Biosafety in Microbiology Laboratory

1. BIOSAFETY: PREVENTING LAB-ACQUIRED INFECTIONS Bacteria Viruses Fungi

Human blood, unfixed tissue Human cell lines Recombinant DNA
2. WHY IS BIOSAFETY IMPORTANT? Laboratories recognize hazards of processing
infectious agents Guidelines developed to protect workers in microbiological and medical
labs through engineering controls, management policies, work practices
3. STANDARD MICROBIOLOGICAL PRACTICES NOT permitted in laboratories:
Pipetting by Handling contact lenses Smoking Drinking Eating Storing food and
drink mouth
4. BIOSAFETY LEVELS Precautions so people researching or trying to identify organisms do
not become infected While handling or testing clinical specimens, workers could
accidentally infect themselves or coworkers Labs must adhere to very specific safety
regulations to work with organisms that pose a threat to human health
5. LABORATORIES DIVIDED ON BASIS OF NATURE OF MICROBES Labs divided into
4 biosafety levels; protective practices increase with each Biosafety Level 1 labs - work
with least dangerous agents, require fewest precautions Biosafety Level 4 labs - have
strictest methods because dealing with agents that are most dangerous to human health
6. BARRIERS - PRIMARY BARRIERS Primary barriers: physical barriers or personal
protective equipment between lab worker and pathogen Gloves, masks, special breathing
apparatuses
7. BARRIERS SECONDARY BARRIERS: Secondary barriers: structural aspects of the
laboratory that make working environment safer against infection Sinks for handwashing,
special containment areas, special air ventilation patterns
8. UNIVERSAL PRECAUTIONS Include hand hygiene, gloves, gown, masks, eye protection,
face shields, safe injection practices Require that all equipment or contaminated items are
handled to prevent transmission of infectious agents Special circumstances may require
additional precautions Protective clothing, special site decontamination
9. RISK GROUPS, BIOSAFETY LEVELS, PRACTICES AND EQUIPMENTBSL Laboratory
type Laboratory Safety equipment practices 1 Basic teaching, Good microbiological None
research techniques Open bench work 2 Primary health Good microbiological Open bench
PLUS services; diagnostic techniques, biological safety cabinet for potential services,
research protective clothing, aerosols biohazard sign 3 Special diagnostic As BSL 2 PLUS
Biological safety cabinet and/or other services, research special clothing, primary devices for
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all activities controlled access, directional airflow 4 Dangerous As BSL 3 PLUS Class III
biological safety cabinet, pathogen units airlock entry, shower positive pressure suits, double
ended exit, special waste autoclave (through the wall), filtered air
10. LEVELS OF CONTAINMENT BL1 - microorganisms that dont consistently cause disease
in healthy adults E. coli K12, S. cerevisiae, polyomaviru s Basic laboratory Standard
Microbiological Practices
11. BIOSAFETY LEVEL 1 (BSL-1) Agents not known to cause disease in healthy adults
Some organisms may cause disease in immunocompromised individuals Agents include
Bacillus subtilis, Naegleria gruberi, infectious canine hepatitis virus, non- pathogenic E. coli
species
12. BIOSAFETY LEVEL 1 (BSL-1) Standard practices required: frequent handwashing door
that can be kept closed when working; limits on access to the lab space when working; no
smoking, eating, drinking, storage of food in laboratory; care to minimize splashes and
actions that may create aerosols (tiny droplets); decontamination of work surfaces after
every use after any spills;
13. BIOSAFETY LEVEL 1 (BSL-1) Standard practices (continued): decontamination of
laboratory wastes; use of mechanical pipettes only (no mouth pipetting); "sharps"
precautions, including special containers for disposing of needles and other sharp objects;
maintenance of insect/rodent control program; use of personal protective equipment (lab
coats, latex gloves, eye protection or face shields) Open bench top sink for hand washing
14. BIOSAFETY LEVEL 2 (BSL-2) Agents associated with human disease Generally required
for any human-derived blood, bodily fluids, tissues in which infectious agent may be
unknown Agents include measles virus, Salmonella species, pathogenic Toxoplasma,
Clostridiu m botulinum, hepatitis B virus
15. LEVELS OF CONTAINMENT BL2 - microorganisms of moderate potential hazard,
transmitted by contact, ingestion, punctu re Salmonella, herpesvir us, human blood Basic
laboratory Standard Practices PLUS
16. BIOSAFETY LEVEL 2 (BSL-2) Primary hazards: accidental needle sticks exposure to
eyes and nose (mucous membranes) ingestion of infectious materials Agents do not cause
lethal infections, are not transmissible via airborne route (do not cause infection if tiny
droplets become airborne and are inhaled, which might occur if the material were spattered)
Agents are pathogens for which immunization or antibiotic treatment is available Extreme
care should be taken with contaminated needles and sharp lab instruments
17. RISK GROUP 2Pathogenic for humansUnlikely a serioushazardTreatment andpreventive
measuresavailableLimited risk of spreadof infection
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18. BIOSAFETY LEVEL 2 (BSL-2) Standard practices include BSL-1 plus: policies to restrict
access to lab; biohazard warning signs posted outside lab; surveillance of laboratory
personnel with appropriate immunizations offered; biosafety manual with definitions of
needed waste decontamination or medical surveillance policies; supervisory staff who have
experience working with infectious agents and specific training for laboratory personnel in
handling these agents
19. BIOSAFETY LEVEL 2 (BSL-2) Primary barriers: biosafety cabinets or other approved
containment devices Personal protective equipment: lab coats, gloves, face protection as
needed Protective clothing removed when personnel leave laboratory area Cabinets
thoroughly decontaminated daily and monitored for radiation for personal protection
Secondary barriers: BSL-1 barriers plus autoclave for glassware
20. LEVELS OF CONTAINMENT BL3 - microorganisms that cause serious disease,
transmitted by inhalation M. tuberculosis, yellow fever virus, hantavirus, Y. pestis (plague)
Containment lab: double door entry; directional airflow; all work in biosafety cabinet
21. BIOSAFETY LEVEL 3 (BSL-3) Agents with potential for respiratory transmission, may
cause serious and potentially lethal infection May Mycobacterium Agents includebe
studied at BSL-2 for diagnosis tuberculosis, St. Louis encephalitis virus, Francisella
tularensis, Coxiella burnetii()
22. TB DIAGNOSTICS AND LABORATORY STRENGTHENING Care of patients with
tuberculosis starts with a quality assured diagnosis, obtained by growing and identifying
Mycobacterium tuberculosis from clinical specimens and conducting DST of the organism to
confirm or exclude resistance. Uptake of TB diagnostic technologies requires appropriate
laboratory infrastructure and adequate policy reform at country level to enable their effective
use in TB screening and diagnostic algorithms Laboratory infrastructure, appropriate
biosafety measures and maintenance Equipment validation and maintenance Specimen
transport and referral mechanisms Management of laboratory commodities and supplies
Laboratory information and data management systems Laboratory quality management
system are a priority.
23. MDR TB , XMDR-TB AND BIOSAFETY With growing incidences of MDR-TB and
XMDR-TB it is highly essential all Microbiology laboratories must install Grade 3 Biosafety
cabinets to prevent exposure to Infection. It necessary precautions are not taken a fraction of
Medical and Technical personal will be infected with grave consequences.
24. RISK GROUP 3Pathogenic, causeserious diseaseEffective treatmentand preventivemeasures
usuallyavailableLittle person-to-person spread
25. BIOSAFETY LEVEL 3 (BSL-3) Standard practices include BSL-2 plus: strictly controlled
access to the lab; specific training for lab personnel in handling potentially lethal agents;
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decontaminating all waste; changing contaminated protective lab clothing, decontaminating

lab clothing before laundering; institutional policies regarding specimen collection and
storage from workers to establish exposure
26. BIOSAFETY LEVEL 3 (BSL-3) Primary barriers: Similar to BSL-2 personal protective
equipment Respiratory equipment if risk of infection through inhalation Secondary
barriers: All BSL-2 barriers Corridors separated from direct access to lab Access through
self-closing double doors Air handling systems to ensure negative air flow (air flows into
the lab) Air pumped into lab not re-circulated in building
27. LEVELS OF CONTAINMENT BL4 - microorganisms that cause lethal disease,
Maximum Ebola virus, Marburg virus with no known treatment or vaccine containment
lab; positive pressure ventilated suits (moon suits)
28. RISK GROUP 4Lethal, pathogenicagentReadily transmittable direct, indirectEffective
treatment andpreventive measuresnot usually available
29. BIOSAFETY LEVEL 4 (BSL-4) Dangerous and exotic agents with high risk of lifethreatening disease, aerosol-transmitted Related agents with Agents (all viruses) include
Marburgunknown risk of transmission virus, Ebola virus, viruses that cause Congo-Crimean
hemorrhagic fever, Lassa fever
30. BIOSAFETY LEVEL 4 (BSL-4) Primary hazards: respiratory exposure to infectious
aerosols mucous membrane exposure to infectious droplets accidental sticks with needles
or other sharp objects contaminated with infectious material For example In late 1960s, 25
laboratory-acquired Marburg infections, including 5 deaths Workers studying infected
monkeys from Uganda First documented naturally-occurring human case occurred in 1975
31. BIOSAFETY LEVEL 4 (BSL-4) Personnel must receive specialized training in handling
extremely dangerous infectious agents, containment equipment and functions Access to lab
is restricted: immunocompromised persons are never allowed to enter the lab Standard
practices include BSL-3 plus: strictly controlled access to the laboratory; changing
clothing before entering and exiting lab (showering upon exiting recommended);
decontaminating all material exiting facility
32. BIOSAFETY LEVEL 4 (BSL-4) Primary barriers: Biosafety cabinets used at other
biosafety levels Full-body, air-supplied, positive pressure personnel suit Secondary
barriers: All physical barriers at BSL-3 isolated zone or a separate building; dedicated
supply and exhaust, vacuum, decontamination systems; a recommended absence of
windows (or sealed and resistant to breakage)
33. ACTIVITY SPECTRUM OF SELECT DETERGENTS AND DISINFECTANTS Myco BG+
BG- Spores Yeast Virus Prions BAlcohol 70 ++ ++ ++ 0 + + 0Aldhydes +++ +++ ++ + ++
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+ ++ 0Ammonium IV +++ + 0 0 + + 0Anilides + 0 NP NP 0 NP 0Chlorhexidine +++ ++ 0 0

+ + 0Cl compounds +++ +++ ++ ++ ++ ++ + (a)Iodine (+ der.) +++ +++ ++ ++ ++ ++ 0Hg
compounds ++ ++ 0 0 + 0 ou + 0Phnols : Variable activity depending on components
(b)Hexachlorophne +++ + 0 0 + 0 0(a) Bleach (6%) during 60 min at 20C ; (b) discussion
on efficacy of phnol on prions
34. LABORATORY LOCATIONS BSL-1: high schools, community colleges, municipal
drinking water treatment facilities BSL-2: local health departments, universities, state
laboratories, private laboratories (hospitals, health care systems), industrial laboratories
(clinical diagnostic companies) BSL-3: state health departments, universities, private
companies, industry, federal government (NIH, CDC) BSL-4: only 15 facilities in the US 9
federal (CDC, NIH), 4 university (Georgia State University, University of Texas Medical
Branch), 1 state, 1 private Renovations underway at several labs, new facilities proposed at
additional sites
35. STANDARD MICROBIOLOGICAL PRACTICES NEVER recap, bend, or break needles
discard needles or sharps into biological waste bags discard needles into regular trash
36. BIOSAFETY IS EVERYONES CONCERN Laboratorians have long recognized hazards of
processing infectious agents Biosafety guidelines developed to protect workers in
microbiological and medical labs through a combination of safeguards including engineering
controls, management policies and work practices. Issue described differences between
biosafety levels Help you understand process labs may have to undertake to identify
microorganism, why every lab cannot test for every organism
37. REFERENCES UNC School of Public Health Laboratory Safety Levels WHO guidelines
on laboratory training for Field Epidemiologists