Biosafety in Microbiology laboratory

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Biosafety in Microbiology laboratory
BIOSAFETY : Preventing lab-acquired infections
 Bacteria
 Viruses
 Fungi
 Human blood, unfixed tissue
 Human cell lines
 Recombinant DNA
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
Standard Microbiological Practices
NOT permitted in laboratories:
 Eating
 Drinking
 Smoking
 Handling contact lenses
 Pipetting by mouth
 Storing food and drink

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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
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
Barriers : Primary barriers
• Primary barriers: physical barriers or personal protective equipment between
lab worker and pathogen
• Gloves, masks, special breathing apparatuses
Barriers Secondarybarriers:
• Secondary barriers: structural aspects of the laboratory that make working
environment safer against infection

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• Sinks for hand washing, special containment areas, special air ventilation patterns
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

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Levels of Containment
 BL1 - microorganisms that don’t consistently cause disease in healthy adults
– E. coli K12, S. cerevisiae, polyomavirus
– Basic laboratory
– Standard Microbiological Practices

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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
Standard practices required:
– frequent handwashing
– door that can be kept closedwhen 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;
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
Biosafety Level 2 (BSL-2)
Agents associated withhuman disease

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– 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,
Clostridium botulinum, hepatitis B virus
Levels of Containment
BSL-2 : microorganisms of moderate potential hazard, transmitted by contact, ingestion,
puncture
– Salmonella, herpesvirus, human blood
– Basic laboratory
– Standard Practices PLUS
 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 occurif the material were spattered)
 Agents are pathogens for which immunization or antibiotic treatment is available

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 Extreme care should be taken with contaminated needles and sharp lab
instruments
Risk Group 2
Pathogenic for humans Unlikely a serious hazard Treatment and preventive measures
available Limited risk of spread of infection
 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
 Primary barriers: biosafety cabinets or other approved containment devices
 Personal protective equipment: lab coats, gloves, face protectionas 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

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Levels of Containment :
BSL-3 - 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
Biosafety Level- 3 (BSL-3):
Agents with potential for respiratory transmission, may cause serious and potentially
lethal infection
– May be studied at BSL-2 for diagnosis
Agents include Mycobacterium tuberculosis, St. Louis encephalitis virus, Francisella
tularensis, Coxiella burnetii
TB diagnostics and laboratory strengthening
Care of patients with tuberculosis starts with a quality assured diagnosis, obtained by growing and
identifying Mycobacteriumtuberculosis 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.

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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 precaution's are not taken a fraction of Medical
and Technical personal will be infected with grave consequences.
Risk Group 3
Pathogenic, cause serious disease Effective treatment and preventive measures usually available
Little person-to-person spread.
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;
– decontaminating all waste;
– changing contaminated protective lab clothing, decontaminating lab clothing
before laundering;

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– institutional policies regarding specimen collection and storage from workers to
establish exposure.
 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 .
Levels of Containment
 BSL-4 - microorganisms that cause lethal disease, with no known treatment or vaccine
 Ebola virus, Marburg virus
 Maximum containment lab; positive pressure ventilated suits (moon suits)
Risk Group 4
– Lethal, pathogenic agent Readily transmittable
– direct, indirect Effective treatment and preventive measures not usually available

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Biosafety Level- 4 (BSL-4)
• Dangerous and exotic agents with high risk of life-threatening disease, aerosol-
transmitted
• Related agents with unknown risk of transmission
• Agents (all viruses) include Marburg virus, Ebola virus,viruses that cause Congo-Crimean
hemorrhagic fever, Lassa fever
• 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
 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

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 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
 Primary barriers:
 Biosafety cabinets used at other biosafety levels
 Full-body, air-supplied, positive pressure personnel suit
 Secondarybarriers:
 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)
Laboratory Locations
 BSL-1: high schools, community colleges, municipal drinking water treatment
facilities

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 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
Standard Microbiological Practices
 NEVER
– recap, bend, or break needles
– discard needles or sharps into biological waste bags
– discard needles into regular trash
Biosafety is everyone's 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 .