Basic Microbiology 202 Lecture PDF

Basic Microbiology 202 Mahbuba Rahman, PhD Assistant Professor Department of Biochemistry and Microbiology NSU Biosafety level and risk group of microorganism Risk Groups Risk Groups are classifications that describe the relative hazard posed by inf...

Basic Microbiology 202 Mahbuba Rahman, PhD Assistant Professor Department of Biochemistry and Microbiology NSU Biosafety level and risk group of microorganism Risk Groups Risk Groups are classifications that describe the relative hazard posed by infectious agents or toxins in the laboratory. The risk group to which an infectious agent or toxin is assigned is the primary, but not only, consideration used in a biological risk assessment to determine the appropriate biosafety level in which a worker can handle the infectious agent or toxin. Other considerations used in a biological risk assessment include the ability of an infectious agent or toxin to cause disease, the way in which the infectious agent or toxin causes disease, the activities performed in the laboratory, the safety equipment and design elements present in the laboratory, and the health and training of the laboratory worker. (continued next slide)  Risk group levels do not always correspond to biosafety levels. For example, a specific research project’s biological risk assessment for the use of human immunodeficiency virus (HIV), a Risk Group 3 agent, may correctly determine that HIV can be handled under Biosafety Level 2 conditions. Risk groups are designated from 1 (the lowest risk) to 4 (the highest risk) https://www.phe.gov/s3/BioriskManagement/biosafety/Pages/ Risk-Groups.aspx The WHO (World Health Organization) has classified infectious microorganisms by risk groups and the following list provides an overview of the risk levels when working with different pathogens. WHO risk group 1 – minimal risk Microorganisms that usually do not cause human disease, such as Escherichia coli K12 or Lactobacillus. WHO risk group 2 – moderate risk Microorganisms that cause treatable or self-healing diseases and are difficult to contract via aerosol in a laboratory setting, such as salmonella or measles virus. WHO risk group 3 – high risk Highly contagious microorganisms that cause serious diseases, such as TB caused by M. tuberculosis. WHO risk group 4 – very high risk Highly contagious microorganisms that cause serious diseases, even epidemics, with high mortality rate, such as Ebola virus or Lassa fever virus. II BIOSAFETY IN THE LABORATORY Microbiological laboratories are rooms or buildings equipped for scientific experimentation or research on microorganisms. Many microbiological laboratories work specifically with pathogenic microorganisms, some of which are highly dangerous, especially in association with the AMB (aeromicrobiological) pathway. Also, many types of equipment, such as centrifuges and vortexes, that are commonly used in microbiological laboratories can promote the aerosolization of microorganisms. Thus, laboratories and specialized equipment used in these laboratories (e.g., biosafety cabinets) are designed to control the spread of airborne microorganisms. There are essentially four levels of control designed into laboratories, depending on the type of research being conducted. These levels of control are termed biosafety levels 1–4, with 1 being the lowest level of control and 4 the highest level of control. Within these laboratories, biosafety cabinets are essentially isolation chambers that provide safe environments for the manipulation of pathogenic microorganisms. Biological Safety Cabinets Biological safety cabinets (BSCs) are among the most effective and commonly used biological containment devices in laboratories that work with infectious agents. There are two basic types of biosafety cabinets currently available (Class II and Class III), each of which has specific characteristics and applications that dictate the type of microorganisms it is equipped to contain. Properly maintained biosafety cabinets provide safe environments for working with microorganisms. Class II biosafety cabinets are characterized by having considerable negative-pressure airflow that provides protection from infectious bioaerosols generated within the cabinet, and Class III biosafety cabinets are characterized by total containment. Class I cabinets are also in existence, but they are no longer produced and are being replaced by Class II cabinets for all applications.  Class II biosafety cabinets, of which there are several types, are suitable for most work with moderate-risk Pathogens. Class II BSCs differ from Class I cabinets in that they allow only air from a HEPA-filtered (sterile) supply to flow over the work surface. A Class II type A2 BSC is shown in Figure. An internal fan draws room air (supply air) into the cabinet through the front opening and then into the front intake grill. After passing through the grill, the supply air is drawn upwards and through a HEPA filter before flowing downwards over the work surface. As the air flows downwards at about 6–18 cm above the work surface it splits so that approximately one half of the volume of the air passes through the front exhaust grill and the other half passes through the rear exhaust grill. Any aerosol particles generated at the work surface are immediately captured in this downward airflow and passed through the front or rear exhaust grills, thereby providing the highest level of product protection. The air is then discharged through the rear plenum into the space between the supply filter and exhaust filter located at the top of the cabinet. Owing to the relative size of these filters, 60–70% of the air recirculates through the supply HEPA filter back into the work zone; the remaining 30–40% passes through the exhaust filter into the room or outdoors. Air from a Class II type A2 exhaust can be recirculated to the room or discharged to the outside of the building through a thimble connected to a dedicated duct; it must NOT be discharged through the building’s exhaust system. Youtube link https://www.google.com/search?sca_esv=cd1c023c2da61779& sca_upv=1&rlz=1C1CAFC_enQA926QA926&q=labelled+biosafet y+cabinet+diagram&tbm=vid&source=lnms&fbs=AEQNm0Aa4s jWe7Rqy32pFwRj0UkWd8nbOJfsBGGB5IQQO6L3J_86uWOeqw dnV0yaSF- x2jqhDR2NB58jThfZD3wixUPwSzOqLO65NSJe5gSIGb1tXpSMjcU dYSTWLilYwsf095amyY9LLwzqb3PXy1Ofij7CeBpf0HmHplF001_8 jfjaqI9kkdc&sa=X&ved=2ahUKEwi06- qS_9WIAxWDR2wGHVw_KUkQ0pQJegQIERAB&biw=1643&bih =976&dpr=0.9#fpstate=ive&vld=cid:b8f34013,vid:PfUohZu7l2k, st:0 The Class III biosafety cabinet ( Fig) is a completely enclosed environment that offers the highest degree of personnel and environmental protection from bioaerosols. Class III cabinets are used for high-risk pathogens (mentioned in earlier slides). All operations in the work area of the cabinet are performed through attached rubber gloves. Class III cabinets use complete isolation to protect workers. All air entering the cabinet is filtered using a HEPA filter, and the air leaving the cabinet is filtered by two HEPA filters in series. The exhaust may also include biocidal treatment such as incineration following the HEPA filtration to further ensure complete biological inactivation. In addition to these safeguards, Class III cabinets are connected with airtight seals to all other laboratory equipment (such as incubators, refrigerators, and centrifuges) that is needed for working with the pathogens while using the cabinet. The Class III cabinet must also be connected to autoclaves and chemical dunk tanks used to sterilize or disinfect all materials entering or exiting the cabinet. Youtube link https://www.google.com/search?q=biosafety+level+3&sca_esv =cd1c023c2da61779&sca_upv=1&rlz=1C1CAFC_enQA926QA92 6&biw=1643&bih=976&tbm=vid&ei=MuHvZvPYLtel2roPxIvCqQ s&ved=0ahUKEwjzuYOwnNaIAxXXklYBHcSFMLUQ4dUDCA0&ua ct=5&oq=biosafety+level+3&gs_lp=Eg1nd3Mtd2l6LXZpZGVvIhF iaW9zYWZldHkgbGV2ZWwgMzIFEAAYgAQyBRAAGIAEMgUQABi ABDILEAAYgAQYhgMYigUyCxAAGIAEGIYDGIoFMgsQABiABBiGAx iKBTIIEAAYgAQYogQyCBAAGIAEGKIEMggQABiABBiiBEirJFAAWKk gcAB4AJABAJgBzQGgAYUXqgEGMC4xNi4xuAEDyAEA- AEBmAIRoAK3F8ICChAAGIAEGEMYigXCAg4QABiABBixAxiDARiK BcICCxAAGIAEGLEDGIMBwgIIEAAYgAQYsQOYAwCSBwYwLjE2LjG gB4Jg&sclient=gws-wiz- video#fpstate=ive&vld=cid:b5ff233c,vid:5isVPg_Sx5w,st:0 Biosafety Laboratories Biosafety laboratories are carefully designed environments where infectious or potentially infectious agents are handled and/or contained for research or educational purposes. The purpose of a biosafety laboratory is to prevent the exposure of workers and the surrounding environment to biohazards. There are four levels of biohazard control, which are designated as biosafety levels 1 through 4. Biosafety level 1, as defined by the Centers for Disease Control ( U.S. Department of Health and Human Services: CDC-NIH, 1993 ), indicates laboratories where well-characterized agents that are not associated with disease in healthy adult humans are handled. In general, no safety equipment is used other than sinks for hand washing and only general restrictions are placed on public access to these laboratories. Work with the microorganisms can be done on bench tops using standard microbiological techniques. A good example of a biosafety 1 laboratory is a teaching laboratory used for undergraduate microbiology classes. Biosafety level 2 indicates an area where work is performed using agents that are of moderate hazard to humans and the environment. These laboratories differ from biosafety 1 laboratories in that the personnel have specialized training in the handling of pathogens and access to the work areas is limited. Many procedures that may cause aerosolization of pathogenic microorganisms are conducted in biological safety level Class II cabinets or other physical containment equipment to protect the laboratory workers. Biosafety level 3 indicates laboratories where agents that can cause serious or fatal disease as a result of AMB exposure are handled. As with biosafety 2, all personnel are specifically trained to handle pathogenic microorganisms. All procedures involving these infectious agents are conducted in biological safety level Class II cabinets or other physical containment devices. These facilities also have permanent locks to control access, negative airflow, and filtered ventilation in order to protect the public and the surrounding environments. With certain pathogens used in biosafety 3 laboratories, Class III safety hoods may also be used and clothes must be changed before leaving the premises. Biosafety level 4 is the highest level of control and is indicated for organisms that have high potential for life threatening disease in association with aerosolization. To work in these facilities, personnel must have specialized training beyond that required for biosafety levels 2 and 3. Biosafety level 4 laboratories are 100% isolated from other areas of a building and may even be separated from other buildings altogether. Work in these areas is confined exclusively to Class III biological safety cabinets.