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Biological Safety

Standard Operating Procedures

Building: Combs Research Building
Room: 222
Department: Markey Cancer Center
PI: Andrew Pierce, PhD.

Section 1: Process, Hazardous Biological Material

Section 2: The purpose of the Biological Safety module is to be used as a guideline to ensure the laboratory worker remains cognizant of the biological material stored and used in the labs and their associated hazards. In this lab, we will be working with human and bacterial cell cultures as well as modifying their DNA by an array of assays. Fetal Bovine Serum and antibiotics such as Puromycin, Hygromycin B, Penicillin-Streptomycin, and G418 (Geneticin) will also be used for various cultured experiments.

Section 3: Microbiological laboratories pose a number of special circumstances that set them apart from other types of laboratories. Most of these circumstances exist because the work conducted within them often involves organisms that are infectious to humans. The purpose of this manual is to define the appropriate kinds of facilities and work practices for microbiological laboratories to reduce the probability of infection of laboratory personnel. All principal investigators, faculty, staff and students working with infectious agents or recombinant DNA are expected to abide by the provisions of this manual. The Institutional Biosafety Committee (IBC) has adopted the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories as the University's official biosafety guidelines for the use of infectious agents. Copies may be obtained from the office of Hazardous Materials Management, 208 Kelly Building. Recombinant DNA research is regulated by NIH guidelines (Guidelines for Research Involving Recombinant DNA Molecules), also available from the office of Hazardous Materials Management. Research involving infectious agents or recombinant DNA must be reviewed by John Lowry, the Biological Safety Officer, and the IBC. All proposals, regardless of funding source, are subject to this review. The IBC will determine the proper hazard classification and will recommend approval or disapproval.

A potentially hazardous biological specimen may be any one type or combination of the following types of specimens, when such a specimen consists of live, frozen or lyophilized material that may possess the potential to produce an infection or genetic alteration in humans, animals or plants. See below:

Potentially hazardous biological specimens do not include live animals that are healthy and presumed to be non-infectious, but do include unhealthy animals that are known or suspected to be infectious. Biological specimens are not considered to be potentially hazardous when the specimens have been chemically, thermally, or otherwise treated to render the specimens, or components thereof, permanently non-viable, permanently inactivated, or permanently incapable of insertion or infection into living cells.

Section 4: When working with biological agents, one must incorporate the use of PPE, personal protective equipment. At a minimum, all lab personnel should be wearing a lab coat or lab apron, gloves, and safety glasses. Full-face shields must be worn when conducting a procedure where splashing is a potential; although, we will mostly require using a cell culture hood with a glass shield for the majority of our biological material usage.

Section 5: The engineering controls for this lab will be the use of cell culture hoods to prevent the use and reduce the exposure to biological material and chemical safety shower/eyewash showers. Safety Shower/Eyewashes Safety showers and/or eyewashes are required in labs where chemical and biological materials are used. PPD is charged with testing the eyewashes and shower units. A log of those checks can be obtained by contacting your respective PPP (LC or MC). Chemical shower and eyewash shower station is located by the sink in the lab.

Section 6: The biological material will be kept in several places in this lab at any given time. Actively growing bacterial and human cell cultures will mainly be stored in the cell culture hood room in the 37C CO2 incubators unless shaking is desired. When a shaker is needed for biological cultures, we will either use the warm room on the 3rd floor of the Combs Research Building or the floor model shaker on the 2nd floor across the hall from the lab. Other places that biological materials may be stored will be the 4C refrigerator, -20C freezer, -80C freezer, or the -180C cryogenic freezer all located in this laboratory. All cell cullture media and constituents will be stored in the 4C refrigerator in the cell culture hood room and labeled according to the date the bottles are opened. Access to the laboratory biological materials will be limited to those working in this lab.

Section 7: A spill that is confined to the interior of the cell culture hood should present little or no hazard to personnel in the area. However, chemical disinfection procedures should be initiated at once while the cabinet ventilation system continues to operate to prevent escape of contaminants from the cabinet.

If potentially hazardous biological material is spilled in the laboratory, the first essential is to avoid inhaling any airborne material by holding the breath and leaving the laboratory. Warn others in the area and go directly to a wash or change room area. If clothing is known or suspected to be contaminated, remove the clothing with care, folding the contaminated area inward. Discard the clothing into a bag or place the clothing directly in an autoclave. Wash all potentially contaminated areas as well as the arms, face and hands. Shower if facilities are available. Reentry into the laboratory should be delayed for a period of 30 minutes to allow reduction of the aerosol generated by the spill. Advance preparation for management of a spill is essential. A "spill kit"--including leak-proof containers, forceps, paper towels, sponges, disinfectant, eye protection and rubber gloves--should be readily available. Respirators should be available for certain hazards.

If the emergency involves personal injury or biological contamination, call 911 from any campus phone and ask for an ambulance to be sent to the area. Be sure to state the type of contaminant on the victim. The caller should remain available to brief emergency responders on the type of contamination and proper procedures for handling the material.

For a general minor spillage occuring in the open lab follow these instructions:
1. Alert people in immediate areas of the spill.
2. Don appropriate protective equipment.
3. Cover the spill with paper towels or other absorbent materials.
4. Carefully pour a freshly prepared 1:10 dilution of household bleach around the edges of the spill and into the spill. Avoid splashing. Allow 20 minute contact period.
5. Use paper towels to wipe up the spill, working from the edges into the center .
6. Clean spill area with fresh paper towels soaked in disinfectant.
7. Place paper towels into a biohazard bag for disposal.

Section 8: To decontaminate when a spillage has occured, spray or wipe walls, work surfaces, and equipment with a disinfectant. A disinfectant with a detergent has the advantage of detergent activity, which will help clean the surfaces by removing both dirt and microorganisms. Use sufficient disinfectant solution to ensure that the drain pans and catch basins below the work surface contain the disinfectant. Lift the front exhaust grill and tray and wipe all surfaces. Wipe the catch basin and drain the disinfectant into a container. A solution of 1% Alconox in water is applied to the soiled area (allow 20 minute contact period) and then wiped free with a towel. To finalize the decontamination process, 70% ETOH should be applied to the spill area and also wiped clean with a towel. Discard all towels/spill pads and gloves into the biohazard container when completed.

Section 9: Separation and labeling of infectious biological waste (which may include red bagging, universal biohazard symbol, etc.) must be done at the point of generation. During collection, storage and transportation, all waste must be managed such that the integrity of the packaging is preserved and that rapid microbial growth and putrefaction is inhibited. Sharps containers should be rigid, impervious and puncture-resistant; plastic bags should be tear-resistant, leak-resistant and sturdy enough to withstand handling.
Whenever possible, infectious waste should be treated to render it non-infectious and non-recognizable as to its former character.

To dispose of any hazardous waste you must completely fill out a hazardous waste ticket for each container. Tickets must be filled out by the person who generates the waste, not a staff assistant or student employee who does not have knowledge of chemicals or has not been through the hazardous waste class. Tickets should be filled out as completely as possible.

One ticket should be filled out for each container. If you have a box of vials or small containers that are all of the same chemicals, then only one ticket is needed for the box. For different chemicals or biological waste, one ticket will be needed for each container. For animal carcasses, one ticket is needed for each bag or box with the total weight listed for the container.

Mail or bring the top or white copy of the hazardous waste ticket to the HMM office. Attach the other two copies (yellow and pink) by securely taping the pink copy to each container. Once the ticket is received it will be entered into the computer tracking system and HMM personnel will come to your area and pick up the waste. No waste materials will be picked up without a properly filled out hazardous waste ticket. The University is required by law to track its hazardous waste from the point it becomes a waste to its point of ultimate destruction. These tickets are our means of fulfilling this requirement.

Section 10: Materials Safety Data Sheets, MSDS, for all chemicals may be found on the following web address:

Section 11: Protocols

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