BIOL 333 Microbiology Laboratory PDF

Department of Biological Sciences Faculty of Science What is Biosafety and Why is it Important? Beirut Arab University ❖ Biosafety:...

Department of Biological Sciences Faculty of Science What is Biosafety and Why is it Important? Beirut Arab University ❖ Biosafety: It is a system for the safe handling of toxic and dangerous biological and chemical substances. In Medicine: It refers to the levels of lab containment protocols, measured as Bio Safety Level (BSL) 1, 2, 3, & 4 in BIOL 333: rising order of danger. Microbiology Laboratory ❖ Importance of Biosafety: Laboratories recognize hazards of processing infectious agents. Guidelines developed to protect workers in Prepared by Lama Fayad microbiological and medical labs through engineering Presented by Malak Mezher & Zeina Darwish controls, management policies, work practices. 3 Work in a Microbiological Laboratory Safety in a microbiological laboratory substantially differs from that in other (chemical or physical) laboratories because, in addition to hazardous chemicals, substances and operations that pose a laboratory work-related risk, there is a risk of infection when working with microbes. ❖ Lab-Biosafety: Preventing acquired infections LAB 1: ✓ Bacteria ✓ Viruses Biosafety in Microbiology ✓ Fungi ✓ Human blood, unfixed tissue Laboratory ✓ Human cell lines ✓ Recombinant DNA 4  Standard Microbiological Practices Behavior and Work in a Microbiological Laboratory ❖ NOT permitted in laboratories: The basic biosafety practices, especially at biosafety level one and two laboratories are as follows: Eating ✓ Students should get appropriate training on the potential hazards at the start of the laboratory course. Drinking ✓ Training is necessary and should be documented. Smoking ✓ Access to the laboratory should be limited to formal students and laboratory personnel. Handling contact lenses ✓ Persons who are at increased risk of infection (pregnant women, nursing mothers, and Pipetting by mouth immunocompromised people) should be excluded from laboratory work. Storing food and drink ✓ 100 % cotton laboratory coat and other protective equipment should be used. ✓ Gloves should be worn, particularly when the hand skin is injured. NEVER ✓ Laboratory safety glasses and face shield should be worn. ✓ Recap, bend, or break needles. ✓ Confine long hair and loose clothing. ✓ Discard needles or sharps into biological waste bags. ✓ Closed shoes should be worn in the laboratory. ✓ Discard needles into regular trash. ✓ Nails should be kept clipped and neat. ✓ Respect the requirements of the spill control and clean-up instructions. ✓ Do not work alone in the laboratory if the conducted procedures are hazardous. Always make a work plan and handle infectious materials with extreme care. 5 7 Biosafety is Everyone’s Concern Behavior and Work in a Microbiological Laboratory Laboratorians have long recognized hazards of processing infectious agents. ✓ Decontaminate the workbench before and after work. ✓ Do not pipette by mouth, use pipetting devices. Biosafety guidelines developed to protect workers in microbiological and medical labs through a ✓ Avoid the use of sharp objects (syringe, needles, and broken glass) combination of safeguards including engineering controls, management policies and work practices. ✓ Do not speak when working with cultures. Issue described differences between biosafety levels. ✓ Wash your hands regularly (especially following work). ✓ Do not take pen and ink or any other objects into your mouth. Help you understand process labs may have to undertake to identify microorganism, why every lab ✓ Do not store food in the laboratory. cannot test for every organism. ✓ Do not handle contact lenses in the laboratory. Persons wearing contact lenses should wear eye protection (protective safety goggles). ✓ Cultures, stocks, and wastes should be decontaminated (autoclaving) before disposal. ✓ Spills and accidents should be immediately reported to the laboratory supervisors. ✓ Chemicals and equipment should be always properly labelled. ✓ Aseptic work with cultures is practically always performed with gas burners on. The opening of glass culture vessels and metal caps are flamed between operations. 6 8  Behavior and Work in a Microbiological Laboratories Divided on Basis of Nature Laboratory of Microbes ✓ Be aware that in a microbiological laboratory, an extremely broad range of chemical hazards is Labs are divided into 4 biosafety levels; protective practices increase with each level. represented because of the variety of chemicals used (solvents, acids, bases, carcinogens, and Biosafety Level 1 labs - Work with least dangerous agents, require fewest precautions. mutagens). Biosafety Level 4 labs - Have strictest methods because dealing with agents that are most dangerous to ✓ Be sure of the proper use of chemicals! human health. 9 11 Biosafety Levels Barriers There are precautions so people researching or trying to identify organisms do not become infected. ❖ Primary barriers: Are physical barriers or personal protective equipment While handling or testing clinical specimens, workers could accidentally infect themselves or between lab worker and pathogen. coworkers. Gloves, masks, special breathing apparatus. Labs must adhere to very specific safety regulations to work with organisms that pose a threat to human ❖ Secondary barriers: health. Structural aspects of the laboratory that make working environment safer against infection. Sinks for handwashing, special containment areas, special air ventilation patterns. 10 12  Risk Groups, Biosafety Levels, Practices, Biosafety Level 1 (BSL-1) and Equipment ❖ Standard practices required: BSL Laboratory type Laboratory practices Safety equipment Frequent handwashing. Door that can be kept closed when working. 1 Basic teaching, research Good microbiological None Limits on access to the lab space when working. techniques No smoking, eating, drinking, or storage of food in laboratory. Open bench work Care to minimize splashes and actions that may create aerosols (tiny droplets). 2 Primary health Good microbiological Open bench PLUS Decontamination of work surfaces after every use after any spills. services; diagnostic techniques, biological safety cabinet for potential aerosols Decontamination of laboratory wastes. services, research protective clothing, Use of mechanical pipettes only (no mouth pipetting). biohazard sign “Sharps" precautions, including special containers for disposing of needles and other sharp objects. Maintenance of insect/rodent control program. 3 Special diagnostic As BSL 2 PLUS Biological safety cabinet and/or other primary Use of personal protective equipment (lab coats, latex gloves, eye protection, or face shields). services, research special clothing, devices for all activities controlled access, directional airflow 4 Dangerous As BSL 3 PLUS Class III biological safety cabinet, positive pressure pathogen units airlock entry, shower exit, suits, double ended autoclave (through the wall), special waste filtered air 13 15 Biosafety Level 1 (BSL-1) Biosafety Level 2 (BSL-2) ❖ Standard practices include BSL-1 plus: Policies to restrict access to lab. Basic laboratory. Biohazard warning signs posted outside lab. Standard Microbiological Practices. Surveillance of laboratory personnel with appropriate immunizations Open bench top sink for hand washing. offered. Biosafety manual with definitions of needed waste decontamination ❖ Levels of containment: or medical surveillance policies. Supervisory staff who have experience working with infectious Microorganisms that don’t consistently cause disease in healthy agents and specific training for laboratory personnel in handling adult, like non-pathogenic Escherichia coli K12, these agents. Saccharomyces cerevisiae, polyomavirus, Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus. ❖ Levels of containments: Some organisms may cause disease in immunocompromised Agents associated with human disease. individuals. Generally required for any human-derived blood, bodily fluids, and tissues in which infectious agent may be unknown. Agents of moderate potential hazard include measles virus, Salmonella species, pathogenic Toxoplasma, Clostridium botulinum, hepatitis B virus. 14 16  Biosafety Level 2 (BSL-2) Biosafety Level 3 (BSL-3) ❖ Barriers: Double door entry, directional airflow, all work in biosafety cabinet. Primary barriers: ✓ Biosafety cabinets or other approved containment devices. ❖ Levels of containment: ✓ Personal protective equipment: lab coats, gloves, and face protection as needed. Microorganisms that cause serious disease, transmitted by inhalation, like M. tuberculosis, yellow fever ✓ Protective clothing removed when personnel leave laboratory area. ✓ Cabinets thoroughly decontaminated daily and monitored for radiation for personal protection. virus, hantavirus, Y. pestis (plague). Secondary barriers: Agents with potential for respiratory transmission, may cause serious and potentially lethal infection. ✓ BSL-1 barriers plus autoclave for glassware. May be studied at BSL-2 for diagnosis. ❖ Primary hazards: ❖ Risk group 3: Accidental needle sticks. Exposure to eyes and nose (mucous membranes). Pathogenic, cause serious disease. Ingestion of infectious materials. Effective treatment and preventive measures usually available. Agents do not cause lethal infections, are not transmissible via airborne route (do not cause infection if Little person-to-person spread. 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 19 Biosafety Level 2 (BSL-2) Biosafety Level 3 (BSL-3) ❖ Mycobacterium tuberculosis: Mdr – Tb , XMdr – Tb, and biosafety ✓ With growing incidences of MDR-TB and XMDR-TB, it is highly essential all Microbiology laboratories must ❖ Risk group 2: install Grade 3 Biosafety cabinets to prevent exposure to Pathogenic for humans. Infection. Unlikely a serious hazard. ✓ Its necessary precautions are not taken a fraction of Treatment and preventive measures available. Medical and Technical personal will be infected with Limited risk of spread of infection. grave consequences. 18 20  Biosafety Level 3 (BSL-3) Biosafety Level 4 (BSL-4) ❖ Standard practices include BSL-2 plus: ❖ Standard practices include BSL-3 plus: Strictly controlled access to the lab. Personnel must receive specialized training in handling extremely dangerous infectious agents, Specific training for lab personnel in handling potentially lethal agents. containment equipment and functions. Decontaminating all wastes. Access to lab is restricted: immunocompromised persons are never allowed to enter the lab. Changing contaminated protective lab clothing, decontaminating lab clothing before laundering. Strictly controlled access to the laboratory. Institutional policies regarding specimen collection and storage from workers to establish exposure. Changing clothing before entering and exiting lab (showering upon exiting recommended). Decontaminating all material exiting facility. ❖ Barriers: Primary barriers: ❖ Barriers: ✓ Similar to BSL-2 personal protective equipment. Primary barriers: ✓ Respiratory equipment if risk of infection through inhalation. ✓ Biosafety cabinets used at other biosafety levels. Secondary barriers: ✓ Full-body, air-supplied, positive pressure personnel suit. ✓ All BSL-2 barriers. Secondary barriers: ✓ Corridors separated from direct access to lab. ✓ Access through self-closing double doors. ✓ All physical barriers at BSL-3. ✓ Air handling systems to ensure negative air flow (air flows into the lab). ✓ Isolated zone or a separate building. ✓ Air pumped into lab not re-circulated in building. ✓ Dedicated supply and exhaust, vacuum, decontamination systems. ✓ Recommended absence of windows (or sealed and resistant to breakage). 21 23 Biosafety Level 4 (BSL-4) Activity spectrum of selected detergents and ❖ Levels of containment: disinfectants Microorganisms that cause lethal disease, with no known treatment or BG+ BG- MycoB Spores Yeast Virus Prions vaccine. Dangerous and exotic agents with high risk of life-threatening disease, Alcohol 70° ++ ++ ++ 0 + + 0 lethal, pathogenic, transmittable. Aldehydes +++ +++ ++ + +++ ++ 0 Related agents with unknown risk of transmission. Ammonium IV +++ + 0 0 + + 0 Agents (all viruses) include Marburg virus, Ebola virus, viruses that cause Anilides + 0 NP NP 0 NP 0 Congo-Crimean hemorrhagic fever, Lassa fever Effective treatment and preventive measures not usually available. Chlorhexidine +++ ++ 0 0 + + 0 Maximum containment lab, positive pressure ventilated suits (moon suits). Cl compounds +++ +++ ++ ++ ++ ++ + (a) Iodine (+ der.) +++ +++ ++ ++ ++ ++ 0 ❖ Primary hazards: Respiratory exposure to infectious aerosols. Hg compounds ++ ++ 0 0 + 0 ou + 0 Mucous membrane exposure to infectious droplets. Phenols Variable activity depending on components (b) Accidental sticks with needles or other sharp objects contaminated with Hexachlorophene +++ + 0 0 + 0 0 infectious material. (a) Bleach (6%) during 60 min at 20°C ; (b) discussion on efficacy of phénol on prions 22 24  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. 25 THANK YOU