A. Guidelines for Good Laboratory Practices at BSL1 and BSL2

• Bulleted items indicate additional requirements for work at BSL2.

1. Immediately notify the laboratory supervisor or Principal Investigator (PI) in case of an accident, injury, illness, or overt exposure associated with laboratory activities. When deemed necessary by the laboratory supervisor proceed to the Student Health Center for appropriate medical surveillance and/or treatment.

2. Be aware that access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures or specimens is in progress. Laboratory should have doors to control access.

• Only personnel advised of the special hazards and meeting any specific entry requirements, i.e., appropriate immunizations, serum sampling, are permitted in the laboratory. Read and follow all biosafety procedures provided by the PI.
• Be aware that any possession or use of select biological agents or toxins requires federal government registration and inspection; restricted lab access; written and strictly followed safety and security plans; personnel background checks and training; accurate records and/or reporting of agent use, transfer, loss, or destruction. Any plans for obtaining such materials must be discussed with the Biosafety Officer and approved by the Biosafety Committee.
• Ensure that when infectious agents are in use in the laboratory, a biohazard sign is posted on the lab access door. This sign identifies the agent(s) in use, the biosafety level, any required immunizations, the PI’s name and telephone number, and any PPE that must be worn in the laboratory.
• Understand that the PI must ensure that all laboratory personnel receive appropriate training on hazards associated with the agents involved; the necessary precautions to prevent exposures; and exposure evaluation procedures.

3. Wash hands frequently and always after handling viable material or animals, after removing gloves, and before leaving the laboratory. A sink for hand washing is present in each laboratory.

• Consider foot, knee, or automatically operated hand washing sinks.
• Know the location of a readily accessible eyewash station.

4. Do not eat, drink, smoke, chew gum, handle contact lenses, or apply cosmetics in the laboratory. Persons wearing contact lenses in the laboratory should also wear goggles or a face shield.

5. Do not bring any food, medications, or cosmetics, into the laboratory for storage or later use. Food is stored outside the work area in cabinets or refrigerators designated specifically for that purpose

6. Do not pipette by mouth; only mechanical pipetting devices are permitted.

• Do not bring animals unrelated to experiments into the laboratory.

7. Perform all procedures carefully to minimize the creation of splashes or aerosols.

8. Establish and follow policies for safe handling of sharps.

• Use a high degree of caution when handling any contaminated sharp item, such as needles and syringes, slides, pipettes, capillary tubes, and scalpels.
• Restrict needles and syringes or other sharp instruments in the laboratory for use only when there is no alternative, such as for parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Substitute plasticware for glass whenever possible. Handle broken glassware with brush and dustpan, tongs, or forceps - not directly with hands.
• Use only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) for injection or aspiration of infectious material.
• Use, whenever possible, syringes which re-sheathe the needle, needleless systems, and other safety devices.
• Do not bend, shear, break, recap, or remove used needles from disposable syringes or otherwise manipulate such units by hand before disposal. Dispose of needles and syringes in the puncture resistant container provided in the laboratory for this purpose. Place full containers in an autoclave bag and sterilize before disposal in medical waste boxes.

9. Use of lab coats, gowns, or other designated laboratory uniform is recommended to prevent contamination or soiling of street clothing.

• Wear lab coats, gowns, smocks, or other provided protective garments while in the lab. When leaving the lab, remove and leave coats and other protective clothing in the lab for either disposal or laundering. BSL2 may require autoclaving before laundering.

10. Wear gloves if the skin on the hands is broken or if a rash is present. Protective eyewear should be worn for procedures that involve anticipated splashes of microorganisms or other hazardous materials to the face.

• Wear gloves when manipulating infectious materials or agents or when hands must otherwise contact contaminated surfaces. Remove and change gloves when overtly contaminated or when torn or punctured. Do not wear contaminated gloves outside the lab. Do not wash or reuse disposable gloves. Consider alternatives to latex gloves to prevent allergic response.
• Wear appropriate face protection (goggles, mask, face shield or other splatter guard) for anticipated splashes or sprays of infectious materials to the face when agents must be handled outside the BSC.

11. Decontaminate equipment and work surfaces at completion of work, at the end of the day, and following spills of viable materials. If a spill occurs, cover the spill with paper towels and soak the towels with a 1 to 10 dilution of chlorine bleach or other suitable disinfectant. Allow the material to soak for approximately 20 minutes before discarding materials in biohazard bag. Bench tops are impervious to water and resistant to solvents, acids, alkalis, and chemicals used for surface decontamination. Laboratory surfaces and spaces between fixtures are designed to be easily cleaned; no carpets or rugs.

12. Work on open bench tops is permitted; use of special containment equipment such as a biological safety cabinet is not generally required for agents assigned to BL1.

• Work in the open laboratory is permitted, except that a properly maintained biological safety cabinet is required whenever:

Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or embryonate eggs. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.

Be aware that air sampling studies have shown that most of the common manipulations of bacterial and viral cultures in research laboratories release aerosols of viable organisms. This must be considered when evaluating need for use of the biological safety cabinet or other physical containment device.

13. Dispose of all regulated medical wastes (potentially biohazardous) and associated wastes as outlined in the provided BIOLOGICAL LABORATORY WASTE STREAMS (see Table 3).

• Cover containers of all cultures, tissues, specimens of body fluids, or other potentially infectious waste to prevent leakage during collection, handling, processing, storage, transport, or shipping.

14. Have an insect and rodent control program in place. Ensure screens are fitted on exterior windows that open into the lab.

15. Ensure that illumination is adequate for all activities, avoiding reflections and glare that could impede vision.

B. Biological Safety Cabinets (BSCs)

1. Types of BSCs

BSCs are classified as Class I, Class II or Class III cabinets. When properly maintained and operated, they effectively contain and capture microbial contaminants and infectious agents using HEPA (High Efficiency Particulate Air) filters. (See Figure 1.) Biosafety cabinets should not be confused with clean benches which only protect the material being worked with and are not suitable for work with infectious or toxic material. (Although clean benches, like BSCs, have HEPA-filtered air, with clean benches the air flows over the experimental material toward the user rather than being drawn away.) BSCs should also not be confused with conventional fume hoodsand laminar flow hoods that do not filter microorganisms.

Class I BSCs provide personnel and environmental protection, but not product protection. (See Figure 2).

Note: A chemical dunk tank may be installed which would be located beneath the work surface of the BSC with access from above. The cabinet exhaust needs to be connected to the building exhaust system.

Figure 4. Class III BSC. Connection to building exhaust system required.

2. Working in a BSC

• Turn the cabinet on for at least 10 - 15 minutes prior to use, if the cabinet is not left running.
• Disinfect work surface with 70% alcohol or other suitable disinfectant.
• Consider the materials necessary for the planned work in the cabinet.
• Place items into the cabinet so that they can be worked with efficiently without unnecessary disruption of the air flow, working with materials from the clean to the dirty side.
• Wear appropriate personal protective equipment. At a minimum, this will include a buttoned laboratory coat and gloves.
• Adjust the working height of the stool so that the worker's face is above the front opening.
• Delay manipulation of materials for approximately one minute after placing the hands/arms inside the cabinet.
• Minimize the frequency of moving hands in and out of the cabinet.
• Do not disturb the airflow by covering any of the grillwork with materials.
• Work at a moderate pace to prevent the air flow disruption that occurs with rapid movements.
• Wipe the bottom and side of the hood surfaces with disinfectant when work is completed.

NOTE: Be very careful when using small pieces of materials such as kimwipes in the hood. These can be blown into the hood and disrupt the motor operations.

3. Certification of the BSC

Certification is a series of performance tests on the BSC to confirm that it will provide the user and experimental material the protection for which it is designed. The air flows, filters, and cabinet integrity are checked to ensure that the cabinet meets minimum performance standards. Certification is arranged through the Safety and Environmental Health Department and provided by an outside vendor.

BSCs intended for user protection must be certified by the Safety and Environmental Health Department:

• After they are received and installed (before use with infectious materials)
• After filter changes
• Annually

Biological safety cabinets intended only for protection of the experimental material are certified at the discretion of the Principal Investigator.

BSC’s that are potentially contaminated with infectious agents may require decontamination. BSC decontamination (using the paraformaldhyde gas production process) is also provided by an outside vendor and needs to be done:

• Before any maintenance work requiring disassembly of the air plenum, including filter replacement
• Prior to cabinet recertification
• Before moving the cabinet to a new laboratory

C. Decontamination

1. Definitions

Decontamination is a process or treatment that renders an instrument or environmental surface safe to handle. A decontamination procedure can be as simple as clean-up with detergent and water or as thorough as sterilization. Sterilization, disinfection, and antisepsis are all forms of decontamination.

Sterilization is the use of physical or chemical processes to destroy all microbial life, including highly resistant forms, such as bacterial spores.

Disinfection is the elimination of essentially all pathogenic non-spore forming microorganisms but not necessarily all microbial forms from work surfaces and equipment. Effectiveness is influenced by a number of factors, including: types and numbers organisms; amount of organic matter; the object being disinfected; the disinfectant being used; exposure time, temperature and concentration.

Antisepsis is the application of a liquid antimicrobial to skin or other living tissue to inhibit or destroy microorganisms. Examples include hand washing with germicidal solutions or swabbing skin before an injection.

2. When to Decontaminate

All material and equipment contaminated with or containing potentially infectious agents should be decontaminated:

• Upon completion of procedures involving the use of biologically-active materials
• In the event of spills of such materials
• At least daily
• Before being washed, stored, or discarded

In most UND laboratories, decontamination is accomplished by steam heat sterilization in an autoclave , or by surface application of or placement in a chemical disinfectant solution , such as 1:10 bleach solution or its equivalent.

3. Autoclave Use

Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250 oF for a designated time) is the preferred and most convenient method to rapidly destroy all forms of microbial life. However, to do this, the autoclave process must reach proper temperature and time and also prevent the entrapment of air in the bag or container of treated material.

• Material to be sterilized must come into contact with live steam.
• Bags or containers should be left open during autoclaving or water (~200ml) should be added to sealed bags to generate steam.
• Heat indicator tape should be used with each autoclave load to indicate that sterilization has been completed.
• Autoclave sterility monitoring should be conducted on a regular basis using biological indicators (such as B. stearothermophilus spore strips) placed among treated materials and at locations throughout the autoclave. The spores, which are more resistant to heat than most microbials, provide validation of general microbial destruction when they are effectively inactivated (250 oF for 13 minutes) by autoclave operation.

4. Chemical Disinfectant Use

The most practical use of chemical disinfectants is for surface decontamination and, when used in sufficient concentration, as a decontaminant for liquid wastes prior to final disposal down the drain.

GENERAL RECOMMENDATIONS:

Liquid Decontamination

• Add liquid chlorine bleach to provide a final 1:10 dilution
• Let stand at least 20 minutes
• Discard down the drain

Surface Decontamination

• Wipe with 1:10 dilution of chlorine bleach, or
• Wipe with iodophor disinfectant (per label concentration), or
• Wipe with 70% alcohol

See Table 4, Table 5, Table 6 for further information on disinfectants.

D. Exposure to Infectious Agents

In the event of an exposure to an infectious agent or material, the following guidelines should be used:

1. Intact skin

• Remove contaminated clothing
• Vigorously wash contaminated skin for 1 minute with soap and water

2. Broken, cut or damaged skin or puncture wound

• Remove contaminated clothing
• Vigorously wash contaminated skin for 5 minutes with soap and water
• Seek medical attention at the Student Health Center

3. Eye

• Immediately flush eyes for at least 15 minutes with water, preferably using an eyewash; if no eyewash is available, pour water on the eye(s) for 15 minutes, rinsing from the nose outward to avoid contamination of the unaffected eye.
• Hold eyelids away from your eyeball and rotate your eyes so that all surfaces may be washed thoroughly.
• Seek medical attention at the Student Health Center

4. Ingestion or Inhalation

• Seek medical attention at the Student Health Center
• Do not induce vomiting unless advised to do so by a health care provider

E. Biological Material Spills

1. Spills and Preparing for Them

In the event of a spill of biological material, the individual(s) who caused the spill is responsible for the clean-up. The PI must contact the Safety & Environmental Health Department.

• Minimize the consequences of any spill of biological material by performing all work on plastic-backed liner to absorb spills

• Have a simple spill kit on hand including:

• Chlorine bleach or some other concentrated disinfectant

• A package or roll of paper towels

• Autoclavable bags

• Rubber gloves

• Forceps for pick-up of broken glass

2. Spills Inside a Biological Safety Cabinet

3. Small Spill of Material Outside of a Biological Safety Cabinet (Spill that can be covered by a few paper towels)

a. Wearing gloves and a lab coat, cover the spill with paper towels and gently apply disinfectant, proceeding from the outer edge of the spill to its center. Leave in place for 20 minutes.

4. Large Spill of BSL2 Material (>500ml) Outside of a Biological Safety Cabinet

1. Biohazardous Waste (Regulated Infectious Waste)

Some wastes associated with biological materials must be disposed of in special ways because they may have been contaminated with infectious organisms or agents. These potentially infectious or biohazardous materials are defined by ND regulations as Regulated Infectious Waste (Chapter 33-20-12 of the North Dakota Administrative Code) . These wastes include the following:

• Cultures and stocks. Cultures and stocks of infectious agents and associated biologicals, including cultures from medical and pathological laboratories; cultures and stocks of infectious agents from research and industrial laboratories; wastes from the production of biologicals; discarded live and attenuated vaccines; and culture dishes and devices used to transfer, inoculate, and mix cultures.

• Pathological waste. Human pathological waste, including tissues, organs, and body parts and body fluids that are removed during surgery or autopsy, or other medical procedures, and specimens of body fluids and their containers.

• Human blood and blood products. Liquid waste human blood; products of blood; items saturated or dripping with human blood; or items that were saturated or dripping with human blood that are now caked with dried human blood (including serum, plasma, and other blood components, and their containers).

• Sharps. Sharps that have been used in animal or human patient care or treatment or in medical, research, or industrial laboratories, including hypodermic needles, syringes (with or without the attached needle), pasteur pipettes, scalpel blades, blood vials, needles with attached tubing, and culture dishes (regardless of presence of infectious agents). Also included are other types of broken or unbroken glassware that were in contact with infectious agents, such as used slides and cover slips.

• Animal waste. Contaminated animal carcasses, body parts, and bedding of animals that were known to have been exposed to infectious agents during research (including research in veterinary hospitals), production of biological, or testing of pharmaceuticals.

• Isolation waste. Biological waste and discarded materials contaminated with blood, excretion, exudates, or secretions from humans who are isolated to protect others from highly communicable diseases, or isolated animals known to be infected with highly communicable diseases.

• Unused sharps. Unused, discarded sharps, hypodermic needles, suture needles, and scalpel blades.

For disposal of these wastes, the lab personnel:

Other wastes generated in these facilities that are not contaminated with biological agents or materials are not treated as biohazardous and may be discarded in the regular trash container, or into other specially designated waste containers. These include such items waste glass, gloves, unused plates or tubes, fly media or embryo plates, etc. Table 3 Biological Laboratory Waste Streams on page 16 outlines the disposal options.

2. Animal Bedding Waste

Animal bedding is bagged by animal care personnel and placed in specially provided gray carts for movement to the pick-up location. Bags should be filled only to a depth and weight that will allow for effective tying of the bag by animal facility staff and for ease of handling by one person. For example, several partially-filled bags should be tied and placed in the gray carts rather than one or two full bags (bag weight should not exceed 40 pounds). This will help to prevent repetitive motion injury to staff and help to prevent bags from being ripped open while being handled.

The carts are maintained clean and in sanitary condition by the animal facility staff.

3. Animal Carcasses and Non-regulated biological waste

Freezers are provided in each animal facility for storage of carcasses that have been bagged and sealed. Freezers are cleaned and defrosted as necessary by animal laboratory personnel to keep them in a sanitary condition.

Non-regulated biological waste cannot include animal tissues containing pharmaceuticals or toxins and cannot include animal tissue used in the following:

• in the diagnosis, treatment or immunization of humans or animals
• in research pertaining to the above
• in the production or testing of biologicals. Biologicals are preparations made from living organisms and their products, including vaccines, cultures etc. intended for use in diagnosing, immunizing or treating of humans or animals, or in research pertaining thereto.

All non-regulated biological waste for disposal in the local landfill must be sealed in plastic bags which are then sealed inside cardboard boxes or other sturdy, opaque containers.  

Contact the Safety Office at 777-3411 if you have questions about your waste being non-regulated.

The waste should not be placed in a dumpster. To make arrangements to have your waste removed directly to the landfill contact Paul Clark, Associate Director of Facilities at 777-3005.

4. Animal Waste from BSL2 Animal Rooms

Rodents housed in BSL2 space are considered to be potentially infectious because as part of the research protocol they are infected with Biosafety Level 2 (BSL2) animal and/or human infectious agents. Animal bedding, carcasses, and tissue are placed in biohazard bags by the research staff. All animal bedding is autoclaved before being placed in medical waste boxes by animal care staff and disposed of in the medical waste stream. Bagged animal carcasses and tissue are placed in the provided storage freezers.

For a list of etiologic agents that require specific packaging see Table 7.

Packaging and shipping of biological materials must be done in a way that ensures the contents will not leak and that the package will arrive in good condition.

The definitions below apply to the packaging and shipping instructions that follow:

Etiologic agent means a viable microorganism or its toxin which causes, or may cause, human disease.

Diagnostic specimen means any human or animal material including, but not limited to, excreta, secretion, blood and its components, tissue, and tissue fluids, etc., which is reasonably believed to contain an etiologic agent, and is being shipped for purposes of diagnosis.

Biological product means a biological prepared and manufactured in accordance with regulations that govern the manufacture of vaccines, reagents, etc.

Interstate shipping means shipping to or from the continental US including to other NJ locations.

Packaging

All biological materials including diagnostic specimens and biological products that may contain an etiologic agent must be packaged to withstand leakage of contents, shocks, pressure changes, and other conditions incident to ordinary handling and transportation (passage through cancellation machines, sorters, conveyors, etc). Contents should not leak to the outside of the shipping container, even if leakage of the primary container occurs.

Specific packaging requirements apply to materials which are known to contain, or reasonably believed to contain certain etiologic agents. See here for etiologic agents. For such materials, the following procedures apply (See Figure 1):

Volume not exceeding 50 ml:

Volume greater than 50 ml:

Packaging with Dry Ice

Labeling

The outer shipping container of all materials containing etiologic agents which are being shipped or transported must bear a special label, as illustrated in Figure 2. These labels are available from your laboratory supply vendor.

H. Shipping and Transportation Methods and Requirements

Registered Mail or the Equivalent

For a list of etiologic agents that use registered mail or an equivalent system which provides the sender with immediate notification of receipt see Table 8

Federal Express or UPS

For such shipments, internationally or domestically, follow the International Air Transport Association (IATA) Dangerous Goods Regulations. (Receipt of shipment notice is not required since the shipment is traceable through the specific carrier).
Apply appropriate labels to the outer shipping container for packages containing dry ice and/or infectious substances as shown in Figures 3 and 4, respectively.
Contact the specific carrier's dangerous goods agent prior to shipment for any additional packaging and labeling requirements.

Only individuals who are certified by the University of North Dakota’s Safety and Environmental Health Office may ship or receive packages of biohazardous material. For assistance contact the Safety and Environmental Health Office at 777-3341.

The University of New Hampshire maintains a web site at: http://www.unh.edu/ehs/shipping/ that includes the “ Shipment of Biological Materials Manual” , the “Guide to Shipping with Dry Ice” , a flow chart entitled “ Classification Guide for Diagnostic Specimens and Infectious Substances” , and sample forms and labels to assist in the shipment of biohazardous material.

There are numerous Federal regulations that must be met when transporting biohazards, failure to comply with these regulations can result in major fines that would be levied on the individuals involved.

Damaged Packages

When evidence of leakage or any other damage to packages bearing an Etiological Agents/Biomedical Material label is discovered, the carrier must promptly isolate the package and notify the Director, Centers for Disease Control and Prevention (CDC), 1600 Clifton Road N.E., Atlanta, Georgia 30333 or by telephone (404) 633-5313.

Notice of Delivery

In the event that a package sent by UND is not received by the recipient within 5 days following the anticipated delivery of the package, the sender must notify the Director, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, Georgia 30333 or by telephone (404) 633-5313.

Importation of infectious agents, etiologic agents and vectors that may contain them is governed by federal regulation. In general, an importation permit is required for any infectious agent known to cause disease to humans. This includes but is not limited to bacteria, viruses, rickettsia, parasites, yeasts and molds. In some instances, an agent which is suspected of causing human disease also requires a permit.

Importation permits are issued by the U.S. Public Health Service (USPHS) only to the importer, who must be located in the United States. The importation permit, with the proper packaging and labeling, will expedite clearance of the package of infectious materials through the USPHS Division of Quarantine and Release by U.S. Customs.

Instead of an importation permit, a Letter of Authorization may be issued by the Centers for Disease Control and Prevention (CDC) after review of an "Application to Import an Etiological Agent". The letter is issued for materials that are judged to be noninfectious, but which might be construed to be infectious by U.S. Customs inspection personnel. Letters of Authorization may be issued for items such as formalin fixed tissues, sterile cell cultures, clinical materials such as human blood, serum, plasma, urine, cerebrospinal fluid, and other tissues or materials of human origin when there is no evidence or indication that such materials contain an infectious agent. Letters of Authorization are in effect for two years, and do not require a shipping label to be issued by CDC.

Importation permits and Letters of Authorization are issued by the CDC, Import Permit Program, 1600 Clifton Road NE, Mailstop E-79, Atlanta, Ga. 30333 after review of a completed application form. Application forms may be obtained by calling CDC at (404)498-2260. CDC can also be contacted on the Internet at http://www.cdc.gov/od/ohs/biosfty/imprtper.htm. Completed forms may be returned to CDC by mail or FAX at 404-498-2275. Application to CDC for the importation permit should be made 10 working days in advance of the shipment date to allow time for processing, issuance and delivery of the permit and shipping labels to the permittee.

Facilities transferring or receiving select biological agents must be registered with the CDC or USDA and comply with elaborate regulatory requirements.

Other Permits