BT102 Lab Manual

Contents · P. No · Practical

P. No. Biosafety levels Sterilization techniques (Physical/Heat method of sterilization) Sterilization techniques (Chemical method of sterilization) Sterilization techniques (Mechanical/Filtration methods) Culturing of bacteria in liquid medium Culturing of bacteria on solid medium Types of media and culturing Gram staining of bacteria Bacterial cell count Biochemical tests Colony and cell morphology Growth Kinetics Testing sensitivity to antimicrobial substances

Practical No: 1 · Biosafety levels · Biosafety

Safety from exposure to Infectious Agents

A biosafety level is a set of biocontainment precautions required to isolate dangerous biological agents in an enclosed laboratory facility.

Importance:

• Laboratorians recognize hazards of processing infectious agents
• Guidelines developed to protect workers in microbiological and medical labs through engineering controls, management policies, work practices
• 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

Biosafety levels (1-4):

There are four 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 Primary barriers: Physical barriers or personal protective equipment between lab worker and pathogen. For example: Gloves, masks, lab coat, goggle, shoes cover Secondary barriers: Structural aspects of the laboratory that make working environment safer against infection. For example: Sinks for hand washing, special containment areas, special air ventilation patterns

• Work is generally conducted on open bench tops using standard microbiological practices.
• Special containment equipment or facility designs is not required nor generally used.
• Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology.

Standard Microbiological Practices:

• Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas where there is reasonable likelihood of exposure to potentially infectious materials.
• Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
• Mouth pipetting is prohibited; mechanical pipetting devices are used.
• All procedures are performed carefully to minimize the creation of splashes or aerosols.
• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method.
• Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory.
• Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.
• Special containment devices or equipment such as a biological safety cabinet is generally not required for manipulations of agents assigned to Biosafety Level 1.
• It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination.
• Gloves should be worn if the skin on the hands is broken or if a rash exists.

Laboratory Facilities:

• Each laboratory contains a sink for hand washing.
• The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill extremely difficult to achieve.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
• Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible for cleaning.
• If the laboratory has windows that open, they are fitted with fly screens.
• Risk Group 2 infectious agents are pathogens that can cause human or animal disease but, under normal circumstances, are unlikely to be a serious hazard to laboratory workers, the community, livestock, or the environment.
• Infections by risk group 2 organisms are not considered to be a serious hazard. They are a moderate individual risk and limited community risk.
• Laboratory exposures rarely cause infection leading to serious disease; effective treatment and preventive measures are available and the risk of spread is limited.
• Examples of infectious agents in this risk level are E. coli, many influenza viruses, some fungi like ringworm; human herpes simplex viruses; Transmissible Gastro-enteritis of swine; Mouse Hepatitis Virus; and a few parasites.
• Primary hazards: accidental needle sticks, exposure to eyes and nose, ingestion of infectious materials
• Agents do not cause lethal infections that 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

Procedures required to contain risk group 2 organisms:

• The laboratory should be separated from all other activities.
• A biohazard sign should be present and visible. (policies to restrict access to lab)
• All the surfaces in the laboratory should be readily cleanable
• An autoclave should be present.
• A certified HEPA filtered class 1 or 2 biological cabinet should be available for the manipulation of organisms.

Gloves and a laboratory coat should be worn at all times in the laboratory

Fig 1.1: Biohazard Symbol

• Risk Group 3 infectious agents are pathogens that usually cause serious human or animal disease, or which can result in serious economic consequences, but do not ordinarily spread by casual contact from one individual to another (high individual risk, low community risk), or that can be treated by antimicrobial or antiparasitic agents.
• Risk Group 3 pathogens include bacteria such as anthrax, Q Fever, tuberculosis, and viruses such as hanta viruses, Human immunodeficiency viruses (HIV - all isolates), eastern and western equine encephalitis viruses.

Procedures required to contain risk group 3 organisms:

• Laboratory staff must be fully trained in the handling of pathogenic and other hazardous material, in the use of safety equipment, disposal techniques, handling of contaminated waste, and emergency response.
• Standard operating procedures must be pasted in visible spot.
• Equipment must include an autoclave and a certified HEPA filtered class II biological safety cabinet
• All activities involving infectious materials to be conducted in biological safety cabinets or other appropriate combinations of personal protective and physical containment devices.

Designed for use with:

• Highly toxic/infectious agents
• Agents that are at a very high risk for forming infectious aerosols
• Life threatening agents

Examples: Ebola virus, Marburg virus, Lassa virus

Special Practices:

❖ Only those that work in the BSL-4 lab will be allowed entry ❖ A logbook must be signed each time one comes in or out of the lab with the date and time