure of lab workers, other persons and outside environment to biohazardous agents
Key elements of containment include:
Laboratory practice and technique
Safety equipment (primary barriers and PPE)
Facility design and construction (secondary barriers)
Risk Assessment of the work to be done with a specific agent or under specific circumstances determines the appropriate combination of these elements to employ
__________________________________________________________WSLH

7. General Lab Requirements: Safety
Knowledgeable supervisor
Knowledgeable personnel
Aware of potential hazards
Proficient in practices &
techniques
Lab specific biosafety manual

11. Laboratory safety

12. Special procedures Decontaminate work surfaces
Report spills and accidents
No animals in laboratories

18. Common Sense Rules
Wash hands & remove protective clothing before eating, drinking, smoking, handling contact lenses, applying lip balm or cosmetics
Keep hands away from eyes, nose, mouth while cleaning
Frequent hand washing is best defense against spreading infection

19. Elements of Containment
Safety Equipment (Primary barriers and PPE)
Available for each possible route of exposure
Aerosol: BSCs, covered centrifuge carriers, loop incinerators or disposable loops, PPE
Percutaneous: sharps disposal; retractable needles
Mucous membrane contact: goggles or safety glasses, face shields, gloves
Ingestion: automatic pipetting devices
Aerosol: BSCs, covered centrifuge carriers, loop incinerators or disposable loops, PPE(respirators, PAPRs)
Percutaneous: sharps disposal; retractable needles
Mucous membrane contact: goggles or safety glasses, face shields, gloves
Ingestion: automatic pipetting devices
Aerosol particles are created by
any activity that imparts energy into a liquid or semiliquid material, such as shaking,
pouring, stirring or dropping liquid onto a surface or into another liquid. Other
laboratory activities, such as streaking agar plates, inoculating cell culture flasks with
a pipette, using a multichannel pipette to dispense liquid suspensions of infectious
agents into microculture plates, homogenizing and vortexing infectious materials, and
centrifugation of infectious liquids, or working with animals, can generate infectious
aerosols
__________________________________________________________WSLH

20. Elements of Containment
The Biological Safety Cabinet
The single most important piece of safety equipment in the laboratory!
Importance of the Biological Safety Cabinet
The principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures
Provides protection to the operator, the laboratory environment and work materials
Which type is for you?
__________________________________________________________WSLH

21. 4 biosafety levels Biosafety Levels
Consist of combinations of lab practices and techniques, safety equipment and lab facilities
Purpose: To categorize risk associated with infectious agent and define the appropriate safety practices, equipment and facilities for handling the agent safely
Appropriate BSL determined by:
Microbiological agent Risk Group
Mode of transmission
Procedural protocols
Experience of staff
Likelihood of aerosol generation
Work involves use of amplified agent
Other?
__________________________________________________________WSLH

22. Risk group classification
Individual risk
Community risk 1
no, low 2
moderate
low 3
high 4

23. Risk Group 1 Unlikely to cause animal or human disease
Non pathogenic agent

24. Risk Group 2 Pathogenic for humans Unlikely a serious hazard
Treatment and preventive measures available
Limited risk of spread of infection

25. Risk Group 3 Pathogenic, cause serious disease
Effective treatment and preventive measures usually available
Little person-to-person spread

26. Risk Group 4 direct, indirect Lethal, pathogenic agent
Readily transmittable
direct, indirect
Effective treatment and preventive measures not usually available

27. Risk groups, biosafety levels, practices and equipment
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
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, special waste
Class III biological safety cabinet, positive pressure suits, double ended autoclave (through the wall), filtered air

28. Recommended Biosafety Levels for Infectious Agents
BSL
Agents
Microbiology
Practices
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers) 1
Not known to consistently cause disease in healthy adults
Standard Microbiological Practices
None required
Open bench top sink required 2
Associated with human disease, hazard equals percutaneous injury, ingestion, mucous membrane exposure
BSL-1 plus: Limited access, biohazard warning signs, “sharps” precautions, biosafety manual defining waste decontamination & medical surveillance policies
Class I or II BSCs or other physical containment for manipulations of agents that cause splashes or aerosols of infectious materials, PPE: lab coats, gloves, face protection as needed
BSL-1 plus: Autoclave 3
Indigenous or exotic agents with potential aerosol transmission; may have serious or lethal consequences
BSL-2 plus: Controlled access; Decontamination of all waste & lab clothing before laundering; Baseline serum
Class I or II BCSs or other physical containment for all manipulations; PPE: protective clothing; gloves; respiratory protection as needed
BSL-2 plus: Physical separation from corridors; Self-closing, double-door access; Exhausted air not recirculated; Negative airflow into laboratory 4
Dangerous/exotic agents with high risk of life-threatening disease, aerosol-transmitted infections; or related agents with unknown risk of transmission
BSL-3 practices plus: Clothing change before entering, shower on exit, all material decontaminated on exit
All procedures in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure personnel suit
BSL-3 plus: Separate building or isolated zone, dedicated supply, exhaust, vacuum, & decon systems; other requirements.
__________________________________________________________WSLH

29. Disinfection
Disinfection requirements depend on the experimental work and nature of the agents being handled
Contact time and concentration for disinfectants are specific for each material and manufacturer
Efficacy is a function of surface, contact time, product, dilution

30. Using detergents Use licensed detergents/disinfectants
Follow manufacturers’ recommendations
Wear adequate personal protective equipment
Perform hand hygiene
Avoid difficult to clean surfaces/ materials (carpet, wood)
Apply disinfectant to a large area - wiping, soaking
Avoid aerosolizing specimens while handling
Mention cleaners and persons who handle the laundry etc

31. Disinfection with household bleach
Work areas
disinfect with 0.5% bleach after every procedure
Contaminated supplies
soak with 0.5 % household bleach for 30 minutes
wash in soapy water
sterilize as necessary
Sodium hypochlorite is a “special” disinfectant.
pros - household bleach – its
Cheap
Readily available – in large quantities
It smells (what has a psychological function = effective
as well as it is detectable/controllable by its smell)..
Problems with bleach ….
It affects the quality of fabric and corrodes metals
Its effectiveness decreases if organic matters are on the items
Can not be used on the human body..
Photo: WHO

32. Risk Assessment Whose responsibility? Technically, the lab director
Practically, the bench microbiologist
Primary factors to consider:
Agent hazards
Laboratory procedures planned
Potential for aerosol generation
Consider facility, equipment needed; appropriate PPE
Capability of the staff
Training, technical proficiency, good habits
Known vs. unknown agent risk
For known or suspected agent, consult BMBL agent summary statements, other references
For unknown agent…
How is all of this applied, practically , in the clinical lab?
As stated before, Risk Assessment of the work to be done with a specific agent or under specific circumstances determines the appropriate combination of the key elements of containment and thus should define the biosafety level needed that can prevent LAIs
Consult Section 2 of BMBL or Chapter 5 of Biological safety: Principles and Practice.
Lab director, in collaboration with institutional biosafety committee, biological safety professional and infection control personnel
Agent hazards defined by:
Infectivity, pathogenicity, infective dose, routes of transmission(natural and “lab-induced”), concentration of agent, environmental stability, availability of prophylaxis/treatments, host range, geographic or species of origin
Understanding route of transmission particularly important in assessing lab risk.
APHA Communicable Disease Manual particularly good reference
A known agent might be one already identified with follow up work done on a pure culture or a specimen form a known outbreak
Unknown agent refers to any clinical specimen from patient submitted to lab without linkage to outbreak
__________________________________________________________WSLH

33. General Safety Rules
1. Listen to or read instructions carefully before attempting to do anything.
2. Wear safety goggles to protect your eyes from chemicals, heated materials, or things that might be able to shatter.
3. Notify your teacher if any spills or accidents occur.

34. General Safety Rules
4. After handling chemicals, always wash your hands with soap and water.
5. During lab work, keep your hands away from your face.
6. Tie back long hair.

35. General Safety Rules 7. Roll up loose sleeves.
8. Know the location of the fire extinguisher, fire blanket, eyewash station, and first aid kit.
9. Keep your work area uncluttered. Take to the lab station only what is necessary.

36. General Safety Rules
10. It is suggested that you wear glasses rather than contact lenses.
11. Never put anything into your mouth during a lab experiment.
12. Clean up your lab area at the conclusion of the laboratory period.
13. Never “horse around” or play practical jokes in the laboratory.

37. Glassware Safety
1. Chipped or cracked glassware should not be used. Show it to the teacher.
2. Broken glassware should not be disposed of in a classroom trashcan. There is a special glass disposal container for it.
3. When pouring liquids into glassware, make sure the container you are pouring into is resting on a table at least a hands breadth from the edge.

38. Glassware Safety
4. If a piece of glassware gets broken, do not try to clean it up by yourself. Notify the teacher.
5. Do not place hot glassware in water. Rapid cooling may make it shatter.

39. Chemical Safety
1. Wear protective goggles whenever heating or pouring hazardous chemicals.
2. Never mix chemicals together unless you are told to do so (and then only in the manner specified).
3. Never taste any chemicals (you should never taste anything in the lab).

40. Chemical Safety
4. If you need to smell the odor of a chemical, waft the fumes toward your nose with one hand. Do not put your nose over the container and inhale the fumes.

41. Chemical Safety
5. Follow the instructions of your teacher when disposing of all chemicals.
6. Wash your hands after handling hazardous chemicals.

42. Heating Safety
1. Use tongs and/or protective gloves to handle hot objects.
2. Never reach across an open flame or burner.

43. Heating Safety
3. Always point the top ends of test tubes that are being heated away from people.
4. When heating a test tube, move it around slowly over the flame to distribute the heat evenly.

44. Heating Safety
5. Only glassware that is thoroughly dry should be heated.
6. Heat glassware by placing it on a wire gauze platform on a ringstand. Do not hold it in your hand.

45. First Aid Injury: Burns
To Do: Immediately flush with cold water until burning sensation is lessened.

46. First Aid Injury: Cuts, bruises
To Do: Do not touch an open wound without safety gloves. Pressing directly on minor cuts will stop bleeding in a few minutes. Apply cold compress to bruises to reduce swelling.

47. First Aid Injury: The eyes
To Do: Flush eyes immediately with plenty of water for several minutes. If a foreign object is lodged in the eye, do not allow the eye to be rubbed.

48. Spillages Clear up spillage promptly
You will already have determined how to do this as part of your risk assessment
Dispose of any hazardous material as toxic waste
Messy workers are usually poor workers!!

49. Unacceptable Lab Clothing
Skirts
Shorts
Open-toed shoes
Sandals
Jackets
Bulky shirts or sweaters
Floppy or loose sleeves

50. Laboratory Equipment
Never use any laboratory equipment unless you are trained & have been authorised to do so
As well as injuring yourself you may cause very costly damage

51. Laboratory hygiene Never eat, drink or smoke in a laboratory
Never apply cosmetics
Never touch your face, mouth or eyes
Never suck pens or chew pencils
Always wash your hands before you leave and especially before eating

52. First Aid
All laboratory workers should undergo simple first aid training
For ALL chemical splashes, wash with plenty of water for 10 minutes
Control bleeding with direct pressure, avoiding any foreign bodies such as glass
Report all accidents to your supervisor or departmental safety officer

53. MSDS Material Safety Data Sheets
These are data sheets that tell you the chemical and safety properties of the various chemicals we will use in the lab.
You must look up MSDS information for the safety section of your Pre-Lab in order to:
Know how to use the chemicals safely.
Know what to do if there is a spill or accident.
Be able to do the lab.

54. MSDS Diamond
The MSDS information is put into a short-hand form for quick and easy reference on labels and posters.
You also see MSDS Diamonds on transport vehicles and containers.
Red: Flammability
Yellow: Reactivity
White: Specific Hazard
Blue: Health Hazard

55. MSDS Diamond

56. MSDS Diamond
Test your Knowledge by writing in your notes what you think this MSDS Diamond is telling you about a chemical:

57. MSDS Diamond Answers: Red: Flammability Yellow: Reactivity
A 4 in the flammability diamond = VERY Flammable
Yellow: Reactivity
A 2 in the reactivity diamond = Reacts Violently with Water
White: Specific Hazard
No Water in specific hazard diamond = Don’t use water, this also corresponds with the Reactivity diamond
Blue: Health Hazard
A 1 in the health hazard diamond = slightly hazardous to your health

58. When in doubt – ASK!!!
Do not carry out a new or unfamiliar procedure until you have been fully trained & understand the precautions necessary for safe working
DO NOT GUESS!!!!

59. When Accidents Happen:
REMAIN CALM!
This sounds like common sense, but when accidents happen remaining calm is crucial!
Report ALL accidents IMMEDIATELY, no matter how small.

60. Indian biosafety rules and regulations
to advice, facilitate and regulate modern biotechnology work at different stages to achieve the objectives of protecting environment including human and animal health from the unintended adverse effects of GMOs and products thereof.

61. Recombinant DNA Advisory Committee (RDAC)
Review developments in Biotechnology at National and International level.
Recommend suitable and appropriate safety regulations for India in r-DNA research, use and applications.

62. Institutional Biosafety Committee (IBSC)
To note, examine and approve proposals involving r-DNA work; to ensure adherence of r-DNA Safety Guidelines of Government; inspection of containment facilities at R&D and production units and to inform the RCGM about the facilities;
To prepare emergency plan according to guidelines;
To approve experiments utilizing the organisms and genetic elements from Risk Group-I and II organisms up to laboratory fermentation 20 ltrs capacity with intimation to RCGM; for using organisms falling in Risk Group III & above, recommend to RCGM for approval to conduct laboratory studies; 
To  recommend for import/ exchange of GMOs/LMOs/Transgenic seeds, vectors, gene constructs, plasmids, etc., for research purposes;
To inform DLC and SBCC as well as GEAC about the experiments where ever needed; to act as a nodal point for interaction with statutory bodies; to ensure experimentation at designated location taking into account of approved protocols etc.

63. IBSC contd...
To examine the description of the target gene and source; nucleotide sequence  and amino acid sequence of target gene and  the target protein; the composition of  the vector used; schematic diagram of the expression cassette; restriction map of vector indicating the location of the target gene; cloning strategy;  description of the host cell line including genera and species; risks involved  in handling of cell line; methods of maintenance of cell line; classification of  the host cell line as per the guidelines;
To approve category I & II experiments, as per the Guidelines 1998 of DBT, up to green house level with intimation to RCGM in category III & above experiments, RCGM to approve conduct lab & green house studies. To recommend all open field experiments in any of the category, for any purpose (biosafety studies, seed increase experiments, agronomic studies, etc) for the approval of RCGM.
To examine protocols for toxicity/allergenicity studies as per national and international guidelines and their recommendations to RCGM

64. Review Committee on Genetic Manipulation (RCGM)
Monitor safety aspects of ongoing rDNA projects involving Genetically Engineered (GE) organisms/ hazardous microorganisms. All ongoing projects including high risk category and confined field experiments shall be reviewed by RCGM to ensure that adequate precautions and containment conditions are complied with as per the Guidelines and Standard Operating Procedures (SOPs) issued by DBT from time to time.
The RCGM shall lay down procedures restricting or prohibiting production, sale, importation and use of such GE organisms or products thereof for research and applications as mentioned in the schedule of Rules, 1989.
RCGM shall bring out manuals and guidelines specifying procedure for regulatory processes with respect to activities involving GE organisms in research, use and application including industry with a view to ensure safety to human, animal and environment health.
The RCGM would issue the clearance letters/permits for import or exchange of genes, DNA fragments, vectors, plasmids, cosmids, etiologic agents and transgenic organisms or germplasm(s) including transformed calli, seeds, plants and plant parts for research use only. It will also take note of all such commercially available agents which are acquired from commercial sources through Institutional Biosafety Committee (IBSC)

65. The RCGM shall be the regulatory body for receiving and reviewing the applications to conduct confined field trials (such as event selection trials, Biosafety Research Level I trials (BRL I), pollen flow studies, strip trials or any other trial involving GE organisms) and recommend appropriate studies to be conducted for data generation for biosafety assessment as per clause 4, as per the decision of the Genetic Engineering Approval Committee (GEAC) or its authorization.
The RCGM, on case-by-case basis, can authorize applicants to use bioreactors to produce sufficient material/end-products of GE organisms required for conducting pre-clinical studies and other relevant data generation including the use of capacity over and above as specified by Recombinant DNA Safety Guidelines, It will authorize to produce sufficient quantities of GE organism/plant product(s) for generating safety data in appropriate animals as per National and International Guidelines, as appropriate on case by case basis.
The RCGM, if required, can generate, examine or invite the research projects, proposals for capacity building and training courses in biosafety, creation of information systems/ data banks in electronic media, websites etc. for financial support and recommend the same to the DBT of specific projects for furthering the cause of generating specific biosafety data related to use of GMOs and strengthening infrastructure facilities & dissemination of biosafety rules, regulations and guidelines in the country.
The RCGM may also invite, induct or appoint special experts in their individual capacities on a case by case basis.
The RCGM may constitute subgroups of its members or subcommittees involving experts for specific functions or seek advice on specific matters from other external experts.
The RCGM or its constituted subgroups/subcommittees may visit periodically the experimental sites where r-DNA projects and activities involving GE organisms/ hazardous microorganisms are being pursued to ensure that adequate safety measures have been taken as per the guidelines and compliance of SOPs

66. Genetic Engineering Appraisal Committee (GEAC)
Statutory Body under the Ministry of Environment & Forests for approval of activities involving large-scale use of hazardous living microorganisms and recombinants in research and industrial production from the environmental angle
Responsible for approval of proposal relating to release of genetically engineered organisms and products Into the environment including experimental field trials as per the provisions of Rules 1989.
Responsible for approval of proposals involving the use of living modified organism falling in the risk category III and above in the manufacture/import of recombinant Pharma products or where the end product of the recombinant Pharma products per se is a living modified organism.

67. State Biotechnology Co-Ordination Committee (SBCC)
Powers to inspect, investigate and to take punitive action in case of violations of statutory provisions through the State Pollution Control Board or the Directorate of Health etc.  
To review periodically the safety and control measures in various institutions handling GMOs.  
To act as nodal agency at State level to assess the damage, if any, due to release of GMOs and to take on site control measures.
District Level Committee (DLC)
Have powers to inspect, investigate and report to the SBCC or the GEAC about compliance or non compliance of r-DNA guidelines or violations.  
To act as nodal agency at District level to assess the damage, if any, due to release of GMOs and to take on site control measures.  

68. Monitoring - cum - Evaluation Committee (MEC)
To undertake field visits at the experimental site(s) approved by the RCGM for the purpose of collecting  scientific information on the comparative agronomic advantages of the transgenic plants.
To advise, time to time, the RCGM on the risks and benefits from the use of the transgenic plants put into evaluation. The committee to suggest new experimental design(s) to RCGM and also assist in collecting, consolidating and analyzing the field data for evaluating the environmental risks emanating from the transgenic plants. 
                                                           
To apprise the RCGM on those transgenic crops which would find environmentally safe and economically viable for recommending the same to Genetic Engineering Approval Committee (GEAC) for consideration for release into the environment.
To undertake field visits at the experimental site(s) approved by the GEAC for the purpose of collecting scientific information, based on specific request made by the GEAC. The Committee to submit its report to GEAC directly with information to RCGM.

69. APPLICATION FORMATS
Proforma for submitting application to IBSC/RCGM for Import/Exchange of GMOs/LMOs and products thereof for research and development purposes 
Proforma for submitting application to IBSC/RCGM to carry out research for development of transgenic plants
Application to GEAC under Rules 1989  seeking approval for environmental release of transgenic crops, as per DBT Guidelines 1998
Application to GEAC seeking approval to conduct large scale field trials on transgenic crops
Proforma for submitting application to IBSC/RCGM to conduct confined field trials of regulated genetically engineered plants