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1.2: Biosafety levels and PPE

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    Learning Outcomes

    • Recognize distinctions between Biosafety Levels (BSL) identify PPE used for working in the lab under BSL classifications  
    • Demonstrate and assess the proper use of Personal Protective Equipment (PPE) and be prepared to safely conduct research


    BioSafety Levels


    Pyramid showing the four BSLs with the lowest risk microbes at the bottom, representing BSL-1, and the highest risk microbes at the top, representing BSL-4.

    Figure 1: Biosafety levels and risk. Image from CDC.


    Handling microbes requires specialized laboratory facilities and techniques.  Biosafety is the application of safety precautions that reduce a scientists’ risk of exposure to a potentially infectious microbe and limit contamination of the work environment. Bacteria pose varying degrees of risk both in a controlled laboratory environment and in their natural settings. Therefore, the level of containment necessary for working safely with bacterial cultures also varies according to a system that classifies microbes into one of four biosafety levels (BSL), which provides minimum standards for safe handling of microbes at each level. BSLs are defined and containment practices are detailed by the Centers for Disease Control and Prevention (CDC) for laboratories in the United States. The full document, “BiosafetyinMicrobiologicalandBiomedicalLaboratories,” can be viewed in its entirety at

    We designate most of our labs under 4 special hazard categories called biosafety levels (BSLs).  Each level has specific controls for containment of microbes and biological agents.  Each biosafety level builds on the controls of the level before it.  All of these levels follow “standard microbiological practices” : which are those practices common to all labs, which include not eating, drinking, or applying cosmetics, washing hands after working in the lab, routinely decontaminating work area. 

    BSL1: If you work in a lab that is designated a BSL-1, the microbes there are not known to consistently cause disease in healthy adults and present minimal potential hazard to laboratorians and the environment. An example of a microbe that is typically worked with at a BSL-1 is a nonpathogenic strain of E. coli.  Work can be done on an open lab bench, requires a sink.  Requires personal protective equipment (PPE) such as lab coats, gloves, eye protection.

    BSL 2: BSL-2 builds upon BSL-1. If you work in a lab that is designated a BSL-2, the microbes there pose moderate hazards to laboratorians and the environment. The microbes are typically indigenous and associated with diseases of varying severity. An example of a microbe that is typically worked with at a BSL-2 laboratory is Staphylococcus aureus. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosols in a lab setting, such as Clostridium difficile, most Chlamydiae, hepatitis A, B, and C, influenza A viruses, Salmonella. BSL-2 differs from BSL-1 in that: laboratory personnel have specific training in handling pathogenic agents and are directed by scientists with advanced training; access to the laboratory is limited when work is being conducted; extreme precautions are taken with contaminated sharp items; and certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. The Microbiology Teaching labs at NC State are designated as BSL-2 laboratory space.

    BSL-3: is required for work involving indigenous or exotic agents, and they can cause serious or potentially lethal disease that are transmitted through the air (via aerosols).  Respiratory transmission is the inhalation route of exposure. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents, and must be supervised by scientists competent in handling infectious agents and associated procedures. Lab personnel are under medical surveillance and  might receive immunizations for microbes they work with.  All procedures involving the manipulation of infectious materials must be conducted within Biosafety Cabinets (BSCs) (shown at the top image), or other physical containment devices, or by personnel wearing appropriate personal protective equipment (eg respirators). A BSL-3 laboratory has special engineering and design features that prevent the release of microorganisms to the environment. Facilities have hands free sink, exhaust air cannot be recirculated, entrance is through two sets of self closing and clocking doors.  Microbes that are worked on in BSL3 facilities are Mycobacterium tuberculosis which cause tuberculosis, etc.

    BSL-4 labs builds on the containment requirements of BSL-3 and is the highest level of biological safety.  BSL-4 labs is required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, aerosol transmission or unknown risk of transmission.  The microbes in BSL4 labs cause infections that are frequently fatal and generally there are no vaccines or treatments for these infections. There are only a small number of such labs in the U.S (<10) and the world.  Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents. Access to the laboratory is controlled by the laboratory supervisor. All handling of agents must be performed in a gas tight Class III Biosafety Cabinet or by personnel wearing a positive pressure protective suit. BSL-4 Laboratories have special engineering and design features to prevent microorganisms from being released into the environment. The lab is in a separate building or isolated restricted zone of the building, and has a dedicated supply and exhaust air, as well as vacuum lines and decontamination systems.  Personnel mush change clothing before entering and shower upon exiting.  Most of the pathogens worked on are viruses: Crimean-Congo hemorrhagic fever caused by Ebola, Junin, Lassa, Machupo, Marburg viruses , and tick-borne encephalitis virus complex (including Absettarov, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian Spring-Summer encephalitis).


    Personal Protective Equipment (PPE)

    Personal Protective Equipment, or PPE, is the clothing and equipment that forms the last line of defense between you and harmful materials in the laboratory environment. It’s essential that you know what you should be wearing, when you should be wearing it, and how it should be stored, cleaned, maintained and disposed of. (1)

    Basic PPE provided in the Microbiology laboratory includes: Disposable gloves, lab coat, safety glasses. We will have disposable face masks.  

    Disposable gloves serve as a barrier between your hands and any chemical, biological, or physical hazards that can enter your body through your skin.  These gloves cannot protect you from all barriers, and most disposable gloves we use in a microbiology lab are made of some synthetic material, such as nitrile or latex.  Any prolonged exposure to a chemical agent can permeate these types of gloves, so it is critical that you safely remove your gloves if you are exposed to a chemical spill and it comes in contact with your gloves.  Gloves should be changed when they are contaminated by biological or chemical hazards.   There are many other types of gloves, such as ones for handling very hot items out of the autoclave, or for very cold items out of a -80degC freezer or for handling liquid nitrogen.  The type of glove you use depends on the work you are doing in the lab.

    Lab coats protect the user's skin and personal clothing from accidental contact with biological or chemical hazards. They also prevent the spread of contamination outside of the lab (provided they are not worn outside the lab).  Lab coats serve as a removable barrier in the event of an a spill or splash of hazardous substances. They should fit the user well (not too large or it will get caught on things and not too small as it may hinder movement), be long-sleeved, a secure cuff, knee-length or longer, fire resistant material, high buttons to provide exposure of the chest or neck area.  They come in a variety of materials and provide varying degrees of protection. There are splash resistant coats, static free coats, chemical resistant coats and flame resistant coats.  Most lab coats we use in a microbiology teaching lab are made of a synthetic material that is fire resistant.  Always select a coat that provides the type of protection that is appropriate for your needs. 

    Safety glasses/goggles provide a barrier to your eyes and prevent exposure to chemical (eg chemical reagents) physical (eg dust, flying objects), and biological (eg bacterial culture splashes) hazards.  Most microbiology labs have standard safety glasses or goggles that wrap around the eyes and avoid splashes, they may have features such as UV light barriers, impact features, and some are designed to be worn over your regular glasses, these are called "Over the glasses" (OTG) safety glasses.  There are also labs that have high intensity light sources such as UV light, lasers and the appropriate safety glasses must be used. 

    Face shields should be worn whenever the entire face needs protection. Such as when there is a potential that an aerosol of chemical or biological hazardous material may be created or whenever chemical or biohazards could splatter, or whenever there is the potential for flying particles or sparks. A face shield should always be worn whenever handling tissue samples or animals where there is the potential for infectious transmission. Safety glasses or goggles should always be worn underneath a face shield for maximal protection.

    Masks that are disposable may need to be worn, especially during the COVID-19 pandemic and will limit the transmission of infectious agents. Disposable masks come in a variety of materials and levels of protection.  Typical facemasks are loose-fitting, disposable masks that cover the nose and mouth, such as surgical masks and nuisance dust masks. Facemasks are not approved by the National Institute for Occupational Safety and Health (NIOSH) for protection against any regulated hazardous material.

    Facemasks help stop droplets from being spread by the person wearing them. They also keep splashes or sprays from reaching the mouth and nose of the person wearing the facemask and are therefore useful when cleaning up spills of infectious materials.  They are not designed to protect you against breathing in gases, vapors, or very small particles. Facemasks should be used once and then disposed of.  There are different types of masks used for different purposes, specialty disposable face masks such as the N95 or KN95 masks are respirators which are approved by NIOSH for use against certain selected airborne particulates when used as part of a respiratory protection program.  


    Watch video 1

    Video 1: proper usage of PPE.  Your instructor will explain how to locate, use, and store your PPE in the lab.

    Watch Video 1: WHO: Good Microbiological Practices & Procedures.  Personal Protective Equipment (PPE) (19:41)  URL:



    1. WHO. World Health Organization Biosafety Series.

    1.2: Biosafety levels and PPE is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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