Biosafety

Biosafety levels and Cabinet in laboratory

1. Dr. Malathi M 20/11/2014 Biosafety levels & biosafety cabinet

2. Topics to be discussed : What is biosafety ? Why we need ? Levels of biosafety Biosafety cabinet Types Decontamination

3. Biosafety is the application of safety precautions that reduce a laboratorians risk of exposure to a potentially infectious material and limit contamination of the work environment and ultimately the community { CDC }

4. Why we need biosafety ???? Lab has hazards of processing infectious agents Accidental threat to workers and environment To have adherence with safety regulations while dealing with highly infectious agents

5. BIOSAFETY LEVEL 1 Microbes not known consistently to cause disease in healthy adults and present minimal potential hazard to lab and environment Eg : non pathogenic strain of E.coli

6. BSL – 1 practices: Standard microbiological practices are followed Work can be performed on an open table or bench PPE{Personal protective equipment} needed Sink – hand washing Lab – doors seperate

7. Biosafety level 2 Microbes that possess moderate hazards to laboratorians Eg : Staphylococcus aureus

8. BSL – 2 practices: Access to lab is restricted when work is being conducted PPE , face shields, eye goggles Biosafety cabinet Autoclave/Decontamination proper Self closing doors Sink with eyewash apparatus readily available

9. Biosafety level 3 Serious / potentially lethal disease through respiratory transmission Eg : Mycobacterium tuberculosis

10. BSL 3 - practices Laboratorians – under medical surveillance and receive immunisation Access to lab restricted & controlled PPE with respirators BSC Sink with eyewash Exhaust air – not recirculated Self closing doors with automatic locking

11. Biosafety level 4 Highest level of biological safety Dangerous and exotic microbes Eg : Ebola , marburg viruses

12. BSL 4

13. BSL 4 - practices Change clothes before entering Shower upon exiting Decontaminate all materials before exiting Class III BSC Separate building for lab Vacuum lines and decontamination systems

14. BSL 4 lab

15. biosafety cabinet

16. Introduction Biosafety cabinets ( BSCs ) are primary means of containment , developed for working safely with infectious micro-organisms BSCs are only one overall part of biosafety program , which requires consistent use of good microbiological practices primary containment equipment p rimary containment facility design

17. To be precise, “BSCs are designed to provide personnel, environmental and product protection when appropriate practices and procedures are followed” Adapted from CDC-BMBL- 5 th Edition/1999 Appendix A – Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets

18. Historical perspective Early prototype clean air cubicles (clean filtered air was blown directly at the working surface inside a cubicle – this places the personnel in a contaminated air stream) Concept of small workstation (non-ventilated cabinets – wood/stainless steel) Ventilated cabinets (lack of controlled/ adequate air flow leading on to mass airflow) Class I HEPA filter were introduced (undergoing modifications till date)

19. HEPA – High efficiency particulate air filter It removes the most penetrating particle size (MPPS) of 0.3 μm with an efficiency of at least 99.97 % The typical HEPA filter is a single sheet of borosilicate fibers treated with a wet-strength water-repellant binder Hepa filter

20. The filter medium is pleated to increase the overall surface area , with pleats being separated by corrugated aluminum tubes This separation is mainly to prevent collapse It removes particulate matter by three mechanisms interception, impaction, diffusion The filtering efficiency depends upon fiber diameter, filter thickness and face velocity These filters are fitted either in the exhaust or air supply system to remove particulate matter

21. http:// en.wikipedia.org /wiki/HEPA

22. Importance of a Biosafety cabinet Provide protection to the personnel handling infectious material environment by preventing the release of microbes product (e.g. in handling cell cultures)

23. BIOSAFETY CABINET - I Provides personnel and environmental protection, but no product protection Exhaust system – HEPA filter Class I BSC – unfiltered room air is drawn in through the work opening and across the work surface Inward airflow – Minimum velocity – 75 linear feet / minutes

24. To enclose equipment ( Eg . Centrifuges, harvesting equipment, small fermenters ) For procedures with potential to generate aerosols ( tissue homogenation , culture aeration) Class I BSC is hard-ducted Cabinet air is drawn through a HEPA filter as it enters the cabinet exhaust plenum.

25. REQUIREMENTS: Open fronted Glass in the upper front An integral tray to contain spills and splashes Inward airflow – 0.7 to 1 m/sec Protection factor – 1.5 * 10 5 Protection factor = number of particles which, if liberated into the air of the cabinet will not escape into the room Filtration from the exhaust air - HEPA

26. CLASS I cabinet

27. Biosafety cabinet class II Product protection Predictable particle behaviour Laminar air flow principle (1960 ) Particle barrier systems Risk of contaminant release into the lab and risk of product contamination

28. Class II

29. CLASS II – Type A1 Internal fan – draws room air – 75lfm velocity Supply air flows through HEPA – particulate free air to the work surface Reduced turbulence Reduced cross contamination

30. Downward moving air – splits into two To the front grille To the rear grille 30% of the air – exhaust HEPA filter 70% of the air – recirculates through HEPA filter back into the work zone of the cabinet

31. Not to be used for work involving volatile toxic chemicals Exhaust the air outside the building ( through use of canopy hood and filter housing ) CLASS II A1 and A2 – never be hard ducted to the building exhaust system

32. CLASS II A1

33. CLASS II A2 ( formerly B3) Inflow air velocity 100lfm all positive pressure contaminated plenums within the cabinet are surrounded by a negative air pressure plenum  ensures leakage

34. CLASS II A2

35. CLASS II B1 For hazardous chemicals and carcinogens Designed and originated with the National cancer institute type 212 ( later called Type B) Definition of Type B1 cabinets: Classic NCI design Type B, and cabinets without supply HEPA filters located immediately below the work surface, or those with exhaust/recirculation down flow splits other than exactly 70/30%

36. Cabinet supply blowers draw room air through the front grille and through HEPA Inflow velocity 100lfm Split in the down flowing air stream just above the work surface 70% air  through the rear grille  exhaust HEPA filter  discharge through building 30% air  Down flow air  front grille

37. CLASS II B1

38. CLASS II B2 Total exhaust cabinet No air recirculation Simultaneous biological and chemical containment Inflow air velocity 100lfm Exhaust 1200 cubic feet/min of room air  expensive cabinet high cost of heavier gauge and higher capacity exhaust fan  hence only for research

39. CLASS II B2

40. CLASS III Highly infectious agents, hazardous operations Gas tight  no leak greater than 1 *10-7 cc/sec with 1% test gas at 3 inches pressure water gauge Non opening view window Passage of materials through a dunk tank Double door pass through box with autoclave

41. Supply and exhaust air  HEPA Negative pressure cabinet No exhaust through the general lab exhaust Long heavy duty rubber gloves attached in a gas tight manner to port in the cabinets

42. Class III

43. Work practices and procedures Checklist of materials and work activity protocol Arm movement slowly Minimum persons Lab coats buttoned fully Proper Stool height

44. Check list Daily check of airflow by airflow indicator and monthly or weekly with an anemometer Ideal air flow – 0.7 to 1 m/s

45. All procedures should be done atleast four inches in from the front grille Only the materials needed for work should be kept inside Wait for minimum of four minutes to switch off the blowers after the work is over

46. Decontamination Disinfectant selection  EPA registration number in the label and list of infectious agents that the disinfectant is effective BSC – ethanol not used as decontamination as it evaporates – no proper contact time – ethanol can be used as a rinsing agent Formaldehyde vapour sterilisation to be done to kill spores

47. Disinfection method A Cabinets with an internal electric power supply Place 25 ml formalin(cabinet with internal volume of 0.38cu.m) t o a vaporizer, or into a beaker on a hotplate Close the cabinet and ensure that the exhause blow back valve is closed Boil away formalin

48. Disinfection method B 35ml formalin in a 100ml beaker inside the cabinet  add 10g potassium permanganate  seal the cabinet Leave the cabinet at least 5 hours , preferably overnight and label DANGER – FUMIGATION IN PROGRESS Open next day and work after 30 min for residual formaldehyde to exhaust

49. Use of Ultraviolet lamps for BSC is not advisable { NIH, CDC }

50. Summary

51. References Appendix A – primary containment for biohazards – CDC article Koneman`s color atlas and T.B of diagnostic microbiology Diagnostic microbiology – Bailey and scott – 13 th edition Practical medical microbiology – Mackie and Mccartney – 14 th edition

52. Thank you……….

53. Questions pls