Biological Safety

http://www.cityu.edu.hk/bch/

Summary of talk

* Why practise Biosafety?

* How dangerous are the organisms?

* How to ensure Biosafety?

* What to do before starting any project?

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Prof. Vrijmoed Lilian

 
 
 

Why practise Biosafety?

* microbes are invisible and unavoidable

* can be present as aerosols - respirablewpe19.jpg (5159 bytes)

* can enter body via broken skin, though unlikely by ingestion in lab

* most microbes in environment are harmless,but can be opportunistic pathogens

* lab workers have higher exposure

 

How dangerous are the microbes?

* guidelines from ACDP (Advisory Committee on Dangerous Pathogens) of HSE in UK.

* microbes are categorised to different hazard levels according to the inherent hazard of organisms.

* laid down guidelines to the different containment levels to compensate the risks posed by the various hazard groups.

* Hazard is the degree of the danger associated with the nature of the organisms.

* Risk is the probability that, in certain given circumstances, the hazard will be expressed as an infection.

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* microbes are categorised into four hazard group using the following criteria:

   - is the organism pathogenic for man ?

   - is it a hazard to laboratory workers ?

   - is it transmissable to the community ?

   - is effective prophylaxis or treatment available ?

Important to recognise hazard group does not allow for additional risks posed to workers involving:

   - pre-existing diseases

   - comprised immunity

   - pregnancy ; and

   - effects or medication

   - allergenic properties

   - toxigenic properties


Hazard Group  1

An organism that is most unlikely to cause human disease.wpe1C.jpg (8477 bytes)

 
 

Hazard Group 2

An organism that may cause human disease which might be a hazard to laboratory workers but is unlikely to spread to the community.

Laboratory exposure rarely produces infection and effective prophylaxis or effective treatment is usually available.

Hazard Group 3

An organism that may cause severe human disease and presents a serious hazard to laboratory workers.

It may present a risk of spread to the community but there is usually effective prophylaxis or treatment available.

Hazard Group 4

An organism that causes severe human disease and is a serious hazard to laboratory workers. It may present a high risk of spread to the community and there is usually no effective prophylaxis or treatment.

 

How to ensure Biosafety?

* How to protect ourselves during work ?

* Reduce unavoidable exposure by CONTAINMENT.

* containment by a system encompassing:

   - laboratory designwpe1E.jpg (6850 bytes)

   - equipment

   - facilities

   - operation procedures and work practices

* 4 containment levels.

* each level deals with the corresponding hazard group of microbes according to the categorisation of ACDP of HSE.

* Containment Level 1 for Hazard Group 1

* Containment Level 2 for Hazard Group 2

* Containment Level 3 for Hazard Group 3

* Containment Level 4 for Hazard Group 4

 

What to do before starting any projects?

RISK ASSESSMENT

 
 

BACTERIA, CHLAMYDIAS, RICKETTSLAS AND MYCOPLASMAS

Hazard Group 3

Bacillus anthracis       V Mycobacterium scrofulaceum (see APP M)
Brucella spp     V Mycobacterium simiae
Chlamydia psittaci    V Mycobacterium szulgai
Coxiella burnetii    V Mycobacterium tuberculosis    V
Francisella tularensis (Types A) Mycobacterium xenopi
Mycobacterium africanum    V Pseudomonas mallei
Mycobacterium avium (see app M) Pseudomonas pseudomallei
Mycobacterium (excl BCG strain) Rickettsia-like organisms
Mycobacterium intracellulare (see App M) Rickettsia spp
Mycobacterium kansasii Salmonella paratyphi A, B, C $
Mycobacterium leprae Salmonella typhi $V
Mycobacterium malmoense Shigella dysenteriae ( Type 1) $
Mycobacterium paratuberculosis (see App M) Yersinia pseudotuberculosis susp pestis(Y pestis) V

Hazard Group 2

Acinetobacter calcoaceticus Legionella spp
Acinetobacter lwoffi Leptospira spp     G
Actinobacillus spp Listeria monocytogenes
Actinomadura spp Moraxella spp
Actinomyces bovis Morganella morganii
Actinomyces israelii Mycobacterium bovis (BCG strain)
Actinomyces hydrophila Mycobacterium chelonei (see App M)
Alcaligenes spp Mycobacterium fortuitum (see App M)
Arizona spp Mycobacterium marinum(see App M)
Bacillus cereus Mycobacterium microti (see App M)
Bacteroides spp Mycobacterium ulcerans (see App M)
Bacterionemia matruchottii Mycoplasma pneumoniae
Bartonella bacilliformis Neisseria spp (spp known to be pathogenic for man)
Bordetella parapertussis Nocarddia asteroides
Bordetella pertussis Nocardia brasiliensis
Borrelia spp Pasteurella spp
Campylobacter spp Peptostreptococcus spp
Cardiobacterium homminis Plesiomonas shigelloides
Chlamydia spp (other than avian strains) Proteus spp
Clostridium botulinum     v Providencia spp
Clostridium tetani     V Pseudomonas spp (other spp known to pathogenic for man)
Clostridium spp (other spp known to be pathogenic for man ) Salmonella spp (other than those in Hazard Group 3)
Corynebacterium diphtheriae    V Serratia liquefaciens
Corynebacterium spp(other spp known to be pathogenic for man) Serratia marcescens
Edwardsiella tarda Shigella spp (other than that in Hazard Group 3)
Eikenella corrodens Staphylococcus aureus
Enterobacter spp Streptobacillus moniliformis
Erysipelothrix rhusiopathiae Streptococcus spp (except those known to be non-pathogenic for man)
Escherichia coli (except those known to known to be pathogenic for man) Treponema pallidum
Flavobactterium meningosepticum Treponema pertenue
Francisella tularensis (Type B) Veillonella spp
Fusobacterium spp Vibrio cholerae (incl El Tor)
Gardnerella vaginalis Vibrio parahaemolyticus
Haemophilus spp Vibrio spp(other species known to be pathogenic for man)
Hafnia alvei Yersinia enterocolitica
Kingella kingae Yersinia pseudotuberculosis subsp pseudotuberculosis
Klebsiella spp
 
 

FUNGI

This list exemplifies the more commonly encountered fungal pathogens and as new pathogens continue to be described it is not exhaustive. The generic and specific epithets used are those commonly employed and the names of perfect states are given in brackets. (There are many synonyms and where a synonym is in use it is essential to ascertain the hazard group: see paragraph 22.)

Hazard Group 3

Blastomyces dematitidis (Ajellomyces dermatitidis) Histoplasma capsulatum var farciminosum (see App N)
Coccidioides immitis Paracoccidioides brasiliensis
Histoplasma capsulatum var. capsulatum (Ajellomyces capsulata) Penicillium marneffei
Histoplasma capsulatum var duboisii

Hazard Group 2

Absidia corymbifera Exophialia jeanselmei
Acremonium flaciforme Exophialia spinifera
Acremonium kiliense Exophialia richardsiae
Acremonium recifei Fonsecaea compacta
Aspergillus flavus Fonsecaea pedrosoi
Aspergillus fumigatus Fusarium solani
Aspergillus nidulans Fusarium oxysporum
Asperillus niger Geotrichum candidum
Aspergillus terreus Hendersonula toruloidea
Basidiobolus haptosporus Leptosphaeria senegalensis
Candida albicans Madurella mycetomatis
Candida glabrata Madurella grisea
Candida guilliermondii Malassezia furfur
Candida krusei Neotestudina rosatii
Candida parapsilosis Phialophora verrucosa
Candida kefyi Piedraia hortae
Candida tropicalis Pneumocystis carinii
Cladosporium carrionii Pseudallescheria boydii
Conidiobolus coronatus Pyrenochaeta romeroi
Cryptococcus neoformans (Filobasidiella neoformans)     S Ricrosporum spp
Cunninghamella elegans Rhizomucor pusillus
Curvularia lunata Rhizopus microsporus
Emmonsia parva Rhizopus oryzae
Emmonsia parva var. crescens Sporothrix schenckii
Epidermophyton floccosum Trichophyton spp
Exophialia dermitidis Trichophyton beigelii
Exophialia werneckii Xylohypha bantiana
 
 

Containment Level 1

Containment Level 1 is suitable for work with organisms in Hazard Group 1. Laboratory personnel must receive instruction in the procedures conducted in the laboratory.

1. The laboratory should be easy to clean. Bench surfaces should be impervious to water and resistant to acids, alkalis, solvents and disinfectants.

2. If the laboratory is mechanically ventilated, it is preferable to maintain an inward airflow into the laboratory by extracting room air to atmosphere.

3. The laboratory must contain a wash-basin or sink that can be used for hand washing.

4. The laboratory door should be closed when work is in progress.wpe1F.jpg (5801 bytes)

5. Laboratory coats or gowns should be worn in the laboratory and removed when leaving the laboratory suite.

6. Eating, chewing, drinking, smoking, storing of food and applying cosmetics must not take place in the laboratory.

7. Mouth pipetting must not take place.

8. Hands must be disinfected or washed immediately when contamination is suspected, after handling viable materials and also before leaving the laboratory.

9. All procedures must be performed so as to minimise the production of aerosols.

10. Effective disinfectants must be available for immediate use in the event of spillage.

11. Bench tops should be cleaned after use.

12. Used laboratory glassware and other materials awaiting disinfection must be stored in a safe manner. Pipettes, if placed in disinfectant, must be totally immersed.

13. All waste material which is not to be incinerated should be rendered nonviable before disposal.

14. Materials for disposal must be transported in robust containers without spillage.

 

Containment Level 2

Containment Level 2 is suitable for work with pathogens in Hazard Group 2. Laboratory personnel must receive instruction and training in handling pathogens, and an appropriate standard of supervision of the work must be maintained.

1. The laboratory should be easy to clean. Bench surfaces should be impervious to water and resistant to acids, alkalis, solvents and disinfectants.

2. Access to the laboratory should be limited to laboratory personnel and other specified persons.

3. There should be adequate space (24 m3) in the laboratory for each worker.

4. If the laboratory is mechanically ventilated, an inward airflow into the laboratory should be maintained by extracting room air to atmosphere.

5. The laboratory must contain a wash-basin which should be located near the laboratory exit. Taps must be of a type that can be operated without being touched by hand.

6. An autoclave for the sterilisation of waste materials must be readily accessible, normally in the same building as the laboratory.

7. The laboratory door should be closed when work is in progress.wpe20.jpg (4943 bytes)

8. Laboratory coats or gowns, which should be side or back fastening, must be worn in the laboratory and removed when leaving the laboratory suite. Separate storage (e.g. pegs) must be provided in the laboratory suite for this clothing.

9. Eating, chewing, drinking, smoking, storing of food and applying cosmetics must not take place in the laboratory.

10. Mouth pipetting must not take place.

11. Hands must be disinfected or washed immediately when contamination is suspected, after handling infective materials and also before leaving the laboratory.

12. In general, work may be conducted on the open bench, but care must be taken to minimise the production of aerosols. For manipulations such as vigorous shaking or mixing and ultrasonic disruption etc, a microbiological safety cabinet (Class I, BS 5726: 1979) or equipment which is designed to contain the aerosol must be used. The cabinet must exhaust to the outside air or to the laboratory air extract system (see paragraph 42 (14) (b)).


13. Effective disinfectants must be available for routine disinfection and immediate use in the event of spillage.

14. Bench tops must be disinfected after use.

15. Used laboratory glassware and other materials awaiting sterilisation must be stored in a safe manner. Pipettes, if placed in disinfectant, must be totally immersed.

16. Material for autoclaving must be transported to the autoclave in robust containers without spillage.

17. All waste materials must be made safe before disposal or removal to the incinerator.

18. All accidents and incidents must be immediately reported to and recorded by the person responsible for the work.

Diagnostic laboratories

All clinical microbiological suites must contain a microbiological safety cabinet (Class I, BS 5726: 1979, or one with an equivalent protection factor). In veterinary practice, a Class I safety cabinet will not be required if diagnostic work is limited to the examination of fixed slides that have been prepared and fixed elsewhere.

 

Containment Level 3

Containment Level 3 is suitable for work with pathogens in Hazard Group 3. Laboratory personnel must have had training in handling pathogenic and potentially lethal organisms and in the use of safety equipment and controls. A high standard of supervision of the work must be maintained.

1. The laboratory must be easy to clean. Bench surfaces and the floor must be impervious to water and resistant to acids, alkalis, solvents and disinfectants.

2. The laboratory must be sealable to permit fumigation.

3. There must be adequate space (24 m3) in the laboratory for each worker.

4. The laboratory should be sited in an area away from general circulation. Access to the laboratory must be limited to authorised personnel. The laboratory door must be locked when the room is unoccupied.wpe21.jpg (7250 bytes)

5. A specific biohazard sign must be posted at the entry to the laboratory and the door must contain a glass panel so that the occupants can be seen.

6. A continuous airflow into the laboratory must be maintained during, work with pathogens by one of the following means:

(a) extracting the laboratory air through independent ducting to the outside air through a HEPA filter;

(b) extracting the laboratory air to the outside air with a fan and HEPA filter sited in a wall or window of the laboratory;

(c) ducting the exhaust air from a microbiological safety cabinet to the outside air through a HEPA filter; or

(d) a safe variation of these provisions (provisions should also be made for comfort factors eg fresh air, temperature control).

In laboratories that have a mechanical air supply system, the supply and extract airflow must be interlocked to prevent positive pressurisation of the room in the event of failure of the extract fan. The ventilation system must also incorporate a means of preventing reverse airflows.

7. A wash-basin must be provided near the exit of the laboratory. Taps must be of a type that can be operated without being touched by hand.

8. An autoclave for sterilisation of waste materials should be situated preferably within the laboratory, but one must be readily accessible in the laboratory suite.

9. The laboratory door must be kept closed when work is in progress.

10. Side or back fastening gowns must be used in the laboratory and they must be autoclaved before being sent for laundering. These gowns must not be used outside the laboratory suite.

11. Gloves must be worn for all work with infective materials and hands washed before leaving the laboratory.

12. Eating, chewing, drinking, smoking, storing of food and applying cosmetics must not take place in the laboratory.

13. Mouth pipetting must not take place.

14. (a) All laboratory procedures with infective materials must be conducted in a microbiological safety cabinet (Class I or Class III, BS 5726: 1979, or unit with equivalent protection factor or performance) except where the equipment to be used provides containment of the potential aerosol (but see also paragraph 21 and notation $ in paragraph 24).

(b) The cabinet must exhaust through a HEPA filter to the outside air or to the laboratory air extract system, and in other respects such as siting, performance, protection factor and air filtration, it must comply with the specifications detailed in BS 5726: 1979. When laboratories are faced with a major problem because of difficulties in arranging for the cabinet to exhaust to the open air, recirculation of exhaust air through two HEPA filters in series may, in exceptional circumstances, be considered as an alternative. In this case the maintenance of a continuous airflow into the laboratory during work with pathogens (see paragraph 42 6 (a) and (b) above) will be of particular importance and such an option must not be adopted without prior consultation with HSE.wpe22.jpg (7199 bytes)

15. The laboratory should contain its own equipment, eg centrifuge (in which sealed buckets must be used), incubator, refrigerator, deep-freeze, vapour phase liquid nitrogen chest etc, so that all infective Hazard Group 3 pathogenic materials are held within the laboratory and nowhere else. Where this is not reasonably practicable (see paragraph 34) material must be transported and stored without spillage in properly labelled robust containers which must be opened only in Containment Level 3 accommodation.

16. Effective disinfectants must be available for routine disinfection and immediate use in the event of spillage.

17. Materials for autoclaving must be transported to the autoclave in robust containers without spillage.

18. All waste materials must be made safe before disposal or removal to the incinerator.

19. All accidents and incidents, spills and exposures to infective materials must be immediately reported to and recorded by the person responsible for the work.

 

 

TABLE 1 Concentration and particle size of aerosols created during representative laboratory techniques [a]

 
Operation No. of
viable
colonies[b]
Particle size[c]
(um)
Mixing culture with :
  Pipette 6.0 3.5
  Vortex mixer 0.0 0.0
  Mixer overflow 9.4 4.8
Use of Waring blender :
  Top on 119.0 1.9
  Top off 1500.0 1.7
Use of sonicator 6.0 4.8
Lyophilized cultures :
  Opened carefully 134.0 10.0
  Dropped and broken 4838.0 10.0

[a] Adapted from references 36 and 56, with permission.

[b] Mean number of viable colonies per cubic foot of air sampled.

[c] Mean diameter of the particle.

 

TABLE 3 Comparison of biological safety cabinets

Cablnets Application
Type Face celocity
(lfpm)
Airflow Pattern Radionuclides/
Toxic Chemicals
Diosafety Level (s) Product
Protection
Class I*, open front 75 In at front; out rear and
top through HEPA filter
NO 2,3 NO
Class II:
    Type A
75 70% recirculated through
HEPA; exhaust through HEPA
NO 2,3 YES
    Type B1 100 30% recirculated through
HEPA; exhaust via HEPA and
hard-ducted
YES
(Low levels/
volatility)
2,3 YES
    Type B2
100 No recirculation; total exhaust
via HEPA and hard-ducted
YES 2,3 YES
    Type B3 100 Same as IIA, but plena under
negative pressure to room and
exhaust air is ducted
YES 2,3 YES
Class III N/A Supply air inlets and exhaust
through 2 HEPA filter
YES 3,4 YES

* Glove panels may be added and will increase face velocity 150lfpm; gloves may be added with an inlet air pressure release that will allow work with chemicals/radionuclide

 

Table 7

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FIGURE 7 Diagram of class I open-front biological safety cabinet. (A) Conventional use; (B) with armhole plate attached; (C) with rubber gloves attached to armhole


Table 14

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FIGURE 14 Simplified diagram of class IIB2 biological safety cabinet. (Adapted from Germfree Laboratories, Miami, Fla.)

 
 
 
 
 
  
 
 
 

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