MICROBIOLOGICAL SAFETY MANUAL

2004

TABLE OF CONTENTS 1. Introduction ………………………………………………………….…… 1 2. Principles of Microbiological Safety …..………………………...….. 1

3. Risk Assessment ………………………….……………………….… 2 4. Biosafety Levels One and Two ……………….…………………… 3 4.1. Biosafety Level 1 (BSL1) ………………………………….. 3 4.2. Biosafety Level 2 (BSL2) ………………………………….. 3 5. Biosafety Level Three …………………………………...…………. 4 6. Good Microbiological Techniques ……………………………….. 6 7. Biosafety Cabinets ……………...………………………………….… 6 7.1. Start-up Procedure ………………………….………..……. 6 7.2. Working in the Cabinet ………………………………….…… 7 7.3. Common Errors to Avoid ……………………………………. 7 8. Select Agents and Toxins ……………………………………….…… 8 9. Recombinant DNA Safety ……………………….……………………. 10 9.1. Recombinant Organism or Molecules ……………………… 10 9.2. Preview by the Recombinant DNA Committee …………….. 10 10. Disinfections and Waste Disposal ……...…………………….…. 10 10.1. Disinfections ………..………………………….………….. 10 10.2. Autoclave Procedures………………….…………………. 11 10.3. Disinfectants ……………………………………………… 11 10.4. Disposal of Waste ……………………………………….. 11 11. Contingency Plans and Spill Control.…………………………….. 12 11.1. Contingency Planning ……………….………………………… 12

11.2. Spill in a Biosafety Cabinet ………………………….……….. 12 11.3. Spill outside the Biosafety Cabinet ………………………… 12

12. Training and Medical Surveillance ……………………..……………….. 13 12.1. Technical Training Requirement ………………………………… 13 12.2. Training Requirement for Non-technical Staff …………………. 13 12.3. Medical Surveillance Program …………………………………… 13

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1. INTRODUCTION

It is OMRF's policy that the Principal Investigator is the responsible individual for all activities that occur in the laboratory. Safety is a shared responsibility among the laboratory staff. Laboratory staff are responsible for following established procedures, cleaning up after each experiment, and reporting problems to their supervisor and the Safety Officer. Safety precautions must be taken to ensure that experiments are conducted in such a way that microorganisms do not escape and contaminate the laboratory.

2. PRINCIPLES OF MICROBIOLOGICAL SAFETY

Throughout this manual, we adopt the concept of Risk Group (WHO Risk Groups 1, 2, 3 and 4). Containment and standard operating procedures are designed for the relative hazards of the infective microorganisms for each risk group. Laboratories may be designated as Biosafety Level 1 (BSL1), Biosafety Level 2 (BSL2), Biosafety Level 3 (BSL3), or Biosafety Level 4 (BSL4), if they meet the criteria set up in Table 2.

Table 1 Classification of infective microorganisms by risk group

Risk Group 1 (no or low individual and community risk)

A microorganism that is unlikely to cause human or animal disease.

Risk Group 2 (moderate individual risk, low community risk)

A pathogen that can cause human or animal disease but is unlikely to be a serous hazard to laboratory workers, the community, livestock or the environment. Laboratory exposure may cause serious infection, but effective treatment and preventive measures are available and the risk of spread of infection is limited.

Risk Group 3 (high individual risk, low community risk)

A pathogen that usually causes serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available.

Risk Group 4 (high individual and community risk)

A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available.

* Table 1 and Table 2 are adopted from the interim guidelines of “Laboratory Biosafety Manual” 2nd Edition (revised) by WHO in 2003.

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Table 2 Relation of risk groups to biosafety levels, practices and equipment

Risk Group

Biosafety Level Laboratory Type Laboratory Practices Safety Equipment

1 Biosafety Level 1 Basic teaching, research

Good microbiological technique

None; open bench top

2 Biosafety Level 2 Primary health services; diagnostic, research

Good microbiological technique plus protective clothing, biohazard sign

Open bench plus biosafety cabinet for potential aerosols

3 Biosafety Level 3 Special diagnostic, research

Using BSL2 plus special clothing, controlled access, directional air flow

Biosafety cabinet and/or other primary devices for all activities

4 Biosafety Level 4 Dangerous pathogen units

Using BSL3 plus airlock entry, shower exit, special waste disposal

Class III biosafety cabinet, or positive pressure suite in conjunction with Class II biosafety cabinet, double-ended autoclave (through the wall), filtered air

Containment is the safe method for managing infectious agents in the laboratory where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers to potentially hazardous agents. There are three elements of containment; good microbiological technique, safety equipment, and facility design. The most important element of containment is strict adherence to standard microbiological practices and techniques. Individuals working with infectious agents or infected material must be aware of potential hazards and be trained and proficient in the practices and techniques required for handling such material safely. The Principal Investigator is responsible for providing or arranging for appropriate training of personnel.

Animal facilities, like laboratories, may be designed primarily according to the risk group of the microorganism under investigation as ABSL1, ABSL2, ABSL3, and ABSL4. You may refer to the OMRF LARC Animal Biosafety Levels for more details.

3. RISK ASSESSMENT

Risk assessment should be performed by individuals most familiar with the specific characteristics of the microorganisms being considered for use, the equipment and procedures to be employed, animal models that may be used, and the containment and facilities available. The Safety Officer or Principal Investigator is responsible for ensuring that adequate and timely risk assessments are performed and for working closely with the Foundation’s various safety

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committees to make sure that appropriate equipment and facilities are available to support the work being considered.

The concept of risk group is one of the most helpful tools available for performing a microbiological risk assessment. However, simple reference to the risk group for a specific agent is insufficient. Consideration must be given to the source of the microorganism (genetic characteristics, pathological, ecological, and physiological traits), whether there will be an alteration of the genome to produce an engineered organism and if there will be expression of the new genome. Questions should be asked whether there will be releases to the environment either intentional or accidental and the subsequent survival, multiplication, and dissemination in the environment. Undesirable effects of the engineered organism on humans, other organisms and the ecosystem should also be evaluated. Finally, consideration must be given to the experimental protocol, addressing the quantity of the organism(s), storage conditions and locations, disposal of materials, and potential aerosol generation. There is a list of microorganisms categorized by type (bacteria, fungi, parasites and viruses) compiled by the American Biological Safety Association, which is useful in determining which biosafety level should be assigned to an experiment involving a specific microorganism.

4. BIOSAFETY LEVELS ONE AND TWO

4.1. Biosafety Level 1 (BSL1)

BSL1 represents a basic level of contamination that relies on standard microbiological practices with no special primary or secondary barriers recommended other than a sink for hand washing. BSL1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause diseases in healthy adult humans.

4.2. Biosafety Level 2 (BSL2)

BSL2 is applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided that the potential for producing splashes or aerosols is low. BSL2 is appropriate when work is done with any human derived blood, body fluid, or tissue where the presence of an infectious agent may be unknown.

Table 3 Standard operation procedure for BSL2 facility Access The biohazard warning symbol must be displayed on the doors of entrance. Only

authorized persons should be allowed to enter the lab and lab doors are kept closed during an experiment.

Personal protection

Lab coats must be worn at all times for work in the lab. Wear gloves and wash hands after handling infectious agents and before leaving the lab. Wear safety glasses and face shields to protect the eyes and face from splashes. No eating, smoking, drinking, and mouth pipetting inside the lab.

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Lab equipment

All experiments should be performed inside the biosafety cabinet.

Medical Surveillance

All lab workers should undergo a pre-employment check at which their medical history is recorded. It is recommended to collect baseline serum samples from lab workers.

Training In house training by the Principal Investigator plays a key role in practicing good microbiological techniques. The Safety Officer can assist in training and with the development of training aids and documentation.

Waste disposal

All infectious materials should be decontaminated, autoclaved or incinerated. All biomedical wastes should be disposed in the biomedical waste container provided. Mark the date of disposal, PI’s name and the lab location on top of the boxes. Do not put more than 40 lbs. waste per box and do not put liquid or water-containing materials in the box. You may refer to the OMRF Waste Management Procedure Manual for details.

Sharps Hypodermic needles should not be recapped, clipped or removed from disposable syringes. The complete assembly should be placed in a sharps container. When they are three-quarters full they should be placed in the biomedical boxes with an “Incineration” sticker on the top of the box. DO NOT put needles in the regular trash.

5. BIOSAFETY LEVEL THREE

The BSL3 laboratory incorporates special engineering features to allow laboratory workers to handle hazardous materials without endangering themselves, the community or the environment. Knowledge of the functional principles and working procedures applicable to the BSL3 laboratory and strict adherence to these rules is required of all individuals who enter and work inside the facility.

The primary distinguishing characteristic of the BSL3 laboratory is that it consists of a series of secondary barriers to prevent biohazardous materials from escaping into the environment. These barriers include one hundred percent exhaust biological safety cabinets, a double-door autoclave, a waste treatment system for each sink, a negative pressure ventilation system which isolates increasingly hazardous work areas by a negative pressure gradient, and high efficiency particle filtration (HEPA) of the exhaust air from the laboratory.

Table 4 Standard operation procedures for BSL3 facility

Access Entry is only by approved card access. The biohazard warning symbol must be posted on the doors of entrance.

Personal protection

Personal protective equipment must be put on after entering the first door in the following recommended order: gowns, booties, mask and gloves. When exiting the facility, exit through sliding glass door to the dirty side of autoclave. Remove protective equipment in the recommended order listed above. Place all disposable protective equipment in trash container by sink. Wash hands before leaving.

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Standard microbiological practices

Work surfaces are to be decontaminated at least once a day and after any spill. All infectious liquids and solid wastes are to be decontaminated before disposal. Use mechanical pipetting, and no eating, drinking, smoking, and mouth pipetting. All procedures must be performed carefully to avoid the generation of aerosols.

Lab equipment Experiments using infectious agents or organisms will be performed in a biosafety cabinet. Emergency shower and eye wash station are installed inside the facility.

Training General training will cover standard microbiological practices, use of personal protective equipment, universal precautions, entry and exit rules, spill control, utility disconnection, medical emergency and fire control and suppression. Specific training provided by the Principal Investigator will cover the biological properties of the infectious disease or microorganism.

Utility disconnection

The red button located on the east wall of the BSL3 laboratory is to be used in the event of an emergency. Once the button has been hit, the HVAC system and electricity will shut down.

Medical emergency

The two-person rule should apply, whereby no individual ever works alone in the facility. In the event of an emergency, dial extension 17272. A first aid kit is available in the facility. Report all cuts and scrapes to your supervisor immediately. Fill out an incident report and submit to the Safety Office.

Medical surveillance

Baseline and additional serum samples may be collected for medical surveillance on all personnel who have access to the BSL3 facility.

Record keeping

Microorganisms under investigation should be secured and preferably locked inside incubators or freezers. Utilization of biological agents should be recorded and the physical inventory of microorganisms must match the book record to prevent theft or loss of infectious materials.

Waste management

All infectious materials must be decontaminated prior to disposal in a biomedical waste box. The BSL3 facility is equipped with a holding tank for all liquid waste. Each time the tap water is used, a bleach solution is mixed with the drainage in a holding tank before being released into the sanitary sewer.

Sharp Hypodermic needles and syringes are used only for injection and aspiration of fluids from animals and diaphragm bottles. Only needle-locking syringes or disposable needle-syringe assemblies are to be used. Extreme care should be used to avoid auto-inoculation and aerosol generation. Needles must not be bent, sheared, replaced in their sheath or removed from the syringe following use. The needle and syringe must be promptly placed in a puncture resistant container.

The maximum containment laboratory; the so-called BSL4 is designed for work with Risk Group 4 microorganisms. Before such a facility is constructed and put into operation, intensive consultation should be held with institutions that have experience in operating a similar facility. OMRF does not have a BSL4 laboratory.

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6. GOOD MICROBIOLOGICAL TECHNIQUES

Human error, poor laboratory techniques and misuse of equipment contribute to the majority of laboratory accidents. It is important to understand safe laboratory techniques and apply them to experiments. Ask your co-workers or supervisor if you have question on how to use equipment. Report any unsafe practice to your supervisor and Safety Officer.

A. Specimen containers may be of glass or preferably plastic. B. When transporting of specimens within the facility, secondary containers should be used

to avoid accidental leakage or spillage. C. For BSL 2 or above organisms, specimens should be unpacked in a biosafety cabinet.

Individuals who receive and unpack specimens should be trained and adopt universal precautions.

D. Mechanical pipetting devices should be used; pipetting by mouth is prohibited. Syringes with hypodermic needles must not be used for pipetting.

E. After use of BSL2 organisms, pipettes should be completely submerged in hypochlorite or other disinfectant before disposal.

F. Discarded specimen tubes containing plasma or blood clots should be placed in the biomedical waste box for autoclaving or incineration.

G. Large particles and droplets released during microbiological manipulations settle rapidly on bench surfaces and on the hands of the operator. Disposable gloves should be worn and preferably changed hourly. Avoid touching mouth, eyes and face during experiments.

H. Personal protective equipment is used in combination with biological safety cabinets and other devices which contain the infectious materials being worked with.

I. Working areas must be decontaminated with a suitable disinfectant at the end of each work period.

J. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Food is stored outside the work area in cabinet or refrigerators designated for this purpose only.

7. BIOSAFETY CABINETS

The biological safety cabinets (BSCs) are the principal devices used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. Open fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The Class II biological safety cabinet also provides protection from external contamination of the materials being manipulated inside the cabinet. The gas-tight Class III biological safety cabinet provides the highest attainable level of protection to personnel and the environment.

7.1. Start-up Procedure

A. Turn on the blower if the unit has not been left running continuously. After turning on the cabinet, let it run for at least three minutes allowing the air to be filtered and clean air to be cycled throughout the cabinet.

B. Turn off the ultraviolet light. Ultraviolet light causes eye and skin damage; it should not be on while there is anyone in the room.

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C. Turn on the fluorescent light. D. Ensure that the drain valve is in the closed position. E. Wipe down the interior of the cabinet with a surface disinfectant before and after all

manipulations. F. Place all material to be used for the procedure inside the cabinet. Within the work area,

segregate the clean and dirty materials. Do not place tape, pencil, paper inside the cabinet, and do not block the front and rear perforated grills.

G. After the equipment is inside the cabinet, ensure that the sash is eight to ten inches height.

H. Wear lab coat and other personal protective equipment as appropriate. I. Hands and arms should be washed thoroughly before and after each work period in the

cabinet. Operators are encouraged to wear long-sleeve gowns or lab coats with tight-fitting cuffs and sterile gloves.

7.2. Working in the Cabinet

A. The operator should be seated. The shoulder should be level with the bottom of the window. An adjustable stool will accommodate people of varying heights.

B. Perform all work on the depressed area of the solid work surface. Try to minimize entering or exiting the cabinet frequently.

C. Because opening or closing doors in the laboratory causes air disturbance, this kind of activity should be kept to a minimum. Schedule uninterrupted work times if possible.

D. Do not pipette without pipette aids. Avoid using floor-type pipette discard canisters. Discard pipettes into a surgical instrument tray or other suitable container within the cabinet.

E. Adopt good aseptic techniques. F. It is recommended that no Bunsen burner should be used inside the cabinet. G. Never operate a cabinet while a warning light or alarm is on; correct the problem first. H. If an accident occurs during cabinet operation, you need to decontaminate all items and

surfaces before any items are removed. I. When the work is finished, allow the cabinet to run for at least three minutes so that the

airborne contaminants are purged from the work area. Decontaminate equipment and work surface and properly dispose of waste.

J. Turn on the ultraviolet light for a short period of time. It is recommended to keep the cabinet running 24 hours a day.

7.3. Common Errors to Avoid

A. Do not use hazardous volatile chemicals in a recirculating biosafety cabinet. B. Do not hang or place a non-sterile item over an area where a sterile product will be

manipulated. C. Avoid sudden movement in and out of the cabinet as well as in the room outside the

cabinet. This disrupts the airflow and comprises safety. D. Do not eat, drink, smoke, chew gum, and store food near the cabinet. If the cabinet is

accidentally contaminated, you may ingest the toxins. E. Do not store equipment or supplies inside the cabinet. F. Do not use the top of the cabinet for storage. The HEPA filter could be damaged and the

balance of the airflow could be disrupted.

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8. SELECT AGENTS AND TOXINS

On October 25, 2001, Congress passed and the President signed legislation affecting the possession of biological agents with bioterrorism potential. These are called select agents. The legislation, called the USA Patriot Act, extends previous legislation that restricted the transport, but not the possession, of these agents. The new legislation restricts possession of the agents. The U.S. Department of Health and Human Services has established requirements to set up a nation-wide database to register all the select agents used in biomedical and clinical research. All individuals working with select agents must pass a background check. All facilities that possess, use, and transfer select agents must be approved by the federal government.

As listed in next page, select agents consist of HHS non-overlap select agents and toxins, high consequence livestock pathogens and toxins / select agents (overlap agents), USDA high consequence livestock pathogens and toxins (non-overlap agents and toxins), and listed plant pathogens. To prepare for the Act’s requirement, OMRF needs to know who is working with select agents including toxins and the nature of the studies contemplated. Based on the information gathered, you will be informed of your responsibilities under the Act. If it is decided your lab has to be registered with CDC or USDA, we will assign a registration number for the study. Periodically, we will write and ask for an update as to your uses of select agents.

USDA HIGH CONSEQUENCE LIVESTOCK PATHOGENS AND TOXINS (NON-OVERLAP HHS NON-OVERLAP SELECT AGENTS AND TOXINS AGENTS AND TOXINS) � Crimean-Congo haemorrhagic fever virus � Akabane virus � Coccidioides posadasii � African swine fever virus � Ebola viruses � African horse sickness virus � Cercopithecine herpesvirus 1 (Herpes B virus) � Avian influenza virus (highly pathogenic) � Lassa fever virus � Blue tongue virus (Exotic) � Marburg virus � Bovine spongiform encephalopathy agent � Monkeypox virus � Camel pox virus � Rickettsia prowazekii � Classical swine fever virus � Rickettsia rickettsii � Cowdria ruminantium (Heartwater) � Foot and mouth disease virus South American haemorrhagic fever viruses � Goat pox virus � Junin � Lumpy skin disease virus � Machupo � Japanese encephalitis virus � Sabia � Malignant catarrhal fever virus (Exotic) � Flexal � Menangle virus � Guanarito � Mycoplasma capricolum/ M.F38/M. mycoides capri Tick-borne encephalitis complex (flavi) viruses � Mycoplasma mycoides mycoides � Central European tick-borne encephalitis � Newcastle disease virus (VVND) � Far Eastern tick-borne encephalitis � Peste Des Petits Ruminants virus � Russian spring and summer encephalitis � Rinderpest virus � Kyasanur forest disease � Sheep pox virus � Omsk hemorrhagic fever � Swine vesicular disease virus � Vesicular stomatitis virus (Exotic) � Variola major virus (Smallpox virus) � Variola minor virus (Alastrim) � Yersinia pestis LISTED PLANT PATHOGENS � Abrin � Liberobacter africanus � Conotoxins � Liberobacter asiaticus � Diacetoxyscirpenol � Peronosclerospora philippinensis � Ricin � Phakopsora pachyrhizi � Saxitoxin � Plum Pox Potyvirus � Shiga-like ribosome inactivating proteins � Ralstonia solanacearum race 3, biovar 2 � Tetrodotoxin � Schlerophthora rayssiae var zeae � Synchytrium endobioticum HIGH CONSEQUENCE LIVESTOCK PATHOGENS � Xanthomonas oryzae AND TOXINS/ SELECT AGENTS (OVERLAP AGENTS) � Xylella fastidiosa (citrus � Bacillus anthracis variegated chlorosis strain) � Brucella abortus � Brucella melitensis � Brucella suis � Burkholderia mallei (formerly Pseudomonas mallei) � Burkholderia pseudomallei (formerly Pseudomonas pseudomallei) � Botulinum neurotoxin producing species of Clostridium � Coccidioides immitis � Coxiella burnetii � Eastern equine encephalitis virus � Hendra virus � Francisella tularensis � Nipah Virus � Rift Valley fever virus � Venezuelan equine encephalitis virus � Botulinum neurotoxin � Clostridium perfringens epsilon toxin � Shigatoxin � Staphylococcal enterotoxin � T-2 toxin

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9. RECOMBINNANT DNA SAFETY

9.1. Recombinant Organisms or Molecules

Recombinant DNA technology involves combining genetic information from different species thereby creating genetically modified organisms that may have never existed in nature before. Initially, this technology was used to clone DNA segments of interest in bacterial hosts in order to produce enough sufficiently pure material for studies. Recently, recombinant DNA molecules have been used to create genetically modified higher organisms such as transgenic and “knock-out” animals and transgenic plants.

Biological expression systems are vectors and hosts cells that fulfill a number of criteria that make them safe to use. A good example of a biological expression system is plasmid pUC 18, which is used as a cloning vector in combination with Escherichia coli K12 cells. Risk assessment must include not only the vector or host system used but also the properties of the DNA to be cloned. In most cases, the inserted DNA sequences are unlikely to alter the biological properties of the host organism, but in some cases they may do so, if they are derived from a pathogenic organism. All these factors should be considered to determine the biosafety level that is required for the safe handling of the genetically modified organism.

9.2. Reviews by the Recombinant DNA Committee

The review will include:

A. An independent assessment of the containment levels required for the proposed research.

B. An assessment of the facilities, procedures, and practices, and of the training and expertise of personnel.

C. Notifying the PI of the results of their review. D. Periodically reviewing biological research being conducted to ensure that the

requirements of the guidelines and other relevant regulations are being fulfilled. E. Emergency plans covering accidental spills and personnel contamination resulting from

such research.

10. DISINFECTIONS AND WASTE DISPOSAL

10.1. Disinfections

All material, equipment, or apparatus contaminated with or containing potentially infectious organisms should be decontaminated before being washed, stored, or discarded. Each individual working with biohazardous materials is responsible for sterilization of material before disposal.

A. Biohazardous material should not be placed in the autoclave overnight in anticipation of autoclaving the next day.

B. All biohazardous material must be placed in an appropriately marked refrigerator or incubator, sterilized, or otherwise confined at the close of each work day.

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C. Precautions should be taken to prevent accidental removal of material from an autoclave before it has been sterilized.

D. Dry hypochlorites, or any other strong oxidizing material, must not be autoclaved with organic material such as paper, cloth, or oil. (OXIDIZER + ORGANIC MATERIAL+ HEAT = EXPLOSION)

E. Laboratory benches and other surfaces where biohazardous material has been handled should be disinfected at the end of each work period.

F. All floors should be disinfected at least monthly or as needed.

10.2. Autoclave Procedures

All biologically active waste should be autoclaved if possible. Treatment conditions to achieve sterility will vary in relation to the volume of material treated, its contamination level, and the moisture content. The most critical factors for insuring the reliability of sterilization by autoclaving are that the waste has reached 121 degrees C for the necessary period of time: 30 minutes for trash, 25 minutes for glassware, and 25 minutes for each gallon of liquid. OMRF currently utilizes a company to haul biomedical wastes, which can be autoclaved or incinerated offsite.

10.3. Disinfectants

A. Phenolic Compounds - They kill vegetative bacteria, including mycobacterium tuberculosis (TB), fungi, and lipid containing viruses. Two examples are phenol and phenolic acid.

B. Quaternary Ammonium Compounds - These are acceptable as general use disinfectants to control vegetative bacteria and non-lipid containing viruses. This is not effective against bacterial spores. Two examples are cetrimide and benzalkonium chloride.

C. Iodophors - These are effective against vegetative bacteria, fungi, and viruses. The mostwidely used iodophors for surgical scrub is providone iodine, which contains 7.5% of iodine.

D. Alcohols - In concentrations of 70 to 95%, alcoholic solutions are good general use disinfectants. They exhibit no activity against bacterial spores.

E. Aldehydes - These are effective against a wide spectrum of bacteria, fungi, and viruses. When used properly they are effective against bacterial spores.

F. Chlorine Compounds - These are recommended for certain disinfecting procedures provided the available chlorine needed is considered. Low concentrations of available chlorine (up to 0.05%) are effective against vegetative bacteria, fungi, and most viruses. Sporicidal action can be attained at about 0.25%.

Be sure to label disinfectant containers with the date the disinfectant was made. The shelf lives of some disinfectants are: iodophors (1 month), chlorine bleach (2 weeks), and activated glutaraldehyde (2 weeks). Before beginning work, check to see that there is disinfectant on hand and that it has not expired. If it has expired, make a fresh solution before beginning work. All disinfectants should be used in accordance with the manufacturer's recommendations.

10.4. Disposal of Waste

A. All material containing human blood or blood products must be disposed in a biomedical waste box for autoclaving or incineration.

B. All cultures containing microorganisms or viruses should be decontaminated prior to disposal in a biomedical waste box, in case of leakage.

C. All contaminated noncombustible waste (broken glassware and sharps) should be contained prior to disposal in a biomedical waste box.

D. All biological liquid waste must be decontaminated prior to disposal in the sanitary sewer.

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E. Broken glass should be placed in a cardboard container or other puncture resistant container prior to being placed in a biomedical waste box.

F. Other Sharps - All needled and needle-syringe assemblies are to be disposed of intact in a puncture resistant sharp container. Needles are not to be recapped, bent, or broken.

G. The sharp container should be disposed in a biomedical waste box.

11. CONTINGENCY PLANS AND SPILL CONTROL

11.1 Contingency Planning

A written contingency plan for dealing with laboratory accidents is a necessity in any facility that works with and stores Risk Group 3 or 4 microorganisms. Emergency procedures should be communicated to all laboratory staff. The telephone numbers of lab supervisor, Safety Officer, campus police, and hospital or ambulance services should be prominently displayed near all telephones. First aid kit, stretcher, fire blanket, and fire extinguisher should be available.

11.2. Spill in a Biological Safety Cabinet

If a spill occurs inside a biological safety cabinet, the decontamination procedure should be initiated at once while the cabinet continues to operate to prevent the escape of microorganisms from the cabinet.

A. Spray and/or wipe walls, work surfaces, and equipment with a disinfectant that is effective against the agent in use.

B. Flood the top work surface tray, the drain pans and catch basins below the work surface with a disinfectant and allow standing for 20 minutes.

C. Remove excess disinfectant from the tray by wiping with a sponge. For Class II cabinets, lift out trays and removable grille work, and wipe off top and bottom surfaces with a disinfectant. Replace in position and drain remaining disinfectant from cabinet base into appropriate container.

D. All cleanup material should be disposed in the biomedical waste box.

11.3. Spill outside the Biological Safety Cabinet

A. If you are working with a BSL3 agent or have spilled more than 100 ml of a BSL2 agent, hold your breath and leave the room immediately.

B. Remove contaminated garments and place in biohazard bag for autoclaving. C. Thoroughly wash hands, face, and any other contaminated body areas. D. Wait for 30 minutes to allow dissipation of aerosols. E. Put on new personnel protection equipment before re-entering the area. F. Place paper towels over the spill and pour disinfectant around and on the paper towels. G. Let disinfectant stand 20 minutes before cleanup. H. Wipe from the outside of the spill toward the center. All disposable material used must be

placed in a biohazard bag for autoclaving and disposal. I. All non-disposable items used in the cleanup need to be decontaminated when the

cleanup is finished.

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12. TRAINING AND MEDICAL SURVELLANCE

12.1. Technical Training Requirements

Individual training for technical staff members will include:

A. Biology of the organism used in experiments with emphasis on potential biohazard B. Good aseptic technique C. Proper techniques for decontamination D. Emergency procedures E. All aspects of the microbiological safety manual.

Before staff members start to work they should:

A. Read and understand the microbiological safety manual B. Enroll in the medical surveillance program, if applicable C. Demonstrate working knowledge of all relevant safety practices, and understanding of the

research they do and its potential hazards.

12.2. Training Requirements for Non-technical Staff

Non-technical staff members should be familiar with the potential hazards associated with biological research in general. All such workers are shown and informed of the meaning of the biohazard and radiation signs. All non-technical staffs are discouraged from entering the BSL3 facility while an experiment is in progress. Glassware washers are informed of all waste disposal procedures, particularly autoclaving methods, as well as emergency procedures for handling spills of biological materials. Safety is a shared responsibility. In order to reduce the risk inherent in research, attention to details is required. Planning and proper execution of procedures often requires additional effort but these extra precautions may prevent problems in the future. Individual workers are directly responsible for cleanup following each experiment.

12.3. Medical Surveillance Program

The medical surveillance program is designed to monitor potential health hazards associated with research and development activity, including recombinant DNA, etiologic agents, and hazardous chemicals. The program is coordinated through the Safety Office and Personnel Office. A serum sample may be drawn from an employee and stored to provide a reference should any medical problem arise. Individuals with severe immunological deficiencies or undergoing treatment with immunosuppressant drugs should be discouraged from working with potentially infectious organisms. Certain other medical conditions may place the worker at increased risk. These conditions will be evaluated to provide the worker with a clear understanding of the associated risk. Individuals undergoing treatment with steroids, and women during pregnancy should consider whether or not they should participate in any potentially hazardous research activities. Consultation with a health professional is available. Personnel using human source materials are urged to be immunized against Hepatitis B. Participation in this program is voluntary and individuals should assess the risks and benefits after consultation with a health care professional.