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United States Department of Agriculture

Agricultural Research Service

Biological Safety Manual
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Biological Safety Manual

BIOSAFETY LEVEL 2 GUIDELINES 5

BIOSAFETY LEVEL 3 GUIDELINES 5

CLASSIFICATION OF ETIOLOGIC AGENTS ON THE BASIS OF HAZARD 6

DISPOSAL OF RESEARCH LABORATORY WASTE 7

UTILIZATION OF THE BIOHAZARD STEP-ON CONTAINERS OR BAG HOLDERS 8

SUMMARY OF WASTE DISPOSAL 9

CENTRIFUGE CONTAINMENT 10

HIGH-SPEED CENTRIFUGES 11

BLENDERS, SONICATORS, MILLS, GRINDERS & CELL SORTERS 12

WATER BATH DISINFECTANTS 13

LABORATORY VACUUM TRAPS 13

REFRIGERATORS, FREEZERS AND DRY ICE CHESTS 14

TRANSPORTATION OF BIOHAZARDOUS MATERIAL 15

HAZARDOUS OPERATIONS (TWO PERSON RULE)AND THE SECURED LABORATORY 17

USE OF DRY POWDERS 18

DECONTAMINATION AND DISINFECTION PROCEDURES 19

STEAM AUTOCLAVING 21

AUTOCLAVE DECONTAMINATION PROCEDURES 22

INDICATORS 22

BIOLOGICAL INDICATORS 23

CONTAINERS 24

SECONDARY CONTAINERS 25

AUTOCLAVE OPERATION (NEW SOP) 25

TRAINING 27

RECORD KEEPING - AUTOCLAVE LOG BOOK 27

AUTOCLAVE LOG BOOK SAMPLES 28

ULTRAVIOLET LIGHT DECONTAMINATION 29

UV LAMP OPERATION 29

TRAINING 30

CHEMICAL DISINFECTANTS 31

CLEAN-UP OF BIOHAZARDOUS SPILLS 34

SHIPMENT OF BIOLOGICAL MATERIALS 37

BIOHAZARD CONTAINMENT EQUIPMENT 41

BIOLOGICAL SAFETY CABINET CATEGORIES 42

USE OF BIOLOGICAL SAFETY CABINETS 44

OPERATION OF BIOLOGICAL SAFETY CABINETS 45

BIOSAFETY CABINET TRAINING MATERIALS 50

CLASS II, TYPE B2 BIOLOGICAL SAFETY CABINETS 50

CLASS II, TYPE B3 BIOLOGICAL SAFETY CABINETS 51

VENDORS OF BIOLOGICAL SAFETY CABINETS 51

TESTING/CERTIFYING BSC 54

ANNUAL CABINET LABOR AND SERVICE CONTRACT 55

MOVING YOUR CABINET 56

BIOLOGICAL SAFETY CABINET (BSC) PERFORMANCE EVALUATION TESTS 56

HAZARD SIGNAGE 59

GENERAL LABORATORY PRACTICES AND PROCEDURES 63

REFERENCES

BIOSAFETY REFERENCES


BIOSAFETY LEVEL 2 GUIDELINES

Biosafety Level 2 (BSL 2) is similar to Biosafety Level 1 and is suitable for work in clinical, diagnostic, teaching,research or production facilities involving agents of moderate potential hazard to personnel and the environment. It differs in that;

(1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists,

(2) access to the laboratory is limited when work is being conducted, and

(3) certain procedures in which infectious aerosols are created are conducted in biological safety cabinets or other physical containment equipment. Refer to Section III -- Recombinant DNA Guidelines.

 

BIOSAFETY LEVEL 3 GUIDELINES

Biosafety Level 3 (BSL 3) is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the aerosol route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other physical containment devices or by personnel wearing appropriate personal protective clothing and devices. No children are permitted in the laboratory.

The laboratory has special engineering and design features. It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetrations, and directional air flow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in laboratories where facility features satisfy Biosafety Level 2 recommendations provided the recommended "Standard Microbiological Practices," "Special Practices", and "Containment Equipment" for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made by the laboratory director on advice of the Biosafety Officer. A specific facility operations manual is prepared, reviewed by the Biosafety Officer, and adopted. Refer to Section III -- Recombinant DNA Guidelines.

 

CLASSIFICATION OF ETIOLOGIC AGENTS ON THE BASIS OF HAZARD

The CDC originally prepared a document which provided a standard for evaluating the hazards associated with the various bacterial, fungal, parasitic, and viral etiologic agents. The list of agents was revised by the NIH for the Recombinant DNA guidelines. A more recent document describes biosafety precautions for use of agents which have resulted in known laboratory infections.

In the CDC documents, human etiologic agents were placed in classes of increasing hazard. The classification is included here as a general reference. Specific examples of agents are listed under each class.

A. Class 1 Agents of no or minimal hazard under ordinary conditions of handling at Biosafety Level 1.

B. Class 2 Agents of ordinary potential hazard. This class includes agents which may produce disease of varying degrees of severity from accidental inoculation or injection or other means of cutaneous penetration but which are contained by the ordinary laboratory techniques described in Biosafety Level 2.

C. Class 3 Agents involving special hazard or agents derived from outside the United States which require a federal permit for importation unless they are specified for higher classification. This class includes pathogens which require special conditions for containment, usually at Biosafety Level 3.

D. Class 4 Agents that require the most stringent conditions for their containment because they are extremely hazardous to laboratory personnel or may cause serious epidemic disease. This class includes Class 3 agents from outside the United States when they are employed in entomological experiments or when other entomological experiments are conducted in the same laboratory area. Biosafety Level 4 facilities are limited to places such as the CDC, NIH, etc.

 

DISPOSAL OF RESEARCH LABORATORY WASTE

A. DEFINITION OF "SHARPS"

"Sharps" are defined by the State of Maryland as; syringes with needles, needles alone, capped needles, scalpels, razor blades, pasteur pipettes, small glass tubes, capillary tubes, butterfly needles, glass pipettes, microscope slides, coverslips, microtome knives, and any other items which can penetrate the skin.

B. DISPOSAL OF LABORATORY WASTE

1. Sharps must never be placed directly into plastic trash bags. Sharps must be discarded into an approved sharps container.

2. Needles must never be left on laboratory furniture, wrapped in paper towels, or covered by other materials.

3. Needles must never be clipped or bent.

4. Needles must not be recapped using both hands.

5. Laboratory waste must never be discarded directly into the general trash or into red bags. All lab waste must be discarded into the red bag-lined biohazard box.

6. Only approved containers are acceptable. Do not substitute glass, metal or plastic jars, bottles or cans.

7. A broken glass box with a red bag liner is not an acceptable substitute for a red bag-lined biohazard box.

C. APPROVED LABORATORY WASTE CONTAINERS

Containers approved for laboratory waste can be purchased from several sources. See Lab Safety Supply catalog.

D. UTILIZATION OF QUART PLASTIC CONTAINERS (TWO, EIGHT GALLON)

See Lab Safety Supply Catalog starting at Page 455.

1. The plastic sharps disposal containers are used for discarding needles attached to syringes or cartridges, needles, small glassware and other sharps (as defined above) at the point of use. The larger, ten-quart plastic sharps disposal container is available for disposal of higher volumes of sharps, including pasteur pipettes and larger-sized sharps.

2. Before use, the plastic sharps containers must be put in the metal stand designed for the container. The screw cap cover is to be left off until the container is ready for disposal. (Parts for mounting unit are in catalog, page 455.

3. When discarded sharps reach the fill level designated on the sharps container (at the constriction), screw on the cap, remove the closed container from the metal stand. (Handle sharps containers with caution, they are puncture resistant, not puncture proof). Discard the closed container into a red bag-lined biohazard box. Also available in Lab Safety Catalog, page 461.

 

E. UTILIZATION OF THE BIOHAZARD STEP-ON CONTAINERS OR BAG HOLDERS

1. The containers with red plastic liner is for disposal of all laboratory waste; including, but not limited to, sharps containers, pipettes, autoclaved waste material, tubes, materials soiled with potentially infectious agents, tissue, or body fluids, calibrated plastic centrifuge tubes, conical tubes and pipettes, glass, and paper towels.

2. All solid, autoclave-decontaminated materials must be discarded into the biohazard box.

3. Each biohazard container used for disposal of laboratory waste must contain a red bag liner or marked bag at least 2 mil thick or equivalent.

4. The red bag liner is removed from the box for in-house autoclaving. Puncture resistant gloves must be worn while removing bag.

5. The biohazard cardboard or plastic container can be reused.

6. When the biohazard box is full, the red bag liner should be carefully closed and sealed with tape.

7. Pasteur pipettes, needles and syringes, and similar "sharps" contaminated with cultures should be placed into a, polypropylene autoclave bag-lined, stainless steel or polypropylene pan with cover containing enough disinfectant solution, such as Wescodyne (diluted 1:10), to cover the pipettes, and then autoclaved. Autoclaved pasteur pipettes, needles and syringes are then discarded into the biohazard box.

F. SAFE DISPOSAL OF SHARPS CONTAINERS

1. Handle all sharps containers with caution, they are puncture resistant, not puncture proof.

2. Filled containers must be closed as appropriate and replaced.

3. All filled, plastic sharps containers are considered to be infectious waste even if filled with non-infectious materials.

4. All properly closed plastic sharps containers must be placed in an approved biohazard box.

5. All plastic sharps containers that have been autoclaved may have "sharps" protruding through the side. The containers must be placed into an approved biohazard box, never directly into a red bag.

G. Biologically contaminated, broken glassware will be discarded into biohazard boxes/bags.

 

H. SUMMARY OF WASTE DISPOSAL

1. All laboratory waste must be discarded into approved red (Or other designation for biohazard) bag-lined biohazard boxes/bags.

2. Cultures of bacteria, fungi, viruses, other microorganisms, and insects must be autoclaved before discarding into a biohazard box/bag.

3. There are several approved biohazard decontamination bags 2 to 3 mil thick, autoclaveable bags,  see  Laboratory Safety Supply Catalog.

4. All sharps (defined as any material capable of piercing skin, including but not limited to needles, razor blades, pasteur pipettes, and capillary tubes) must be discarded into the approved sharps container. 

5. Glass, metal or plastic jars, bottles and cans are not acceptable substitutes for the approved plastic sharps container.

6. Broken glass boxes are not acceptable substitutes for biohazard boxes.

7. Do not, under any circumstances, put laboratory waste outside the building.

 

CENTRIFUGE CONTAINMENT

A. CENTRIFUGE PRECAUTIONS

When used properly, centrifuges should not produce aerosols.  However, an accident resulting in breakage of a centrifuge tube or bottle can result in a massive exposure. Centrifuges should not be used in corridors or other unauthorized locations.

Certain precautions should be considered when using free standing centrifuges:

1. A swinging bucket head should be used in preference to a fixed angle head because there is a lower probability of aerosol generation.

2. Centrifuge models should be chosen which have a sealed rotor, or sealed buckets, or with at least a guard bowl and gasketed cover. Safety centrifuge cups (tube or bottle carrier with sealable cap or "O" gasketed cap) should be used.

3. The centrifuge chamber should be fitted with a HEPA-filtered exhaust system to capture and remove aerosols from the chamber when containment is needed.

4. Centrifuge tubes or bottles should only be filled, loaded into rotors, and removed from rotors from within a biological safety cabinet. This practice provides containment in case a tube or bottle leaks or breaks.

B. CENTRIFUGE TUBES

1. Before use, tubes should be checked for cracks. The inside of trunnion cups should be inspected for rough walls caused by erosion or adhering matter, and glass pieces should be carefully removed from the rubber cushion. A germicidal solution added between the tube and trunnion cup not only disinfects the outer surfaces of both, but also provides a cushion against shocks that might otherwise break the tube. Metal or plastic tubes (other than nitrocellulose) should be used whenever possible.

2. Never overfill a centrifuge tube or bottle, particularly when using a fixed angle head. Avoid filling tubes to the point where the rim of the centrifuge head becomes wet during a run.

3. Decanting from centrifuge tubes should be avoided. If decanting is necessary, the outer rim should be wiped with a disinfectant.

4. If a tube breaks, the centrifuge should be turned off, allowed to stand undisturbed for 15 minutes, opened, and disinfected. The rotor should also be disinfected.

5. After use, tubes, rotors, and centrifuge interiors should be adequately cleaned and disinfected.

 

C. HIGH-SPEED CENTRIFUGES

1. High-speed centrifuge chambers are connected to a vacuum pump. If there is a breakage or accidental dispersion of infected particles, the pump and pump oil will become contaminated. A HEPA filter should be placed between the centrifuge inner chamber and the vacuum pump when containment is needed.

2. High speed rotor heads are prone to metal fatigue. Each rotor should be accompanied by its own log book indicating the number of hours run at top or de-rated speeds. Failure to observe this precaution can result in dangerous and expensive rotor disintegration. Frequent inspections, cleaning and drying are important to ensure absence of corrosion or other traumata which cause rotor cracks. Rubber O-rings and tube closures must be examined for deterioration and be kept lubricated with material recommended by the manufacturer (high temperature silicone vacuum grease). Where tubes of different material are provided (e.g., celluloid, polypropylene, stainless steel), make sure that you are using the tube closure that was designed specifically for the tube being used. These caps are often similar in appearance, but are prone to leakage if put on tubes of the wrong material. When properly designed tubes and rotors are well maintained and handled, leakage should not occur.

3. Cleaning and disinfection of tubes, rotors and other components requires considerable care. No single method is suitable for all items, and the various manufacturers' recommendations must be followed meticulously if rotor fatigue, distortion and corrosion are to be avoided.

4. Manufacturers' safety notices and recalls may not reach you if a transfer of owner/user has occurred unless you notify the manufacturer.

 

D. BLENDERS, SONICATORS, MILLS, GRINDERS & CELL SORTERS

These devices release considerable aerosols during operation.  For maximum protection to the operator during blending or mixing of infectious materials, the following practices should be observed.

1. Operate blending, cell-disruption and grinding equipment only in a biological safety cabinet. Place a towel moistened with disinfectant over the top of the blender.

2. Use safety blenders designed to prevent leakage from the rotor bearing at the bottom of the bowl.

3. If you don't have a leak-proof rotor, inspect the rotor bearing at the bottom of the blender bowl for leakage prior to operation. Test it in a preliminary run with sterile saline or methylene blue solution prior to use with infected material.

4. Autoclave the device and residual infectious contents promptly after use.

5. Glass blender bowls are undesirable for use with infectious materials because of potential breakage. If used, they should be covered with a polypropylene jar to prevent dispersal of glass in case of breakage.

6. A heat-sealed flexible plastic film enclosure for a grinder or blender can be used, but it must be opened in a biological safety cabinet.

7. Before opening, permit the blender to stand for at least 15 minutes after the run to allow the aerosol cloud to settle within the chamber.

8. Clinical or other laboratories handling human blood or tissue should be aware of possible biohazardous aerosols produced by microhaematocrit centrifuges, autoanalyzer stirrers, microtonometers, FACS's, etc.

 

WATER BATH DISINFECTANTS

Water baths and Warburg baths used to inactivate, incubate, or test biohazardous materials should contain a disinfectant.  For cold water baths, 70% propylene glycol is recommended.  WescodyneTM (AMSCO) works well in intermediate temperature water baths. Azides should not be used as a bacteriostatic agent because they may form heavy metal azides. These are shock-sensitive and may explode. Change disinfectant frequently.

 

LABORATORY VACUUM TRAPS

When laboratory vacuum is used to manipulate biohazardous materials, a suitable trap must be employed to insure that building vacuum pump and compressed air lines (they are often linked together in the building mechanical room) do not become contaminated.  Laboratory vacuum service should be provided by the use of small, individual vacuum pumps fitted with high efficiency particulate air (HEPA) filters on the suction side. Central house systems are not recommended. If house vacuum must be used, the system should include in-line HEPA filter as near as practical to each point of use or service cock. An approved reservoir and filtration apparatus for vacuum systems is described below:

Vacuum filtration or aspirating supernatants into collection flasks are common laboratory procedures. During vacuum filtration or aspiration procedures building and/or laboratory vacuum systems should be protected.

A simple bench-top aerosol/fluid trap can protect building/laboratory vacuum systems. The basic vacuum trap consists of a disposable cartridge-type filter or equivalent installed in-line with a collection/overflow vacuum flask system.

The aerosol/fluid trap consists of two vacuum flasks, preferably plastic, (size dependent on amount of fluid that may accidentally be aspirated out of the collection flask), thick walled plastic tubing (to prevent tubing collapse), rubber stoppers, a filter (prevents unwanted potentially biohazardous fluid and aerosols from entering vacuum systems), and a ceramic sparger (ceramic fish tank bubbler) immersed in disinfectant. The sparger disperses aerosols passing out of the collection flask into small bubbles so that adequate contact is made with a disinfectant solution. Use an appropriate  disinfectant solution shown to be effective on the biohazardous material under study. It is essential that anti-foam spray be added to the overflow flask since air bubbling through the disinfectant- immersed sparger may produce foam that could shut off the vacuum if allowed to clog the filter.

When the filter or overflow flask require routine changing, they can be safely removed by clamping the line between the filter and the vacuum source before disconnecting the tubing from the source. The filter and vacuum flask should be decontaminated by autoclaving if they have been in contact with potentially biohazardous material.

 

REFRIGERATORS, FREEZERS AND DRY ICE CHESTS

Refrigerators, freezers and dry ice chests should be checked, cleaned out, and defrosted periodically to remove any ampules, tubes, etc., containing biohazardous materials that may have broken during storage. They must be discarded into a biohazard box. Rubber gloves are recommended for cleaning.

All materials, especially infectious or toxic materials stored in refrigerators or freezers, should be labeled with their scientific name, date stored, and name of the individual storing the material. Do not store flammable solutions in refrigerators because standard domestic refrigerators must not be used for storage of flammable materials.

When storing biohazardous materials in liquid nitrogen, use gasketed, screw-cap plastic ampules whenever possible because these are specially designed for this purpose and reduce the risk of accidental shattering or leakage. Wear protective gloves and a face shield when working with liquid nitrogen storage equipment.

 

TRANSPORTATION OF BIOHAZARDOUS MATERIAL

A. Transportation of potentially pathogenic/oncogenic fluid cultures or toxins and viable, powdered biohazardous materials should be transported, incubated, and stored in easily handled, non-breakable, leak-proof pans, trays, pails, carboy holders, or other secondary containers large enough to contain all the fluid or powder in case of leakage or breakage of the primary container. All inoculated petri plates or other inoculated solid media should be transported in leak-proof containers.

Containers of biohazardous materials to be transferred between buildings must be placed inside non-breakable containers having solid sides and bottoms and tight covers to prevent breakage or spillage during transit.

B. Shipment of biohazardous materials such as cultures or biological toxins and/or diagnostic or clinical specimens which the shipper expects may contain etiologic agents (such as bloodborne pathogens) must conform to 42 CFR Part 72.3 "Interstate Shipment of Etiologic Agents", 1980. Packages must contain a secondary container, absorbent material between the primary and secondary container, and an outer shipping container with proper labeling.

TEST TUBE / VACUTAINER TECHNIQUES

Tubes containing cultures, blood, or other biohazardous materials should be manipulated with extreme care. Studies have shown that a simple procedure such as removing a tube cap or transferring an inoculum can create a potentially hazardous aerosol.

Manipulation of biohazardous material should be conducted in biological safety cabinets. Tubes and racks of tubes containing biohazardous material should be clearly marked. It is the responsibility of the individual employee to insure that tubes containing biohazardous material are properly decontaminated prior to disposal and/or glassware washing.

Whenever possible, use test tube trays with a solid bottom and sides deep enough to hold all spillage from broken tubes.

 

HAZARDOUS OPERATIONS (TWO PERSON RULE) AND THE SECURED LABORATORY

The Lead Scientist is responsible for ensuring that laboratory personnel in the secured lab are in constant contact with outside personnel.

All staff members, including the Physical Plant group, have a key to enter the laboratory. A viewing panel allows outside people to see in.

The interior lab has a phone and emergency contact numbers available.

Intercom systems, and 2 way radios can be used.

 

USE OF DRY POWDERS

The preparation, handling, and use of dry powders of biohazardous materials present hazards of an unusual nature.  The slightest manipulation of such powders may release an aerosol containing high concentrations of infectious or toxic material. Therefore, work with dry powders of biohazardous materials should be done only in biological safety cabinets, or in glove boxes with slight negative airflow. Dry powders are difficult to decontaminate, particularly with liquid disinfectants. Disinfectant applications should be thorough and with longer than usual contact times.

 

DECONTAMINATION AND DISINFECTION PROCEDURES

A. All infectious or toxic materials, and all contaminated equipment or apparatus should be  decontaminated before being washed and stored, or discarded. Autoclaving is the preferred method. Each individual working with biohazardous materials should be responsible for decontamination before disposal.

B. To minimize hazard to firemen or disaster crews, all biohazardous materials should be placed in an appropriately marked refrigerator or incubator, or decontaminated, or otherwise confined at the close of each work day.

C. All autoclaves should be certified for operating efficiency by the frequent use of biological indicator controls. All autoclaves should bear a sign indicating the maximum permissible pressures and last date of certification.

D. Special precautions should be taken to prevent accidental removal of material from an autoclave before it has been sterilized or decontaminated. Simultaneous opening of both doors on a double door autoclave must not occur. Biohazardous materials should not be placed in autoclaves overnight in anticipation of autoclaving the next day.

E. Dry hypochlorites, liquid bleach, and any other strong oxidizing material, must NOT be autoclaved.

F. All laboratory rooms containing biohazardous materials should designate two separate carts or containers labeled:

1. BIOHAZARDOUS - TO BE AUTOCLAVED BEFORE CLEANING

2. NON-INFECTIOUS - TO BE CLEANED

G. All floors, laboratory benches, and other surfaces in buildings where biohazardous materials are handled should be disinfected as often as deemed necessary. The surroundings should be disinfected after completion of operations involving plating, pipetting, centrifuging and similar procedures with biohazardous materials. It is rhe responsibility of the supervisor or CHO (CHO means Chemical Hygiene Officer) to determine that the disinfectant and the time and method of exposure is effective against the biological agent(s) used in the facility.

H. Drains in BL2 and BL1 facilities should be flooded with water or disinfectant at least once each week in order to fill traps and thus prevent the backflow of sewer gases.

I. Floor cleaning procedures which minimize the generation of environmental aerosols should be used. Wet mopping or wet vacuum pickup is recommended. Water used to mop floors should contain a disinfectant or disinfectant-detergent. (Dry mopping or dusting should be avoided). Where wet procedures are not practicable, dry vacuum cleaning with a HEPA filter on the exhaust, sweeping compound used with push brooms, or dry dust mop heads treated to suppress aerosolization may be used.

J. Stock solutions of suitable disinfectants should be maintained in each laboratory for disinfection purposes. The disinfectant should be kept readily available in the use-dilution.

 

STEAM AUTOCLAVING

A. Steam sterilization of clean materials is a dependable procedure for the destruction of all forms of microbial life.  Steam sterilization generally denotes heating in an autoclave employing saturated steam under a pressure of approximately 15 psi to achieve a chamber temperature of at least 121oC (250oF) for a minimum of 15 minutes. The time is measured after the temperature of the material being sterilized reaches 121oC (250oF). The most critical factor in insuring the reliability of this sterilization method other than proper temperature and time is the prevention of entrapment of air that is not replaced by steam.

Some autoclaves utilize a steam activated exhaust valve that remains open during the replacement of air by live steam until the steam triggers the valve to close.  Others utilize a pre-cycle vacuum to remove air prior to steam introduction.

B. Physical controls such as pressure gauges and thermometers are widely used but are considered secondary methods of insuring sterilization. The use of appropriate biological indicators at locations throughout the autoclave is considered the best indicator of sterilization. The biological indicator most widely used for wet heat sterilization is a Bacillus stearothermophilus spore suspension or strip.

C. Sterilization the biological indicator and associated documentation is required by regulation. Maintain information as part of the autoclave log book.

 

AUTOCLAVE DECONTAMINATION PROCEDURES

This establishs guidelines for the effective use of steam autoclaves for the decontamination of cultures and other potentially pathogenic materials and protects personnel and the environment from these potentially infectious materials and waste. This will also meet the waste disposal requirements of the State of Maryland, AAMI, AHA, CDC, and JCAHO.

A. BACKGROUND

Use the following basic guidelines to develop your own autoclave quality assurance program for monitoring autoclave decontamination effectiveness. Effective quality assurance includes: using chemical and biological indicators to check autoclave operation; selecting appropriate containers to hold waste while being decontaminated; using effective decontamination processing times for each load; maintaining proper autoclave use records; providing odor control when necessary; and providing personnel training for the operation of an autoclave.

Use of steam sterilizers (autoclaves) should include validation of decontamination effectiveness. Validation of effectiveness includes monitoring temperature, pressure and cycle duration time for each cycle and providing periodic decontamination challenges (quality assurance) i.e., use of biological indicators. A logbook shall be maintained to record autoclave use. The logbook should be available for inspection by various agencies, authorities, and the NAA Safety Office.

B. INDICATORS

1. CHEMICAL INDICATORS

a. Chemical Color Change Indicators:

Chemical indicators for steam autoclaves change color after being exposed for a few minutes to normal autoclave operating temperatures of 121oC (250oF). Hence, chemical indicators can give you a quick visual reference for heat penetration inside the load. Chemical indicators should be positioned near the center of each load, and toward the bottom front of the autoclave.

CAUTION: Most chemical indicators can only be used to verify that your autoclave has reached normal operating temperature for decontamination, 121oC (250oF); they have no time factor.  Chemical indicators alone are not designed nor intended to prove that organisms are actually killed during a decontamination cycle.

Where to Purchase Chemical Indicators: Chemical indicators are manufactured by many companies and come in a wide variety of sizes, shapes, and colors. Some examples are: "Chemdi Strips" - Amsco, "Dualchek and Single Indicators" - Baxter, "Test-A-Clave" - Baxter, "ATI Indicator Tape" - Thomas Scientific, "ATI Sterilometer-Plus 250" - Thomas Scientific, "Sterikon-Bioindicator" from EM - VWR, and "TSI Time Indicator" - VWR.

b. Tape Indicators:

Tape indicators are adhesive backed paper tape with heat sensitive, chemical indicator markings. Commonly used heat sensitive markings include diagonal stripes (autoclave tape), and/or the word "sterile". These markings only appear when the tape has been exposed for a few minutes to normal autoclave decontamination temperatures.

CAUTION: Tape indicators can only be used to verify that the autoclave is reaching normal operating temperatures for decontamination, 121oC (250oF). Tape indicators alone are not designed nor intended to prove that organisms have actually been killed.

Tape indicators should be used on all material decontaminated by autoclaving to show that the material has been processed. A three to four inch strip of autoclave tape placed on the outside of the autoclave pan, bag, or individual container is sufficient.

2. BIOLOGICAL INDICATORS

Biological indicator systems are designed to demonstrate that an autoclave is capable of killing microorganisms.

Only Bacillus stearothermophilus spores can be used to monitor the effectiveness of steam  autoclaves.

Typical biological indicator systems consist of a vial with spore strips or a small glass ampule of growth medium with spores and indicator dye. Refer to manufacturers' instructions for use. The biological indicator is removed from a load after it has been autoclaved. Then the biological indicator is incubated at 56oC for up to three days. Your control vial, which was not autoclaved, should be turbid after incubation; the successfully decontaminated test vial should remain clear without evidence of turbidity (no growth). If the autoclaved biological indicator is turbid (cloudy, indicating growth) the autoclave did not function properly.  Notify the lead scientist when this happens.

Where to Purchase Biological Indicators: Some manufacturers of biological indicators have incubators available to fit their vials. Some examples of biological indicators are: "Spordex Sprore Strips or Suspensions and "Proof Systems" - Amsco, "Tower Spore" - Baxter and "Spore Strips" from Ravin - Baxter or VWR.

C. CONTAINERS

Materials that are to be decontaminated should be carried to the autoclave in closed, leak proof containers.  Containers used to hold material while being autoclaved are described below.

1. PRIMARY CONTAINERS

Plastic Autoclave Bags:

Polypropylene bags are used to contain materials during decontamination cycles within autoclaves. Also known as "biohazard bags" or "autoclavable bags", autoclave bags come in a wide variety of sizes, shapes and colors. The Lab Safety Supply Catalog has many styles to choose from.

Autoclave bags are usually placed in polypropylene or stainless steel pans during decontamination cycles to catch liquids that may drain out of the bag. Autoclave bags should be left open during decontamination to allow steam to penetrate into the bag. Additional water should be added to the bag's interior to facilitate heat transfer to the items being decontaminated. The amount of extra water added to the bag should be determined experimentally by the autoclave user, based upon the type and size of material being decontaminated. Do not add water if there is a chance that potentially infectious materials may splash out of the bag.

Autoclave bags being filled in a laboratory should be temporarily placed inside a red bag-lined biohazard box. The lab personnel can then take the autoclave bag and biohazard box combination to the autoclave. The autoclave bag should be removed from the box, decontaminated in the autoclave and discarded into the red bag lined biohazard box.

2. SECONDARY CONTAINERS

a. Plastic Containers:

Polypropylene is a durable, inexpensive plastic resin which is commonly used contain material during autoclaving. Polypropylene plastic pans with 6-12 inch sides are favored over polyethylene and polystyrene because polypropylene can withstand autoclaving without melting.

Note: When using polypropylene containers, add extra processing time to the autoclave decontamination cycle because polypropylene does not conduct heat as well as stainless steel.

b. Stainless Steel Containers:

Stainless steel containers are durable and come in a variety of sizes and shapes. Because stainless steel is a good conductor of heat, autoclave decontamination cycle processing time may be reduced. Where waste containment is mandatory, stainless steel containers may be the container of choice because it is durable. Restaurant supply companies are a good source for these pans.

D. AUTOCLAVE OPERATION (NEW SOP)

1. CAUTION: Only personnel with adequate training on autoclave use should be permitted to operate an autoclave. Personnel should wear proper personal protective equipment, ie. heat resistant gloves, eye protection, etc. particularly when unloading the autoclave.

2. Regularly inspect your autoclave components for proper operation. Autoclave door clamps and seals should be inspected for wear and damage. Also remove debris from the autoclave chamber floor drain. If a problem is found, promptly notify your area supervisor who will call facilities or maintenance. DO NOT OPERATE AN AUTOCLAVE UNTIL IT HAS BEEN PROPERLY REPAIRED.

3. At the end of a decontamination cycle make sure that the pressure in the autoclave chamber is near zero before opening the door. Slowly crack open the autoclave door and allow the steam to gradually escape from within the autoclave.

CAUTION: Opening the autoclave door too quickly may result in glassware breakage and/or steam burns on your skin.

Allow materials inside the autoclave to cool for 10 minutes before removing them from the autoclave.

Avoid dead air pockets where steam cannot penetrate (ie., closed screw cap tubes) because temperature within the air pocket may be lower than the saturated steam.

Avoid dry packages, add some water to the load. To avoid creation of infectious aerosols while adding water, trickle water down the sides of the container instead of pouring water directly onto the material in the container or bag.

E. PROCESSING TIMES

1. After loading and starting the autoclave, processing time starts after the autoclave reaches normal operating conditions: Temperature = 121oC (250oF); Pressure = 15 psi.

2. Decontamination conditions vary with type of load, load volume (loose packed or tightly packed), container type (polypropylene, glass, stainless steel), and type of material to be decontaminated. Therefore, processing times will vary according to the conditions of each decontamination cycle. In general, the larger the load, the longer the decontamination time.

* 3. Thirty minutes are needed to decontaminate lab and medical waste, unless a shorter interval has proven effective by testing with biological indicators. Add additional time if polypropylene containers are used instead of stainless steel containers.

4. More time is recommended for decontamination of waste in low sided polypropylene containers with bags half filled and loosely gathered. If bags are tightly closed, add more processing time.

5. If your autoclave is equipped to operate at 132oC (270oF), you may be able to reduce processing time if biological indicator spores placed in the center of the material to be decontaminated are killed during shorter autoclaving times.

6. The US Environmental Protection Agency (EPA) has reported that....."Infectious wastes from departments of health care facilities may be rendered noninfectious by subjecting the waste to autoclave temperatures of 121oC (250oF) and 15 minutes of prevacuum of 15 psi for the following dwell times when proper containers are used:"

EPA Recommended Decontamination Processing (Dwell) Times

TRASH 60 Minutes

GLASSWARE 60 Minutes

LIQUIDS 60 Minutes / Gallon

ANIMAL CARCASSES 8 Hours

ANIMAL BEDDING 8 Hours

F. ODOR CONTROL

Some waste material has an extremely noxious odor, i.e. anaerobic bacteria, feces or decaying organic materials. When decontaminating these materials it may become necessary to add an odor control additive to the load. A few examples of odor control additives are: any absorbent kitty litter, "Odo-Clave", "Decon Decap" - Thomas Scientific.

G. TRAINING

The Lead Scientist or Chemical Hygiene Officer must train and qualify staff for operation of steam autoclaves for decontamination of materials. Qualified autoclave users should understand the time, temperature, pressure relationships required for proper materials decontamination.

Additional training on handling materials to be decontaminated should also be provided. Maintain a permanent record of training provided.

Additional training support is available by contacting the Biosafety Officer.

H. RECORD KEEPING - AUTOCLAVE LOG BOOK

1. A durable notebook should be used as a permanent record of autoclave use. The autoclave log book should be located in an easily accessed location near the autoclave. The autoclave log book should have at least the following information entered;

Autoclave Manufacturer,

Autoclave Serial Number,

Autoclave Room Location,

Date Log Book Started,

Maintenance Work Done, and

Materials Processed.

2. The main section of the autoclave record should include:

Autoclave User,

Date Used,

Materials Decontaminated,

Process Type,

Run Duration (Cycle Time),

Chemical/Biological Indicator Used,

Chemical/Biological Indicator Results, and

Envelope for "Wheel Graphs" or "Data Strips".

I. AUTOCLAVE LOG BOOK SAMPLES

Samples of autoclave process information sheets for your logbook are below. Make extra copies as needed.

TEMPERATURE CHART

(this can be replaced by saving the paper tape from the autoclave)

ROOM #

AUTOCLAVE #

DATE

RUN #

MEDIA

STERILE

ITEMS

TIME

TEMP

NOTES

INDICATOR STRIP

 

STERILIZATION

Dry heat sterilization is less efficient than steam sterilization and requires more time and/or higher temperatures. The specific times and temperatures must be determined for each type of material being sterilized.  Generous safety factors are usually added to allow for the variables that can influence the efficiency of this method of sterilization. The moisture of the sterilization environment as well as the moisture history of organisms prior to heat exposure appear to affect the efficiency of dry heat sterilization.

Sterilization of clean materials by dry heat can usually be accomplished at 160-170oC (320-338oC) for periods of 2-4 hours.

Higher temperatures and shorter times may be used for heat-resistant materials. The heat transfer properties and the arrangement of articles in the load are critical to insuring effective sterilization.

ULTRAVIOLET LIGHT DECONTAMINATION

Under certain conditions of radiation intensity, exposure time, humidity, and temperature, ultraviolet radiation at approximately 254 nanometers will cause eventual death of microorganisms. The radiation at this wavelength causes formation of thymine-thymine dimers and other effects on DNA and RNA. Nucleic acid containing thymine dimers does not replicate properly and lethal mutations are often produced. Ultraviolet light’s greatest effectiveness is against actively growing bacteria. Low pressure mercury vapor lamps usually supplied with biological safety cabinets emit germicidal radiation at a wavelength of 254 nanometers for about nine months. After this time, the lamp may not produce enough germicidal radiation to effectively kill bacteria, even though it appears to be functioning properly.

In general, ultraviolet radiation is used to reduce exogenous contaminants and/or pathogenic microorganisms on exposed surfaces and in the air.

A. UV LAMP OPERATION

1. All UV installations used for disinfection should be checked semi-annually. Periodic examination is necessary because UV bulbs may continue to burn without emitting effective radiation. UV lamps should be replaced when they emit 70 percent or less of their rated initial output.

2. UV lamps installed in biological safety cabinets must be replaced when the 254 nm UV irradiation intensity on the work tray surface of the cabinet is less than 40 microwatts per square centimeter.

3. UV lamps should be cleaned often if located in an unusually dusty area. Lamps should be turned off and wiped with a soft pad moistened with alcohol. Cleaning is the responsibility of the personnel in charge of the laboratory.

4. All exposed UV installations in lighting fixtures and safety cabinets shall be turned on only when no personnel are in the area. Louvered, wall mounted UV equipment may be left on continuously.

5. Each UV installation should be equipped with an outside switch and an appropriate safety sign. Interlocks should be installed where appropriate to turn off UV lamps when room lights are turned on.

6. Biological safety cabinets listed by the National Sanitation Foundation (NSF) after 1992 may not have UV lamps installed because there is no longer a NSF secondary test standard for UV lamps.

B. TRAINING

All personnel should be instructed in the proper use of each UV installation. Such instruction should include emphasis on the following:

1. Do not look directly at UV lamps;

2. Do not loiter in UV airlocks and door barriers;

3. Turn off lamps before cleaning;

4. Wear eye and skin protection if anticipated exposure to UV will be for longer than a few seconds;

5. Protective goggles should transmit less than 4% of 400 nm wavelength light;

6. Particular care needs to be exercised around UV gel transilluminators, they produce considerable radiation.

CHEMICAL DISINFECTANTS

A. PHENOLIC COMPOUNDS

1. Recommend for the killing of vegetative bacteria, including Mycobacterium tuberculosis, fungi and lipid-containing viruses using a concentration of 0.5-2.0%. They are less effective against spores and non-lipid containing viruses.

2. Low solubility in water unless combined with detergent.

3. Stable in storage.

4. Germicidal against Gram-negative and Gram-positive organisms and tubercle bacilli.

5. Less adversely affected by organic matter than other common germicides.

6. Effective over relatively large pH range.

7. Limited sporicidal activity.

8. Prolonged contact deteriorates rubber.

9. Can cause skin and eye irritation.

10. Not for use on food contact surfaces.

B. QUATERNARY AMMONIUM COMPOUNDS

1. Acceptable as general use disinfectants to control vegetative bacteria and non-lipid containing viruses. However, they are not active against bacterial spores or Mycobacteria at the usual use concentrations (1:750).

2. Stable in storage.

3. No odor but act as deodorizers.

4. Use dilution usually non-irritating to skin but prolonged skin or eye contact should be avoided.

5. Effective at temperatures up to 212oF.

6. Effective against Gram-positive organisms, bacteriostatic in high dilutions.

7. Generally ineffective against tubercle bacilli, spores, and viruses.

8. More effective in alkaline than acid solutions.

9. Neutralized by soap and anionic detergents.

10. Effectiveness reduced by organic material.

11. Has built-in detergent properties.

12. Some are active against lipophilic viruses.

C. IODOPHORS

1. Although these show poor activity against bacterial spores, they are recommended for general use in concentrations of 70 to 150 ppm. They are effective against vegetative bacteria and viruses.

2. Combine iodine with non-ionic detergent.

3. Rapid biocidal action.

4. Effective against Gram-negative and Gram-positive organisms, some viruses, and tubercle bacilli.

5. Most effective in acid solutions.

6. Vaporize at 120oF to 125oF -- should not be used in hot water.

7. Effectiveness reduced by organic matter (but not as greatly as hypochlorites.)

8. Stable in storage if kept cool and tightly covered.

9. Iodophors are relatively harmless to man.

10. Iodophors have a built-in indicator. If the solution is brown or yellow, it is still active.

11. Iodophors can be readily inactivated and iodophor stains can be readily removed with solutions of sodium thiosulfate.

12. May tarnish silver, silver plate, and copper.

D. ALCOHOLS

1. In concentrations of 70%, alcoholic solutions are good general-use disinfectants, but they exhibit no activity against some bacterial and fungal spores or tubercle bacilli.

2. Germicidal against a broad spectrum of bacterial species and many viruses.

3. Fast acting.

4. Leave no residue.

5. Compatibly combined with other disinfectants (quaternaries, phenolics, and iodine) to form tinctures, extends alcohol's cidal action.

6. Flammable, not to be used near a flame. Therefore, use of alcohol inside a biological safety cabinet may be a fire safety hazard.

E. ALDEHYDES

1. Effective against wide spectrum of bacteria and viruses.  Sporicidal when used properly (10 hour contact period).

2. Formaldehyde Solutions - At concentrations of 8%, formalin exhibits good activity against vegetative bacteria, spores, and viruses. Its use must be limited and controlled because of its toxic properties.

3. Formaldehyde-Alcohol - Solutions of 8% formalin in 70% alcohol are considered very good for disinfection purposes because of their effectiveness against vegetative bacteria, spores and viruses. For some applications, this may be the disinfectant of choice.

F. CHLORINE COMPOUNDS

1. Recommended for certain disinfecting procedures such as cleanup of blood or body fluid spills when household liquid chlorine bleach is diluted 1:10 with tap water.

2. A 1:10 dilution (5,000 ppm) of bleach has a biocidal effect on M. tuberculosis, S. aureus, other vegetative bacteria, and HIV after 10-20 minutes.

3. For bacterial spores and mycobacteria, higher concentrations of at least 2,500 ppm (1:5 dilution) are needed.

4. Their decay rates and lack of residuals is such that they must be made up fresh. A 1:50 dilution of chlorine bleach stored at room temperature in a closed plastic container will deteriorate to the equivalent of a 1:100 dilution (500 ppm) after one month, if the container is kept closed (Rutola,W.A., Am. J. Infect Control. 1989;17:1).

5. Neutralized rapidly in the presence of organic matter.

6. A 0.5 percent sodium hypochlorite solution (1 to 10 dilution of liquid laundry bleach) is recommended for decontamination of HBV, HIV, and cleanup of biohazardous spills.

7. A fresh solution of 0.05 percent hypochlorite solution (1 to 100 dilution of 5% chlorine liquid laundry bleach) is recommended for general disinfection of surfaces and liquid waste.

CLEAN-UP OF BIOHAZARDOUS SPILLS

A. BIOHAZARDOUS SPILL INSIDE A BIOLOGICAL SAFETY CABINET

Chemical decontamination procedures should be initiated at once while the cabinet continues to operate to prevent escape of contaminants from the cabinet. Be careful with paper towels which can be sucked into the blower fan or HEPA filters.

1. Spray or wipe walls, work surfaces, and equipment with an appropriate disinfectant detergent, (e.g., 1:10 dilution of household bleach and 0.7% soap). A disinfectant detergent has the advantage of detergent activity, which is important because extraneous organic substances frequently interfere with the reaction between the microorganism and the active agent of the disinfectant.

The operator should wear gloves during this procedure.

2. Flood the top work surface tray, and, if a Class II cabinet, the drain pan below the work surface, with a disinfectant and allow to stand 20 minutes.

3. Remove excess disinfectant from the tray by wiping with a sponge or cloth soaked in a disinfectant. For Class II cabinets, drain the tray into the cabinet drain pan, lift out tray and removable exhaust grillework, and wipe off top and bottom (underside) surfaces with a sponge or cloth soaked in a disinfectant. Then replace the grillwork and drain disinfectant from the drain pan into an appropriate container and autoclave according to standard procedures. Gloves, cloth or sponge should be autoclaved and discarded into the biohazard box.

B. BIOHAZARDOUS SPILL OUTSIDE A BIOLOGICAL SAFETY CABINET

1. LARGE SPILL (Onto floor, over 1 liter in size)

a. Do not breathe, leave the room immediately, and close the door.

b. Notify the supervisor, and warn others not to enter the contaminated area. Post the door temporary warning sign.

c. Remove and put your contaminated labcoat and garments into a container for autoclaving and thoroughly wash your hands and face.

d. Wait 60 minutes to allow dissipation of spill-created aerosols by the room ventilation air changes.

e. Put on a long-sleeve labcoat and tighten the cuffs. Place on a mask, and rubber gloves before reentering the room.

f. Pour an appropriate disinfectant solution (1:10 dilution of household bleach) around the spill and allow it to flow into the spill. Paper towels soaked with the disinfectant may be used to cover the area. To minimize aerosolization, avoid pouring the disinfectant solution directly onto the spill.

g. Let stand 20 minutes to allow an adequate contact time.

h. Using an autoclavable dust pan and squeegee, and forceps for sharp materials, transfer all contaminated materials (paper towels, glass, liquid, gloves, etc.) into a autoclave bag lined deep autoclave pan. Cover the pan with a suitable cover and autoclave according to standard directions.

i. The dust pan, squeegee, and forceps should be placed in an autoclave bag and autoclaved according to standard directions. Contact of reusable items with non-autoclavable plastic bags should be avoided -- separation of the plastic after autoclaving can be very difficult.

* Non autoclaveable supplies can be used, but will be discarded after incident.

j. Wash and mop adjacent area and spill area with appropriate disinfectant - detergent solution.

k. Remove and discard protective clothing. Shower with a germicidal soap.

2. SMALL SPILL (Table top quantity of 1 liter or less)

a. Cover spill with paper towels.

b. Flood spill with appropriate disinfectant using care not to cause spatter. Add disinfectant slowly to outer margin of spill and allow it to flow in.

c. Allow disinfectant to act for 20 to 30 minutes before cleaning up with more paper towels.

d. Discard materials (paper towels, gloves, and other wastes from clean-up into an autoclave bag and autoclave.

SHIPMENT OF BIOLOGICAL MATERIALS

INTRODUCTION

Because of potential hazards associated with the shipment of biohazardous materials, state and federal regulations control shipping practices. The regulations for shipment of etiologic agents and recombinant DNA materials state that:

"No person may knowingly transport or cause to be transported in interstate traffic, directly or indirectly, any material, including but not limited to, diagnostic specimens and biological products, which such person reasonably believes may contain an etiologic agent, unless such material is packaged to withstand leakage of contents, shocks, pressure changes, and other conditions incident to ordinary handling in transportation."

A. Shipment of biohazardous materials, within the U.S.A. and through importation and exportation, are subject to a variety of regulations involving five different regulatory agencies:

U.S. Postal Service [39 CFR, part III]

U.S. Public Health Service, [42 CFR, part 72]

U.S. Department of Transportation, [49 CFR, parts 171-179]

U.S. Department of Agriculture, [9 CFR, subchapters D & E]

B. Recombinant DNA molecules contained in an organism or in a viral genome shall be shipped under the applicable regulations of the governmental agencies listed above.

C. For purposes of the NIH Recombinant DNA Guidelines (Federal Register, Vol. 55, No. 41, Thursday, March 1, 1990, pages 7447-7448):

1. Host organisms or viruses will be shipped as etiologic agents regardless of whether or not they contain recombinant DNA if they are regulated as human pathogens by the U.S. Public Health Service [42 CFR, part 72] or as animal pathogens or plant pests under the Animal and Plant Health Inspection Service (APHIS), U.S. Department of Agriculture [Titles 9 and 7 CFR, respectively].

2. Additionally, host organisms and viruses will be shipped as etiologic agents if they contain recombinant DNA.

D. DEFINITIONS (42 CFR Part 72 - Interstate Shipment of Etiologic Agents, 1980)

1. BIOMEDICAL MATERIALS

Materials that are known to contain, or could contain, etiologic agents.

2. DIAGNOSTIC SPECIMENS

Any human or animal material including, but not limited to excreta, secreta, blood and its components, tissue, and tissue fluids "which (the shipper) reasonably believes may contain an etiologic agent" [and may pose a danger to others, if they were exposed to it because of a transportation accident or mishap in handling], even if it is being shipped for purposes of diagnosis, must be packaged according to 42 CFR Part 72.3. Patient specimens that would be expected to contain an etiologic agent should be shipped according to 42 CFR Part 72.3 (Etiologic Agent).

Materials which the shipper reasonably believes does not contain an etiologic agent should ship according to 42 CFR Part 72.2.

3. BIOLOGICAL PRODUCT

A product prepared and manufactured in accordance with provisions of 9CFR Parts 102-104, 21CFR Parts 312 & 600-680, and 42 CFR 72.2.

4. ETIOLOGIC AGENTS [ INFECTIOUS SUBSTANCES ]

A viable microorganism or its toxin that causes or may cause, human or animal disease. A culture or  suspension including purified or partially purified spores or toxins that are themselves etiologic agents. Packaged according to 42 CFR Part 72.3.

E. PACKAGING OF DIAGNOSTIC SPECIMENS AND BIOLOGICAL PRODUCTS

(42 CFR Part 72.2). Must withstand leakage of contents, shocks, pressure changes, etc.

F. PACKAGING OF ETIOLOGIC AGENTS [INFECTIOUS SUBSTANCES] (42 CFR Part 72.3) -DANGEROUS GOODS

1. Primary container is securely closed and watertight tube, vial, ampule, etc.

2. Primary container placed in a durable, watertight secondary container.

3. Several primary containers in one secondary container if total contents of primary containers doesn't exceed 50 ml.

4. Absorbent material around and between primary and secondary containers. Should be  nonparticulate and absorb entire contents of primary container(s).

5. Outer shipping container for secondary containers is constructed of fiberboard, of cardboard, wood, etc., not bags, envelopes, etc.

6. Single primary containers shall not contain more than 1,000 ml of material.

7. Two or more primary containers can be put into one secondary container, if total volume doesn't exceed 1,000 ml.

8. Maximum amount of etiologic agent in one outer shipping container may not exceed 4,000 ml.

9. Dry ice must be placed between secondary container(s) and the outer shipping container.

10. Etiologic agent "Infectious Materials" label must be placed on outer shipping container.

G. SPECIAL HANDLING

1. Some highly infectious materials must be shipped by registered mail with notification of receipt to the sender immediately upon delivery.

2. When the notice of receipt is not received within 5 days following anticipated delivery the sender must notify CDC.

H. FOREIGN QUARANTINE (42 CFR PART 71.54 and 72.3)

1. The CDC regulates importation of all etiologic agents and hosts and vectors of human disease. Failure to have an appropriate import permit may result in confiscation of a shipment at a port of entry.

2. Call CDC at (404) 639-3883 to get a permit application (Form 0.753).

I. ORGANISMS & VECTORS - USDA IMPORTS (9 CFR PART 122.2) (Reserved)

J. For further information on shipping etiologic agents, please contact:

1. Centers for for Disease Control, ATTN: Biosafety Office, 1600 Clifton Road, Atlanta GA 30333, (404) 639-3883;

2. Department of Transportation, ATTN: Office of Hazardous Materials Transportation, 400 7th Street, S.W., Washington DC 20590, (202) 366-4545; or

3. Department of Agriculture, ATTN: Animal & Plant Health Inspection Service, 6505 Belcrest Road, Hyattsville MD 20782, (301) 436-7885 for Animal Pathogens, (301) 436-7612 for Plant Pests.

For additional details on biosafety levels and classification of etiologic agents, refer to the CDC NIH Biosafety in Microbiological and Biomedical Laboratories, 3rd Edition.

DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) June 1994. These NIH Guidelines supersede all earlier versions and shall be in effect until further notice.

 

BIOHAZARD CONTAINMENT EQUIPMENT

A. TYPES OF CONTAINMENT EQUIPMENT

Control of microbiological containment in the laboratory can be achieved by proper use of containment equipment. It is generally accepted that such equipment is most effective if the potential hazard is enclosed at the source i.e., at the work site.

Laboratory containment equipment can be described as either "partial barrier" or "absolute barrier". Partial barrier equipment depends upon negative air pressure and airflow directions to achieve containment. Absolute barrier equipment provides a separated negative pressure working environment when manipulations are done through arm-length rubber gloves.

Examples of partial barriers are Class I Open Face Safety Cabinet and Class II Biological Safety Cabinet. Absolute barriers are Gas-Tight Centrifuge Chamber, Gas-Tight Blender Units, and Class III Gas-Tight Cabinet.

B. USE OF CONTAINMENT EQUIPMENT

1. A suitably ventilated biological safety cabinet is recommended for all procedures with biohazardous materials such as:

opening test tubes, flasks and bottles, doing dissections, using pipettes, making dilutions, inoculating or autopsying animals, grinding tissue, blending cultures, opening lyophile tubes, and operating ultrasonic disintegrators.

2. Prior to use, all biological safety cabinets, laminar flow devices or any other ventilated containment devices shall be tested by qualified personnel to determine the adequacy of the equipment.

3. Frequent washdowns of biological safety cabinets are recommended to prevent buildup of infectious agents. In no instance should such a use of disinfectant solution be considered a sterilization procedure.

 

C. BIOHAZARD CABINET CONTAINMENT EQUIPMENT

Control of microbiological containment in the laboratory can be achieved by proper selection and use of ventilated cabinet containment equipment. It is generally accepted that potentially hazardous materials should be controlled at the source to protect personnel, product, and the environment.

The heart of a ventilated cabinet is the high efficiency particulate air (HEPA) filter. The HEPA filter is positioned within the cabinets' ducting. The HEPA filter removes particulate material, such as dust, fungal spores, bacteria, and viruses from the internal cabinet air. This HEPA-filtered air is then directed to either the laboratory (worker protection) or to the work surface (product protection).

A common error people make with ventilated cabinets is mistaking a chemical fume hood (hood) or clean air bench (CAB) for a specialized cabinet called a biological safety cabinet (BSC).

Only annually certified BSC's should be used with biohazardous materials. The chemical fume hood, or "hood", is specifically designed for use with potentially hazardous chemicals and is not generally used with potentially biohazardous materials. The CAB is designed to provide a clean work area for assembling electronic components.

 

BIOLOGICAL SAFETY CABINET CATEGORIES

A. Class I - Open Face Biological Safety Cabinets:

DESCRIPTION: A partial barrier biological safety (BSC) with unfiltered, non-recirculated inward airflow away from the operator and over the work surface.

USE: Class I BSC's provide worker and environmental protection only. Class I BSC's do not provide product protection. Class I's may be used for manipulations at Biosafety Level 1, 2 and 3 when no experiment (product) protection is required.

Note: To provide environmental protection, Class I BSC exhaust air must pass through a high efficiency particulate air (HEPA) filter before being discharged to the outside atmosphere.

B. Class II Biological Safety Cabinets:

Class II biological safety cabinets (BSC's) are broken down into four "Types" with specific performance characteristics. These Types are: Class II, Type A; Class II, Type B1; Class II, Type B2; and Class II, Type B3.

DESCRIPTION: The Class II BSC's are partial barrier ventilated cabinets. Incorrect technical slang words used for these BSC's are "hood" or "tissue culture hood". The correct technical name is "biological safety cabinet" or the acronym "BSC". The "Type" of BSC dictates the configuration of the cabinet. The BSC configuration determines what percentage of internal BSC air is recirculated within the BSC and what percentage of air is exhausted from the BSC.

Air flows into Class II BSC's through the front work access opening and is drawn away from the operator. This provides personnel protection. Class II BSC's have either one or two HEPA filters within the cabinet, depending on the cabinet Type. Class II, Type A, B1, and B3 BSC's have one supply air HEPA filter and one exhaust air HEPA filter. Class II, Type B2 BSC's typically have one internal supply air HEPA filter. The Class II, Type B2 exhaust air is typically HEPA filtered through an ancillary HEPA filter installed upstream in a dedicated building exhaust system.

The supply HEPA filter provides particle-free vertical laminar airflow over the work surface, which protects the product from contamination.  The exhaust HEPA filter provides particle-free BSC exhaust air.  This prevents potentially biohazardous material used in the BSC from escaping into the environment (laboratory).

USE: Class II Biological Safety Cabinets (BSC's) provide personnel, product, and environmental protection. Class II BSC's must be used for all activities involving low or moderate risk biohazardous agents, etiologic agents, oncogenic viruses, and recombinant DNA experiments requiring Biosafety Level 1, 2, and 3 containment.

USE OF BIOLOGICAL SAFETY CABINETS

A. Know the potential biohazard of the etiologic agent or material to be used in the BSC.

B. Know the Class and Type of biological safety cabinet (BSC) you are about to use prior to using it. Be familiar with the weaknesses and strengths of the Class and Type of BSC selected.

UMES, Microbial Food Safety Lab only uses Class II, Type II A.

C. Procedure to be carried out in BSC's include:

1. Opening test tubes, flasks, and bottles,

2. Dissections or necropsy,

3. Pipetting,

4. Making dilutions,

5. Injecting animals,

6. Grinding tissue,

7. Blending cultures,

8. Opening lyophile tubes, and

9. Operating ultrasonic disintegrators, vortex mixers or other high energy equipment.

D. The Lead Scientist, on advice of the Biosafety Officer, is responsible for ensuring proper personnel safety and use of ventilated cabinets in the lab.

E. At times there may be requirements for special cabinets to protect workers from airborne transmission of biohazardous agents or materials. Cabinets may be required for shaking machines, animal waste containers, and centrifuges.

F. Prior to use, check the status of your BSC. All laminar flow BSC’s must be performance tested ("certified") annually by qualified personnel.

G. Employ special safety work practices designed for the work you are doing. If you are not sure what practices you need to follow, talk to your CHO, Lead Scientist, or call the Biosafety Officer.

H. Disinfect all work surfaces before, during and after BSC use.

 

OPERATION OF BIOLOGICAL SAFETY CABINETS

A. GENERAL SUGGESTIONS

1. Keep your laboratory meticulously clean. Minimize storage of boxes and supplies particularly near a BSC. Never place boxes on top of a BSC.

2. Wash your hands thoroughly before and after working in your BSC. Wearing a clean lab coat and gloves while working in a BSC increases your safety and helps reduce contamination of research materials.

3. The effectiveness of a BSC is a function of directional airflow, inward and downward, through a high efficiency particulate air filter (HEPA). Anything that disrupts the airflow pattern reduces cabinet effectiveness, such as; rapidly moving your arms in and out of the BSC, people walking behind you, down-drafts from ventilation systems and drafts from open laboratory doors.

4. Understand how your BSC works. Plan your work ahead. Protect yourself, your research and your co-workers.

B. STARTING UP YOUR BSC

1. Turn off the ultraviolet (UV) lamp, turn on the fluorescent light, and inspect the air intake grilles for obstructions and foreign materials. Remove any obstructions. Make sure the view screen window is adjusted (if adjustable) to the proper height (usually 10 inches). The window is safety alarmed.

2. Turn on the blower motor and allow it to operate for at least five minutes. This purges the air from the internal BSC cabinetry.

3. Wash your hands and arms with mild hand soap. Next, put on laboratory attire. Put on a pair (or two pairs, depending on procedure and agent being used) of high quality latex gloves. Additional protection from contamination may be provided by wearing disposable sleeve protectors and a second or third pair of latex gloves.

4. Next, disinfect the interior surfaces of the BSC by wiping down with appropriate disinfectant such as 1:10 Wescodyne, 1:10 bleach, or 70% alcohol. (Caution -- 70% Ethanol is highly flammable -- DO NOT USE in the presence of flame or spark.) Commercial products such as Envirocide can be used also. Refer to Lab Safety Catalog, page 453-454.

5. Place a plastic-backed "chux-pad" or bench liner on the dry, disinfected work surface of the BSC. Avoid covering the air intake/exhaust grilles.

6. Install all necessary items for your experiment in the BSC at this time. Keep clean items segregated from dirty items.

a. Minimize the amount of equipment and supplies; overloading the working zone with equipment and supplies may compromise the effectiveness of your BSC.

b. Organize your material so that dirty "contaminated" items are not passed over (cross contaminate) clean items. Work from "clean" to "dirty" areas. A good work layout of materials would position clean items, i.e., pipettes, cultures, flasks, etc. toward the front or either side of the work surface.

Place your waste container and contaminated pipette trays to the rear. You should work at least six inches back from the front of the air intake grille.

7. If you are using a vacuum flask system in your BSC, put a vacuum protection device, a filter and flask, in line to protect the vacuum system.

8. After all equipment and supplies are added to your BSC, allow it to operate for an additional three to four minutes. This will allow the BSC to purge itself of airborne contaminants.

9. Assume a comfortable seating position in front of the BSC. Your chair should be adjusted to a comfortable height that promotes proper posture. When inserting your arms into the BSC remember that they are penetrating a delicate "curtain" of air.

Allow the air curtain to stabilize around your arms before starting work. Avoid making rapid, jerking arm motions. Use smooth motions that avoid disrupting the air curtain.

10. Remember that the BSC air curtain is delicate and can only provide protection from contamination as long as it is not disrupted. The BSC is not a substitute for good microbiological practices and does not automatically provide you with protection from potentially hazardous materials or automatically prevent contamination of the experiment and materials.

11. Follow good microbiological techniques, i.e., holding open tubes and bottles as horizontal as possible.

a. Never mouth pipette. Use mechanical pipetting aids.

b. Do not use vertical pipette discard canisters on the floor outside the BSC. Use horizontal pipette discard pans inside the BSC.

c. It is not necessary to flame items. The flame creates turbulence in the airflow and will compromise sterility. If the lip of a tube or flask is wet, an aerosol may be created when the lip is flamed. Additionally, heat buildup can also damage expensive HEPA filters. Unattended burner pilot lights have created extensive fire damage to BSC's and sometimes entire laboratories.

12. If you need to introduce new items or remove items from the BSC, move your arms in and out slowly to minimize airflow disruption.

13. If you use equipment that creates air turbulence, such as a centrifuge, blender, or sonicator, place the equipment as far back in the BSC as possible (usually 1/3 of the way back from the front intake grill is acceptable). Stop other work while the equipment is operating.

14. Clean up all spills inside the BSC immediately and then wait 3-5 minutes before resuming work, if your laboratory operating procedures allow.

An externally mounted drain valve is located under the drain pan of most BSC's. In the event of an accidental spill, you can remove large volumes of disinfected, spilled materials from under the work surface. The drain valve is not to be connected to a sink or similar drain. A bucket can be used to collect any spilled liquid for decontamination and disposal.

C. CLEANING YOUR BSC

1. When work has been completed, disinfect the exterior surfaces of potentially contaminated materials and supplies with appropriate disinfectant before removing them from the BSC. Remove all materials from the interior of the BSC.

2. Disinfect the interior BSC surfaces, including the inside of the view screen, with an appropriate disinfectant solution.

3. Allow the BSC to operate an additional 10 minutes, then turn the blower motor off. Caution; do not close the view screen when the blower is on, the view screen may crack or shatter.

4. Examine the spill pan beneath the work surface. Clean and disinfect the spill pan at least four times per year or as necessary.

Be careful when removing interior work surfaces. They are heavy and may have sharp edges and corners. Consult your service manual for proper removal/maintenance procedures.

Do not clean the spill pan when a BSC blower is operating.

Paper towels may be accidently sucked into the airstream and can lodge in the blower motor and HEPA filter. Recovering paper towels can only be accomplished by decontamination and disassembly of the cabinet by an authorized service technician.

Do not attempt retrieval yourself.

5. Turn off the fluorescent lamp and turn on, if you wish, the ultraviolet lamp, if your BSC is so equipped.

6. Discard waste materials appropriately.

7. Remove your lab coat and gloves and then wash your hands thoroughly with a mild hand soap.

D. FLAMMABLE OR EXPLOSIVE MATERIALS WITHIN BSC'S

Standard electrical systems of Class II BSC's are not explosion proof. No flammable or explosive materials (chemicals, cleaners, or solvents) should be used in a BSC. Most BSC's are labeled with a warning sign on the front face of the cabinet warning against the use of flammable or explosive materials within it. Refer to your owners manual for proper guidance. Under no circumstances should a chemical's vapor concentration be allowed to approach its level of flammability.

Each BSC type has its own ventilation characteristics. Remember, in the absence of a thimble exhaust duct connection, a BSC's air is recirculated within the laboratory. This can return a vapor or gas contaminant back to the "clean" work space and back into the laboratory until equilibrium is reached. Standard BSC HEPA filters are made of paper and do not remove chemical contaminants from the air. A chemical's equilibrium concentration level depends on the vapor or gas generation rate at the work space and the air exchange characteristics of the BSC and the laboratory facility.

HEPA filters remove only particulates. Gases and vapors will readily pass through HEPA filters and must be controlled by other methods. When planned work involves chemical carcinogens or solvents, it is necessary to evaluate the quantities to be used to determine the amount that might be entrained in the BSC air stream during an accident. By referring to the Threshold Limit Value Lists for chemical substances, from the American Conference of Industrial Hygienists and the Occupational Safety and Health Administration (OSHA), the proper method for dealing with the gases or vapors from chemical substances can be determined.

BIOSAFETY CABINET TRAINING MATERIALS

The Area Safety Office has several audiovisual programs for instruction. Some of these audiovisuals are listed below:

A. Safe use of Biological Safety Cabinets (22 minutes).

 

CLASS II, TYPE B2 BIOLOGICAL SAFETY CABINETS

1. A partial containment biological safety cabinet (BSC).

2. These BSC's provide personnel, product and environmental protection (Figure 2(c).( Next page unmarked)

3. They are referred to as a "Total Exhaust" or "100% Exhaust" BSC's.

4. Type B2 BSC's must be "hard connected" to the building exhaust system. These BSC's rely on the building exhaust system for proper operation. The building exhaust system must be equipped with a bag-in/bag-out HEPA filter caisson to provide environmental protection.

5. These BSC's maintain a minimum average inflow velocity of 100 fpm through the work area access opening.

6. They have HEPA filtered downflow supply air. A blower motor takes in laboratory air and pushes it (positive pressure) through a supply HEPA filter. There is no recirculated air in this BSC;

7. All inflow and work area downflow air is exhausted, with no recirculation within the work area, to the outside through a hard connected building exhaust duct system after passing through a HEPA filter.

8. All internal contaminated ducts and plenums are under negative pressure, or surrounded by directly exhausted (non-recirculated) negative air pressure ducts and plenums.

9. Class II, Type B2 BSC's are suitable for work with low to moderate risk biological agents. They are used widely in toxicology laboratories and similar applications where chemical vapors are present. They must be used for biological agents ed with toxic chemicals and radionuclides.

 

CLASS II, TYPE B3 BIOLOGICAL SAFETY CABINETS

1. These are classified as partial containment biological safety cabinets (BSC's, see figure 2(d).

2. These BSC's provide personnel, product, and environmental protection.

3. Type B3 BSC's can be "thimple conneted" or "hard connected" to the building exhaust system.

4. A minimum average inflow velocity of 100 fpm is maintained through the work access opening.

5. HEPA filtered downflow air comes from a common exhaust plenum that is a mixture of downflow supply air and laboratory supply air.

6. All exhaust air is discharged to the outside after HEPA filtration.

7. All internal biologically contaminated ducts and plenums are under negative pressure, or surrounded by negative pressure ducts and plenums.

8. Type B3 BSC's are suitable for work with low to moderate risk biological agents treated with minute quantities of toxic chemicals and trace quantities of radionuclides that will not interfere with the work if recirculated in the downflow air.

9. OSEH recommends a "thimble" exhaust connection instead of a "hard" exhaust connection for most biomedical research applications.

 

VENDORS OF BIOLOGICAL SAFETY CABINETS

Manufacturer Address / Phone Number

Baker Baker Company, Inc.
Sanford Airport
Sanford ME 04073
(207) 324-8773

LOCAL BAKER DISTRIBUTER:

GTS Scientific, Inc.
P.O. Box 7555
Gaithersburg, MD 20898
(301) 929-1444

Bellco Bellco Glass, Inc.
340 Edrudo Road
Vineland, NJ 08360
(800) 257-7043

Envirco Envirico, Inc.
6701 Jefferson NE
Albuquerque, NM 87109
(505) 345-3561

Forma Scientific Forma Scientific, Inc.
P.O. Box 649
Mill Creek Road
Marietta, OH 45750
(800) 848-3080

LOCAL FORMA DISTRIBUTER

(800) 872-7133 ext. 4648

 

Germfree Germfree Laboratories, Inc.
7435 N.W. 41st Street
Miami, FL 33166
(305) 592-1780

Labconco Labconco Corporation
8811 Prospect
Kansas City, MO 64132
(816) 333-8811

LOCAL LABCONCO DISTRIBUTER:

504 Sunfield Way
Frederick, MD 21702
(800) 821-5525

Microzone (Formerly Canadian Cabinets Company, Ltd.)
25F Northside Road
P.O.Box 11336, Station H
Nepean, K2H7V1 Canada

Nuaire Nuaire, Inc.
2100 Fernbrook Lane
Plymouth MN 55447
(612) 553-1270

LOCAL NUAIRE DISTRIBUTER:

LabRepco.
626 Street Road.
Southampton, PA, 18966
(800) 521-0754

TESTING/CERTIFYING BSC

A. Frequency of testing is not limited to once a year. Ventilated cabinets may be certified as often as required. For example, when a unit is moved or damaged/repaired.

B. Payment for certification must be made at the time that you schedule your service with the vendor. ( this may vary locally)

C. Performance tests/certifications must be conducted only by a qualified contractor.

D. Ventilated cabinet installations associated with new construction or renovation project specifications should include the statement that after installation, each ventilated cabinet must be performance tested to the appropriate performance standard (NSF or 209-D) and be certified to the manufacturer's design performance specifications.

E. Performance testing / certification must be performed:

1. Before initial use.

2. After moving a BSC from one location to another.

3. After replacement of high efficiency particulate air (HEPA) filter(s). BSC HEPA filter replacement can be expected to occur at intervals of every 3 to 5 years.

4. At least annually.

5. After possible BSC damage.

6. Following a large spill or accident inside the BSC.

7. When requested by the Biosafety Officer.

ANNUAL CABINET LABOR AND SERVICE CONTRACT

The Lab will arrange for all cabinets to be certified at least on an annual basis through one approved contractor (sole provider of services). This should be budgeted annually in the ARMPS.

The certification and service repair costs are charged to the individual CRIS account via:

Purchase Order number payable to the contractor;

other method include service contractor or use of landlord services where available.

 

MOVING YOUR CABINET

It is a common practice to move laboratory ventilated cabinets to other locations within the lab or to other laboratories. However, despite the apparent simplicity of the job, there are certain conditions that must be met prior to moving this equipment. These guidelines were developed to insure the safety of people who work with ventilated cabinets.

A. Ventilated cabinets must not be moved unless the Biosafety Officer has approved the new location and "cleared" the cabinet for moving. ( the next available professional is the Area Safety Officer. )

B. Existing ventilated cabinets and ancillary equipment, such as thimble exhaust ducting, gas, electric and vacuum systems, must be "CLEARED FOR MAINTENANCE" by the Area Engineering & Safety , or local supporting engineering department prior to disassembly.

C. Contact your Facilities Management office for instructions on how your ventilated cabinet will be physically moved.

D. Prior to a move, all biological safety cabinets (BSC's) must be professionally decontaminated with formaldehyde gas. Call your Biosafety Officer or safety officer for guidance.

E. After the ventilated cabinet is moved, it must be recertified according to applicable performance standard.

C. BIOLOGICAL SAFETY CABINET (BSC) PERFORMANCE EVALUATION TESTS

The National Sanitation Foundation (NSF) serves as neutral medium in which business and industry, official regulatory agencies, and the public come together to deal with problems involving products,equipment, procedures, and services related to health and the environment. NSF acts as the national authority to which Class II biological safety cabinets are constructed and tested. The NSF-49 performance tests are listed below for your information:

These steps describe aspects of cabinet certification.

1. AIR VELOCITY TEST

This test is performed to measure the velocity of downward airflow.

2. SUPPLY AIR VOLUME TEST

3. WORK ACCESS OPENING AIRFLOW TEST

This test is performed to determine the face velocity of inflow air (supply air) through the work access opening, and the calculated exhaust flow volume.

4. HEPA FILTER LEAK TESTS

This test is performed to determine the integrity of supply and exhaust HEPA filters, filter housings, and filter mounting frames.

5. AIRFLOW SMOKE PATTERN TEST

This test is performed to determine that the airflow along the entire perimeter of the work access opening is inward, airflow within the work area is downward with no dead spots or refluxing, ambient air does not pass on or over the work surface, and there is no refluxing to the outside at the window wiper gasket and side seals.

6. ELECTRICAL GROUND FAULT TEST

This test is performed to determine the electrical leakage and ground circuit resistance to the BSC ground connection, and determine if a potential shock hazard exists.

7. NOISE LEVEL TEST

This test is performed to provide a uniform method for measuring noise levels produced by the BSC.  The need for such a test is based on belief that workers should not be exposed to unnecessary noise. Secondarily, an increase in noise level is a indication of mechanical deterioration or malfunction.

8. ULTRAVIOLET LIGHT INTENSITY TEST

Ultraviolet (UV) lighting, when requested by the purchaser, shall be installed so that it does not reduce the required performance of the BSC. The UV irradiation must be in accordance with American Conference of Governmental Hygienists (ACGH) standards, and the irradiation must not affect any of the construction materials or containment integrity of the BSC.

a. For decontamination purposes, low pressure mercury vapor lamps which emit about 90% of their radiation at 254 nm, are generally used. Cold cathode, low ozone lamps, are preferred.

b. UV lamp replacement is provided by the contractor free of charge as part of the institution's service contract.

c. UV Lamp Safety Considerations:

Exposure of your eyes and skin to direct or strongly reflected UV radiation should be avoided. The effect of UV radiation depends on irradiance level, wavelength, part of body exposed, and individual sensitivity. Overexposure of the eyes results in a painful inflammation of the conjunctiva, cornea, and iris.  Symptoms develop 3 to 12 hours after exposure and usually disappear in a few days. Overexposure of the skin produces a reddening (i.e., erythema) in 1 to 8 hours after exposure.  UV exposure should be minimized as a matter of good practice; eyes should be protected by UV-opaque glasses with side shields and the skin should be protected by cloth or rubber coverings.

d. This test is performed to determine the intensity of ultraviolet (UV) radiation, in microwatts per cm2, on the work surface in the BSC. This test will determine if the UV lamps are providing sufficient UV radiation for decontamination purposes. A secondary function of the test is to clean the lamps so they operate more efficiently.

 

HAZARD SIGNAGE

The hazard warning signs described in this section are to inform personnel and visitors that a hazard exists in the area. Three (3) levels of risk have been established. The degree of hazard is indicated by the admission instructions on the placard. The specific hazard is identified by symbols and/or hazard warnings affixed to the door or at equipment. There are a variety of signs available from several vendors such as Lab Safety Supply.

The levels of risk are defined on the admittance placards as follows:

1. WARNING - ADMITTANCE TO AUTHORIZED PERSONNEL ONLY.

Visitors and personnel not assigned to the area must secure permission to enter from the investigator, supervisor or administrator in charge of the area.

2. CAUTION or WARNING- RESTRICTED AREA - ADMITTANCE TO AUTHORIZED PERSONNEL ONLY. Admittance is forbidden to all except those assigned to the area unless accompanied by the principal investigator or laboratory supervisor. This sign shall also be used to identify specific higher risk locations, i.e., incubators, refrigerators, biological safety cabinets, within areas posted with a restricted area sign.

3. DANGER - DO NOT ENTER - CONTAMINATED AREA or DANGER DONOT ENTER. This no-access sign is to be used in temporary situations, such as following an accident. Areas posted with this sign shall be off limits to all personnel except the ARS employee or Lead Scientist. The sign shall be taken down immediately after the source of danger is removed.

C. RESPONSIBILITY

It is the responsibility of the Lead Scientist to determine the need for hazard warning(s) and to contact the Area Safety and Health Manager to determine the level of hazard in the area.

It is the responsibility of the Lead Scientist to list the names and telephone numbers of two individuals on the admittance placard as emergency contacts.

Labels and Warnings

The biohazard symbol in a sign or posting, without a specific hazard description, is a general biohazard warning to be used where there are multiple biological hazards, where biological wastes are stored, and for mixed biological waste containers. This label, with the restricted area label is generally used to identify refrigerators, incubators, and cabinets where biological agents or materials are stored.

Laboratories and support areas where viral, bacterial, rickettsial, fungal, and parasitic agents requiring containment at biosafety level 2 or greater are used or stored shall be conspicuously posted with the Caution - Infectious Agents sign.

Corrosives

Separate rooms, cabinets or areas where corrosives (concentrated acids or bases) in excess of one (1) day's supply are stored shall be conspicuously posted with the Caution - Corrosive Materials sign(or equivalent).

The toxic chemicals sign shall be posted in all areas or rooms where hazardous chemicals with an American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV) of 5 ppm or less are used or stored. Refer to the MSDS.

The toxic gas sign shall be posted in all areas or rooms where toxic gases with an American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV) of 10 ppm or less are used or stored. A partial list of these toxic gases and their TLV or ceiling level (C) is below:

Arsine (0.05 ppm) Hydrogen sulfide(10 ppm)

Boron trichloride (1 ppm) Methyl mercaptan(0.5 ppm)

Boron trifluoride (1 ppm C) Monomethylamine(5 ppm)

Butadiene (10 ppm) Nitrogen dioxide(3 ppm)

Chlorine (1 ppm) Phosgene(0.1 ppm)

Cyanogen (10 ppm) Phosphine(0.3 ppm)

Diborane (0.1 ppm) Silane(5 ppm)

Dimethylamine(5 ppm) Silicon tetrafluoride(0.1 ppm)

Hydrogen bromide(5 ppm) Sulfur dioxide(2 ppm)

Hydrogen chloride(5 ppm C) Sulfur tetrafluoride(0.1 ppm C)

Hydrogen fluoride(3 ppm C) Trimethylamine(5 ppm)

Hydrogen selenide(0.05 ppm)

All rooms and areas, accessible to personnel, containing, unguarded (exposed, open) electrical  equipment in excess of 600 volts shall be conspicuously posted with the Caution - High Voltage sign.

All rooms or areas, accessible to personnel, with exposed (open) electrical systems or live parts less than 600 volts shall be labeled with the Caution - Electrical Hazard sign.

Labs or maintenance rooms which have one or more approved flammables storage cabinets shall have a Caution- Flammables sign.

The sign, DO NOT EAT, SMOKE OR DRINK indicates that eating, drinking, smoking, handling of contact lenses, and applying cosmetics are not permitted in the posted work area because there is reasonable likelihood of exposure to hazardous chemicals, radioactive materials or  potentially infectious materials.

The EYE PROTECTION REQUIRED sign shall be posted in all areas, accessible to personnel, where there is a reasonable probability of exposure to hazardous chemicals, potentially infectious agents or physical hazards which could result in injury that can be prevented by eye protection shall be conspicuously posted with this sign.

Each area or room, accessible to personnel, in which there is a potential for noise exposure which may equal or exceed an 8-hour time-weighted average sound level (TWA) of 85 dBA. (OSHA standard 1910.95)shall be posted with the hearing protection required sign.

The PROTECTIVE CLOTHING REQUIRED sign shall be posted when work conditions require specific protective clothing which is beyond the standard Laboratory coat. Personal protective equipment selected for this work area shall be based on an evaluation of the task specific conditions and the hazards and potential hazards that are encountered.

In compliance with the Workplace Hazard Assessment - OSHA standard CFR 1910.132.

CAUTION

BIOHAZARD

INFECTIOUS AGENTS

BIOSAFETY LEVEL 2

ADMITTANCE TO AUTHORIZED PERSONNEL ONLY

The above wording on a 10"x 10" sign shall be posted on the doors of laboratories where work is performed with infectious agents that require special conditions for containment. The need for this sign will depend on the nature of the work as well as the pathogenicity of the agent.

Laboratories posted with the BSL 2 sticker are in compliance with the BMBL* requirements for safety equipment and facility design for this level of containment. Laboratory staff have been trained to use the work practices required at the BSL 2 level.

Activities during DAY: Visits in these areas are prohibited unless the visitor has permission from the investigator(s) in charge, who will be responsible for the safety of the visitor while he is in the area. Visitor access to the laboratory must comply with policy.

Activities at NIGHT: Normally, each laboratory shall be "secured" at the end of each work day. Infectious materials shall be stored in refrigerators, incubators, etc.; table tops shall be wiped down with an appropriate disinfectant; contaminated glassware and equipment shall be sterilized or contained in covered pans. All hazard sources shall have been contained for the night.

The area shall be safe for entry by night cleaning crews and other service personnel; however, these personnel must observe specific instructions and precautions and must not work on any equipment with a 5"x 5" CAUTION - BIOHAZARD - RESTRICTED AREA sign.

GENERAL LABORATORY PRACTICES AND PROCEDURES

Routine safety procedures observed in laboratories do help control biohazards. However, there are additional precautions and procedures that must be followed when dealing with known or potentially biohazardous agents. The following paragraphs summarize general safety procedures for biohazard control.

A. EMPLOYEE ACTION

1. Learn the location and operation of fire extinguishers, safety showers, and other safety items in the work area.

2. Learn what actions are to be taken in the event of a spill or accidental exposure; know the location of a stock solution of disinfectant and the procedure for a clean-up of biohazardous spills. Keep fresh disinfectant available at the proper dilution.

3. Notify the responsible investigator or supervisor in case of an accident, injury, exposure, or illness.

B. FOOD, DRINK, AND COSMETICS

1. Food, candy, gum, or beverages for human consumption should not be taken into or consumed in any area where work with biohazardous materials is conducted.

2. Refrigerators, freezers, and cold rooms in laboratories where work with biohazardous materials is conducted should not be used for storage of lunches or other food supplies.

3. Drinking fountains should be the sole source of water for drinking by laboratory personnel.

4. Employees should not wear potentially contaminated laboratory clothing into lunchrooms, cafeterias, or designated eating areas.

5. Applying cosmetics, including hand lotion and lip balm, are prohibited in laboratories.

C. HAND WASHING

1. Hands must be washed throughout the day, at intervals dictated by the nature of the work. The hand washing procedure should include at least a 10 second wash with mild soap and water and thorough rinsing.

2. Hands should be washed promptly after removing protective gloves or soiled protective clothing, before leaving the laboratory area, before eating, and before applying cosmetics.

D. BEARDS AND LONG HAIR

Wearing a beard in biohazardous areas is discouraged; it may become involved in tranmission of infectious agents. When the work requires the use of face masks or respirators, a proper facial fit cannot be obtained without a clean, shaven face. Individuals with long hair are encouraged to wear a suitable hairnet or hair cover.

E. BOOKS AND JOURNALS

1. Every effort should be made to limit the number of books and journals kept in biohazardous areas. Books and journals should not be taken into laboratory rooms where biohazardous agents are being used.

2. Books and journals on loan from libraries should not be taken into hazardous areas.

F. PIPETTES

1. Materials should not be pipetted by mouth. Mechanical pipetting aids must be used for all procedures.

2. Mixtures must not be prepared by bubbling expiratory air through a liquid containing infectious material with a pipette.

3. Infectious material must not be blown out of pipettes.

4. Pipettes used with infectious or toxic materials must be plugged with cotton unless they are being used in a gas tight, Class III biological safety cabinet system.

5. Contaminated pipettes must be placed horizontally in a 15 cm deep, rectangular, stainless steel or polypropylene pan containing enough disinfectant (such as WescodyneTM from AMSCO) for complete immersion. The pan and pipettes should be autoclaved as a unit and replaced by a clean pan with fresh disinfectant.

6. Decontaminated disposable pipettes must be discarded into the biohazard box.

G. SYRINGES

1. Avoid unnecessary use of syringes and needles. Whenever possible, use a blunt needle or cannula on the syringe. Do not use a syringe and needle as a substitute for a pipette.

2. Use of syringes and needles in a biological safety cabinet if possible.

3. Use of syringes of the LUER-LOK type to assure that the needle cannot separate during use.

4. Use of an alcohol pleget around the stopper and needle when removing a syringe and needle from a rubber stoppered vaccine bottle.

5. Expel excess fluid and bubbles from syringes vertically into a cotton pledget soaked with disinfectant, or into a small bottle containing disinfectant-soaked cotton.

6. Swab the site of injection with an appropriate antiseptic before and after injection of an animal.

7. Submerge contaminated, non-disposable glass syringes (with attached needles) in a container of disinfectant fluid (such as Wescodyne) in a biological safety cabinet prior to removal for autoclaving. To minimize accidental injection of infectious material, needles should remain on syringes until after autoclaving. When possible, reusable syringes with attached needles should be placed in a stainless steel pan separate from that holding materials to be discarded after autoclaving.

 

REFERENCES

These publications are good reference sources:

A. 1988 Agent Summary Statement for Human Immunodeficiency Virus and Report on Laboratory Acquired Infection With Human Immunodeficiency Virus. MMWR, April 1, 1988/Vol.37/No. S-4 Supplement.

B. Guidelines for Prevention of Transmission of Human Immunodeficiency Virus and Hepatitis B Virus to Health-Care and Public-Safety Workers. MMWR, June 23, 1989/Vol. 38/No. S-6.

C. H.I.V. Infection Control Guidelines, Maryland Governor's Advisory Council on Aids 5/89.

D. Occupational Exposure to Bloodborne Pathogens; Final Rule. 
Occupational Safety and Health Administration. 29 CFR Part 1910.1030. Federal Register, vol. 56, No. 235, December 6, 1991, p. 64175-64182.

E. Enforcement Procedures for the Occupational Exposure to Bloodborne Pathogens Standard, 29 CFR 1910.1030. OSHA Instruction CPL 2-2.44C, March 6, 1992.

F. Public Health Service Statement on Management of Occupational Exposure to Human Immunodeficiency Virus, Including Considerations Regarding Zidovudine Postexposure Use, Morbidity & Mortality Weekly Report, vol. 39, no. RR-1, January 26, 1990.

G. Working Safely With HIV in the Research Laboratory; Biosafety Level 2/3, Occupational Safety and Health Branch, NIH Division of Safety, June, 1988.

H. Guidelines for Preventing the Transmission of Tuberculosis in Health-Care Settings, with Special Focus on HIV-Related Issues, Morbidity & Mortality Weekly Report, vol. 39, no. RR-17, December 7, 1990.

I. Johns Hopkins Institutions Bloodborne Pathogens Professional Study Guide, February, 1994

 

BIOSAFETY REFERENCES

1. Classification of Etiologic Agents on the Basis of Hazard. (4th Edition, July, 1974). U.S. Department of Health, Education, and Welfare. Public Health Service.

Centers for Disease Control, Office of Biosafety, Atlanta, Georgia 30333.

2. Biosafety in Microbiological and Biomedical Laboratories, Centers for Disease Control, Atlanta, Georgia, National Institutes of Health, Bethesda, Maryland, U.S. Department of Health and Human Services, Public Health Service, HHS Publication No. (CDC) 93-8395, 3rd Edit., May 1993.

3. USDA permit, required for import and interstate transport of pathogens, may be obtained from the Animal and Plant Health Inspection Service, USDA, Federal Building, Hyattsville, MD 20782.

4. National Cancer Institute Safety Standards for Research Involving Oncogenic Viruses. (October, 1974), U.S. Department of Health, Education, and Welfare Publication No. (NIH) 75-790.

5. Guidelines for Protecting the Safety and Health of Health Care Workers. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control, and National Institute for Occupational Safety and Health {Division of Standards Development & Technology Transfer, 4676 Columbia Parkway, Cincinnati, OH, Telephone (513) 533-8287}; DHHS (NIOSH) Publication No. 88-119, September, 1988.

6. Safety & Emergency Procedure Manual, The Johns Hopkins Hospital, Revised August, 1992.

7. Use of Experimental Animals at the Johns Hopkins University, The Johns Hopkins University School of Medicine, Division of Comparative Medicine, Revised September, 1992.

8. Laboratory Biosafety Guidelines, Medical Research Council of Canada and Laboratory Centre for Disease Control, Health Protection Branch, Health and Welfare Canada, Cat. No. MR 21-1/1990E, 1990.

9. Biohazards Management Handbook, Second Edit., D.F. Liberman, Marcell Dekker, Inc., New York, 1995.

10. Laboratory Safety Principles and Practices, Second Edit., D. Fleming, J. Richardson, J. Tulis, D. Vesley, ASM Press, Washington, DC, 1995.

Additional References

1. Laboratory Safety Monograph, NIH.

2. Standard Number 49. Class II (Laminar Flow) Biohazard Cabinetry, National Sanitation Foundation, Ann Arbor, Michigan, Revised May, 1992.

3. Peterson, A.P.G. and Gross, E.E., Handbook of Noise Measurement, General Radio Company, Concord, MA 1984

4. Industrial Noise Manual, 3rd Ed., A.I.H.A., Akron, OH, 1975

5. The Industrial Environment - Its Evaluation and Control, U.S. Dept. of H.E.W., Center for Disease Control, N.I.O.S.H., Cincinnati, OH, 1973

6. IES Lighting Handbook, 5th Ed, Illuminating Engineering Society, New York, NY, 1972.

7. Certification of Biological Safety Cabinets Manual for the Harvard University Workshop sponsored by the National Institutes of Health, latest edition.

8. NSF Listings Class II Biohazard Cabinetry. National Sanitation Foundation. Ann Arbor, Michigan, February 1, 1993.

9. Biosafety in Microbiology and Biomedical laboratories, 3rd Edition. US Department of Health and Human Services and National Institutes of Health, Washington, DC, Revised May, 1993, Publication NO. (CDC) 93-8395.

10. Laboratory Safety: Principles and Practices, B.M. Miller (ed), American Society for microbiology, Washington, DC, 1986.

11. Federal Standard 209E: Airborne Particulate Cleanliness Classes in Cleanrooms and Clean Zones, The Institute of Environmental Sciences, Mount Prospect, IL, 1992. Insert Summary table

 

 


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