PH 152

Biosafety in the Diagnostic Lab

November 14, 2012
Prof. Marohren C. Tobias-Altura
LAB HAZARDS o Biosafety started mainly due to biowarfare
1. Biological  People developing the biological
o Most important weapons also got sick
o Bacteria, viruses, parasites, etc.  So containment equipment (BSCs) and
o Anything capable of replicating, transmitting, and biosafety programs were designed
causing disease
2. Chemical LABORATORY ACQUIRED INFECTIONS (LAI)
o Reagents like acid-alcohol, carbol fuxin (contains  All infections acquired through lab-related activities
phenol which are carcinogenic)  Includes both asymptomatic and symptomatic infections
3. Radiation
o In techniques that use radioactive light or Table 1 – Top 10 LAIs (from 1979 to 1999)
materials (Ex. Radioactive Immunoassays/RIA) Biological Agent Risk Disease
4. Pressure Group
o In relation to gases Brucella spp. 3 Brucellosis
o Ex. hydrogen sulphide gas produced by bacteria, Coxiella burnetii 3 Q Fever
LPG, autoclave, nitrogen, hydrogen, and CO2 Hepa B, C, and D 3* Hepatitis
5. Heat/Cold Salmonella typhi 3* Typhoid Fever
o Heat – alcohol lamp, burners, etc. Francisella tularensis 3 Tularaemia
o Cold – freezer, liquid nitrogen, etc. Mycobacterium tuberculosis 3 Tuberculosis
6. Electrical Trycophyton 2 Dermatomycosis
o Risks in short circuited equipment, octopus plugs mentagrophytes
o Important to note appropriate diameter for Venezuelan equine 3 Venezuelan Equine
extension cords encephalitis virus encephalitis
 If equipment needs HIGH wattage, plug
Chlamydia psittaci (avian) 3 Psittacosis
directly into outlets (bawal sa extension)
Coccidioides immitis 3 Coccidioidomycosis
7. Gravity
o Glass wares with dangerous chemicals should
NOT be placed above eye level Table 2 – Top 10 LAIs according to Laboratory Category
o Bomb/Blast proof containers for reactive reagents Lab Category Number Percentage
o Shelves with special casing or safeguards Research 2307 58.8
against earthquakes Diagnostic 677 17.3
8. Motion Biological Product 134 3.4
o Use carts or trolleys rather than manually Teaching 106 2.7
carrying equipment Unspecified 697 17.8
Total 3921
*** Study from Aug. 20, 2010 to June 29, 2011 on prevalence *** Nice to know the tables pero to be safe, study their order 
of Salmonella typhimurium among lab workers and family
 109 cases across 38 states ROUTES OF TRANSMISSION
 Could have been prevented with biosafety measures 1. Ingestion
o Mouth pipetting
BIOSAFETY o Splashes of infectious material into mouth
 Biological safety is a concept that promotes: o Contaminated fingers entering the mouth
o Safe laboratory practices and procedures o Smoking and eating in workplace
o Proper use of containment equipment by 2. Inoculation
workers in the life science environment o Needle stick accidents
 Purpose: o Cuts from sharp objects
o To prevent occupationally acquired infections o Animal and insect bites and scratches
o To ensure that the organisms released to the 3. Contamination of Skin and Mucus Membranes
environment are dead o Spills or splashes into eyes, mouth, and nose
 History o Spills or splashes on non-intact skin
o Biosafety was pioneered by the Americans o Contaminated surfaces, equipment, articles
o Arnold G. Wedum (1976) 4. Inhalation
 Stated that BOTH good engineering o Numerous procedures that produce aerosols
design and technique are required to
prevent laboratory acquired infections RISK GROUPS
 Opened the 1st Biological Safety  Risk Group 1
Conference o NON-PATHOGENIC to humans and animals

Robles, Salacup, Salindo, See 1 of 5

 Risk Group 2 LAB ACCIDENTS
o May cause infection to an individual but effective  20% are known
treatment and preventive measures are available o Usually from accidents, animal, clinical
o Moderate individual risk, low community risk specimens, discarded glassware, autopsy,
o Exposure Routes: intentional infection, and AEROSOLS
 Inoculation  80% are unknown
 Ingestion o Person doesn’t know the cause of accident
 Membrane, Skin, and Sexual Contact
 Risk Group 3
AEROSOL
o May cause serious infection but doesn't
ordinarily spread  Aerosols are tiny particles or droplets suspended in air
o Treatment and preventive measures available  Biological aerosols (Bioaerosols) are suspensions that
o High individual risk, low community risk were released by or contain living organisms
o Exposure Route:  Size can range from 0.02µm (viruses) to 100µm (pollens)
 usually through inhalation only  Primary cause of LAIs
 Class 3* if pathogen uses other routes
 Risk Group 4
o May cause serious infection (can be FATAL)
o Readily transmitted (directly or indirectly)
o Treatment and preventive measures usually NOT
AVAILABLE
o High individual AND community risk

Table 3 – Infectious Dose and Route of Some Bacteria

Biological Agent Infectious Dose Route of
Inoculation
8
Escherichia coli 10 Ingestion
Escherichia 10 Ingestion
coli O157: H7
5
Bacillus cereus ≥ 10 per gram Ingestion
Campylobacter jejuni ≤ 500 Ingestion
Treponema pallidum 57 Intradermal
Francisella tularensis 10 Inhalation,
Ingestion Fig 1 – Size Ranges of Different Bioaerosols
3
Bacillus anthracis 8-50 Inhalation ,
Ingestion AEROSOL CHARACTERISTICS
Mycobacterium < 10 Inhalation  Large Droplets
tuberculosis and bovis o 100 µm in size
Coxiella burnetii 10 Inhalation o Settle quickly
Salmonella typhi 10
5
Ingestion o Easy to see
Shigella flexneri 180 Ingestion o Will contaminate the surfaces on which they
Treponema pallidum 57 Intradermal come to rest
Vibrio cholera 10
8
Ingestion  Small Droplets
Yersinia pestis 100-500 Inhalation, o ≤ 5 µm
Ingestion o Evaporate rapidly
Smallpox virus (V. 10-100 Inhalation(rare), o Travel further by air currents
major) Ingestion o Microorganism particles remain in a dried state
as "droplet nuclei”
Poliovirus 2 Ingestion
o Note - the smaller the droplet, the greater its
Influenza A2 virus < 790 Inhalation
potential to travel long distance
Venezuelan 1 Subcutaneous
 Proteins (serum, sputum)
encephalitis virus
o Slower evaporation
Hepatitis A virus 10-100 Ingestion,
o Settle more rapidly
intravenous
Adenovirus > 150 Intranasal *** SOP when there is a spill in the lab
Respiratory syncitial > 100 – 640 Intranasal 1. Cover with Tissue
virus 2. Spray with disinfectant (70% alcohol or Lysol)
Plasmodium 10 Intravenous 3. Leave for about 10-15 minutes (contact time between
falciparum contaminant and disinfectant)
Histoplasma 10 (mice) Inhalation 4. Wipe area
capsulatum 5. Dispose of tissue properly (put in disposal bags or
*** For a Cholera outbreak to occur, that means water is containers)
HIGHLY contaminated (since ID is very high) 6. Disinfect again

Robles, Salacup, Salindo, See 2 of 5

Table 4 – Practices and Procedures that Generate
Aerosols
 Subculturing and
streaking
Inoculating loop manipulation
 Cooling a loop on media
 Flaming a loop
 Mixing microbial
Pipetting
suspension
 Pipette spills on hard
surfaces
 Expelling air
 Withdrawing needle from
Needle and Syringe
stopper
 Injecting animals
 Centrifugation
 Using blenders, shakers,
and other mixing
Others equipment
 Pouring/Decanting
 Opening culture
containers
Table 5 – Estimated Aerosol Exposure Dose from Pipetting
Individual Exposed Good Technique Poor Technique
Person pipetting 25 1,200
Another person in <1 30
the room
ENGINEERING CONTROLS
 Refer to equipment and facility design elements
 Eliminates or reduce exposure to a biological, chemical
or physical hazard (e.g. aerosols)
 Place a barrier between workers and hazards
 HIERARCHY of controls
o Elimination
 By removing the problem
o Substitution
 Ex. Using the less dangerous strain
o Engineering Controls
 Ex. Local exhaust ventilation system,
lab fume hoods, enclosures, and shields
o Administrative Controls
 Ex. Job rotation, work instructions, and
safety inspectors
o Personal Protective Equipment (PPE) - lowest
 Ex. Gloves, mask, lab gowns, etc.
TYPES OF CABINETS
1. Biosafety Cabinet (Class II)
o Protects personnel, product, and environment
o Air from outside  forced down  circulated
towards HEPA filter  goes to working area 
re-circulated (down again) OR exits the BSC*
 * final step depends if BSC is Type A or B
o Do NOT use for volatile, toxic chemicals because
HEPA can’t filter such fumes

Table 6 – Estimated Aerosol Exposure from Other Sources

Source Exposure Dose
Blender lid opening 1200
Sonic homogenizer
 Maximum 1200
 Minimum 6
Streaking Petri Dish <1
Dropping flask culture 360
Splash on centrifuge rotor 120

GOOD TECHNIQUES
 Do not blow out the last drop using aspirator bulb
 Drain pipettes gently against the inside wall of tube
 Use pipettes with plugs
 Work over plastic-backed absorbent material
 Do not mix by alternate suction and expulsion in pipette
 Make sure that the lab blender has a gasket lid and leak
proof bearings
 Wait a few seconds before opening a lid after mixing

PRINCIPLES OF LAB SAFETY

 The following have to work hand in hand:
Practice Safety
Technique Equipment

Fig 2 – Airflow of a BSC II, Type A2

2. Clean Bench
o Protects product only
o Notice that air (unfiltered) is expelled towards
Facility the personnel and environment
Design o Use only for sterile, non-volatile, non-toxic
materials (such as saline solutions)

Robles, Salacup, Salindo, See 3 of 5

 Room Air
HEPA filtered Air
 Class III
Contaminated Air
*** Some guidelines when working with BSCs:
Fig 3 – Airflow of a Clean Bench (A) Front Opening, (B)
Sash, (C) HEPA filter, (D) Blower
3. Chemical Fume Hood
o Protects personnel only
o Does NOT protect products and environment
because air going in and out is NOT filtered
o Use for chemicals with toxic fumes
Fig 5 – Bacterial Incinerator
 Biosafety Level 1
Fig 4 – Airflow of a Fume Hood
*** HEPA Filters
 “High Efficiency Particulate Air”
 Removes particles greater than 0.3 µm
 Does NOT capture volatile agents
CLASSES OF BIOSAFETY CABINETS (BSCs)
 Class I
o Protects personnel and environment
o Can be used when working with radionuclides
and volatile toxic chemicals
o Unsterilized room air passes over work surface
and products
Robles, Salacup, Salindo, See
 Class II
o Protects personnel, product and environment
o HEPA-filtered (sterile) air flows over work
surface and products
o Used when working with infectious agents in
Risk Groups 2 and 3
o TYPE A1 and A2
 Ductless
 Differs only in speed:
 A1 - 75 fpm
 A2 – 100 fpm
o TYPE B1 AND B2
 Have dedicated exhaust duct system
 Same speed differences as Type A
o Protects personnel, product and environment
o Provides highest personnel protection
o Used for working with Risk Group 4 agents
o Completely sealed from outside environment
(ito yung may kasama nang gloves yung BSC)
o Entering and exiting air are HEPA-filtered
 Work from left to right (clean workspace  dirty)
 There should be at least 30 cm between the reagents
and 15 cm distance from the front grille
 Trash bag for tissues should be INSIDE
 Avoid using burners inside the BSC (short bursts of
flame only if kelangan talaga)
o Better if bacterial incinerators are used
BIOSAFETY LEVELS
o Basic laboratory
o For Risk Group 1 organisms
o Radioactive sign only
4 of 5
 Biosafety Level 2
o Basic laboratory
o For Risk Group 2 organisms
o Radioactive and biohazard signs
o Autoclave OUTSIDE the room

 Biosafety Level 3
o Containment laboratory
o For Risk Group 3 organisms
o Double doors
o Radioactive and biohazard signs
o Autoclave INSIDE the room
o Complete PPE “We learn lessons thru experience the hard way and
sometimes it’s expensive.” (Altura, 2012)

 Biosafety Level 4
o Maximum containment laboratory
o For Risk Group 4 organisms

REFERENCES:
Lecture notes
Belgian Biosafety Server
WHO Biosafety Manual
http://www.webconferences.com/nihoba/ppt/Risk%20Assessm
ent%2009-21-04_Barkley.pdf
http://www.lbl.gov/ehs/biosafety/manual/html/AppxE.shtml

Robles, Salacup, Salindo, See 5 of 5