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United States Department of Agriculture

Agricultural Research Service

Biosafety SOPs Developed at ERRC
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Standard Operating Procedure for Spiral Plater

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Telephone: [deleted]

SAFETY PRECAUTIONS: Always wear gloves, lab coat and safety glasses when operating this instrument.

OPERATION:This procedure is applicable when the sample is clear, i.e. it does not contain excessive amounts of particulate matter, if it is not too viscous and when conservation of the amount of sample to be used is not relevant.

1. Set PLATER in the automatic operation mode. Carriage ready light will be on. Turn on the vacuum flask.

2. Fill 2 disposable polystyrene sample cups, one with disinfectant and one with sterile distilled water. Make identifying marks on each and place them in the sample holder.

3. Move sample cup holder into position under stylus tip. Open vacuum filling valve.

4. Sanitize the stylus by lowering the tip into the disinfectant and lifting it out twice with the stylus lift arm. The vacuum pulling the solution through the stylus tube followed by air creates a scrubbing action. Repeat the operation with rinse water. Close valve.

5. Pour sample into disposable 5-ml cup and place in sample cup holder. Lower stylus into sample and draw it through the stylus by opening the vacuum valve until a continuous column if liquid is seen in the sight glass.

6. Close vacuum valve. Make sure tip of stylus is still beneath the liquid level in the sample cup. Raise stylus with stylus lift arm. Move sample holder out of way. 7. Place open petri dish on turntable and lower the stylus by means of the stylus lift arm until the latter is well past the point where the stylus tip touches the agar surface.

8. Start instrument by depressing start switch.

9. At completion of plating, the stylus is lifted automatically and the turntable stops. Remove Petri dish and replace cover. It is now ready for incubation. While removing the Petri dish the carriage will return to the starting position. It is now ready for the next sample.

10. After all the samples are run, the instrument should be rinsed well with water and left filled with water when not in use.

11. The vacuum is turned off, and the instrument power is turned off.


WORKING WITH ACRYLAMME GELS

Acrylamide in either the powder or liquid form is a potent neurotoxin and a carcinogen and should be handled with care. It can be inhaled in powder form and is easily absorbed through the skin.

1. Always wear gloves and a lab coat when working with acrylamide

2. Weigh out the powder under a fume hood. Wear a mask and avoid inhaling the powder.

3. Never dispose of acrylamide down the drain.

4. To dispose of acrylamide, cross-link it with bis-acrylamide using temed

(N,N,N', N'- tetramethylethylenediamine) and ammonium persulfate. The polymerized acrylamide is no longer considered toxic, and it can be autoclaved and disposed of with the biohazard waste.

5. Polymerized gels can be autoclaved and disposed with the biohazard waste.

6. NEVER DISPOSE OF GELS IN THE REGULAR TRASH.

Staining and destaining reagents used following electrophoresis contain hazardous chemicals (ie. methanol and acetic acid) and must be disposed of properly:

1. Never pour them down the drain.

2. They should be stored in appropriately marked hazardous waste containers and disposed of through ERRC hazardous waste pick-up.

3. The running buffer for Western blots contains 20% methanol, and should be collected and stored along with the staining and destaining reagents and picked up as hazardous waste.


Standard Operating Procedure For Shipment of Human Pathogens and Related Material

Location:[deleted]

Contact: [deleted]

The shipment of etiologic agents and vectors of human disease is subject to the Public Health Service Foreign Quarantine Regulations (42 CFR, Section 71.156). Permits specifying conditions under which the agent or vector is shipped, handled, and used are issued by the Centers for Disease Control and Prevention.

The interstate shipment of etiologic agents and vectors of human disease is subject to applicable packaging, labeling, and shipping requirements of the Interstate Shipment of Etiologic Agents (42 CFR Part 72). Packaging and labeling requirements for interstate shipment of etiologic agents are summarized below.

1) All human pathogenic bacteria to be shipped within the United States must be properly packaged within a Class 6.2/96 container.

2) These containers can be obtained from the contact listed above.

3) It is preferable that streak or stab cultures in screw cap tubes be shipped. With the Class 6.2/96 container liquid broth cultures can be used.

4) Wrap each tube in bubble wrap and seal closed with self-adhesive tape.

5) Insert each wrapped tube into a plastic divider located within the plastic container.

6) Screw on lid of container, tighten firmly.

7) Insert the securely closed, packed plastic container into corrugated cushioning material located within shipping box.

8) Place a piece of paper with the shipper's name, address, and a 24 hour telephone number inside the shipping box.

9) Close the naps on the shipping box and secure with tape.

10) On the dotted line located on the side of the box write the applicable UN number (pathogenic bacteria are UN 2814), and the proper name (genus and species) of the bacteria.

11) Below the dotted line apply a biohazard label.

12) Write the shipper's name, address, and a 24 hour telephone number on the outside of the box.

13) Contact the shipping service to rind out if they require additional safety precautions and/or additional information concerning the shipment of the bacteria.

Safety Precautions:

- No more than 50 nil of culture should be sent in one container.

-Additional information on the shipment of etiologic agents of human disease and other related materials may be obtained by contacting:

Centers for Disease Control and Prevention
Attention: Biosafety Branch
Office of Health and Safety
Mail Stop F-05 1600
Clifton Road N.E.
Atlanta, GA 30333
Telephone (404) 639-3883


Standard Operating Procedure For Eppendorf Type Centrifuges

Location:[deleted]

Contact: [deleted]

Telephone: [deleted]

Safety Precautions:

Remember to wear eye protection, gloves, and lab coat at all times while operating the centrifuge. Examine the rotor for any damage. Damaged tubes should never be used and should be matched for size to the rotor. Tubes should never be filled more than 2/3 full and caps should always be used when biohazard materials are being used. The unit should always be kept clean. Always close the lid while operating and never open the lid until the unit has stopped spinning.

Instrument Startup and Operation:

Use centrifuge tubes that are matched to the rotor typically 1.5 ml Eppendorf centrifuge tubes. Be sure that all tubes are matched in volume or weight to each other and arranged symmetric-ally in an opposing or triangular pattern. Tubes should be balanced to within one tenth of a gram or less. Close the cover and set one of the speed levels. If vibration occurs immediately turn the unit off. Open lid only after the rotor has come to a stop.

In Case of a Biohazard Spill:

The rotor must be decontaminated using 70% ethanol followed by a 1% solution of a non-alkaline detergent; never use bleach due to its corrosive effect on metal. The chamber should be cleaned with a 1:256 dilution of Quatsyl.

Emergency Shutdown:

Disconnect the power cord or turn off the power switch and allow the unit to come to a stop. Do not attempt to manually stop the rotor.


Standard Operating Procedure for Eppendorf Centrifuge 5403

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Contact:[deleted]

Telephone: [deleted]

SAFETY PRECAUTIONS: The centrifuge must not be operated in a hazardous or flammable environment and must not be used to centrifuge explosive of highly reactive substances. Install the rotors correctly and check that they are securely tightened. Always load the rotors symmetrically. Wear gloves when handling infectious materials in and out of the centrifuge.

Mounting/Dismounting Rotors

1. Mount the rotor onto the drive axle of the centrifuge. Push right down. Where appropriate, turn the rotor nut a little to the left using the Phillips head screwdriver that was supplied with the instrument. Then turn the rotor nut a little to the right using the Phillips head screwdriver until it is hand-tight.

2. The rotor must always be pushed down to the stop before centrifugation is started.

3. Press 'Start' and let the centrifuge run briefly and then press"Stop" to stop it. During this process, the rotor code is read and stored.

4. To release the rotor, turn its nut to the left using the screwdriver. As soon as it moves freely after slight resistance, it can be removed.

OPERATION

1. Switch in the device (main power switch is turned to 'ON")

2. Set the desired data (spin time, rotational speed, temperature) with 'Select' and the 'UP' and 'DOWN" keys.

3. Load the rotor and close the centrifuge lid (control lamp lights up). Press 'START' to start run.

4. At the end of the selected time or if the run is interrupted with 'STOP', the breaking circuit is activated and the rotor brought to a standstill.

5. When the rotor is at a complete stop, the control lamp lights up and the material which has been centrifuged can be removed.


Standard Operating Procedure For Biological Safety Hoods

Location: [deleted]

Contact: [deleted]

Biological Safety Hoods are designed to capture and contain airborne particles released in the course of working with bacterial pathogens, protecting the laboratory worker from infection. Air enters the hood from the surrounding room and is forced through a series of HEPA filters. This filtered air circulates at a sufficient velocity to retain within the safety hood any infectious bacteria which may be released.

Operating Procedures

1) Turn on blower. Allow ten minutes before starting work within the hood.

2) Make sure that glass shield is at proper working level. A buzzer should sound if at an incorrect level.

3) Disinfect the hood surface with a quaternary ammonium surface-active disinfectant such as Quatsyl 256 (Use reconunended dilution). Follow this with a 70% ethanol wash. Allow to air dry.

4) Turn on light and receptacle switches as needed.

Procedure For Shutdown

1) Disinfect hood surface as above.

2) Turn off all switches.

Safety Precautions

-Always wear gloves and a lab coat when worldng in a biological hood.

-Do not use a biological safety hood as a fume hood. Do not dispense acids or solvents inside the hood.

-Always have a disinfectant nearby in case of spills.

-Never rest anything on the air intake grills located at the front of the hood. This impedes the flow of air into the hood.

-Minimize movement in and out of the hood.


HANDLING AND DISPOSAL OF INFECTIOUS WASTE

PURPOSE: To control handling and disposal of processed biological infectious waste, sharps, pipettes and glass-plastic containers at ERRC, ARS, USDA as required by the Pennsylvania Department of Environmental Resources (PADER)

1. Biological infectious waste consists of media preparations with living organisms and their products. Autoclave waste is classified as "infectious waste" by PADER, Tide 25, Municipal Waste Regulation. Only those persons who are properly trained are allowed to handle infectious waste or operate the autoclaves.

2. All biological infectious wastes, sharps, pipettes and containers (culture tubes) will be disposed of as processed infectious waste. This waste will include the following:

a. Petri dishes containing media with microorganisms. Contaminated plates are to be placed in a durable, leakproof autoclave bag that is to be left loosely sealed with autoclave tape. All bags must be transported in a plastic autoclave pan and placed on a cart.

b.Pipettes and pipette tips; including all sources (chemical, biological, microbial, etc.) from all laboratories (bagged and autoclaved)

c. Culture tubes (excluding autoclaved media)(bagged and autoclaved)

d. Sharps - All syringes, needles and Pasteur pipettes, even if they have been used for chemical transfer, will be placed in recommended sharps containers that are available from the chemical storeroom on the ground floor, sealed with autoclave tape, labeled with your name and room number, autoclaved and disposed of with the infectious waste. Syringes and hypodermic needles are considered high risk equipment and should not be used if another means of delivering materials can be substituted. Never attempt to recap or cut hypodermic needles after use.

e. See next page for full ERRC waste disposal regulations

3. Disposal Procedures:

a.Two locations have been established for collecting waste. In the main building, room 2121, Microbial Food Safety Unit (MFS), has been chosen.

b. After autoclaving, all infecfious waste will be weighed and sealed in a box (not to exceed 45 lbs.) with the proper identification (weight, unit and date) on the outside of the box.

c. [deleted] is the supervisor of this operation, and all questions should be addressed to him.

4. Animals, their waste, bedding and anything that has come in contact with them will be autoclaved and disposed of with other infectious waste.


BIOHAZARD WASTE DISPOSAL

ITEM

Regular Biohazard Sharps Glass

trash bag box box

auto sampler vial x

ELISA plates x

filters x

gloves x

micro columns x

petri dishes with media x

petri dishes without media x

pipetter - repeater x

pipettes - glass (not used for cultures) x

pipettes-Pasteur x

pipettes-plastic x

plastic pipet tips X if large x if small

suction bulbs x

swabs x

syringes and needles x

test tubes-glass x

test tubes-plastic x

tubes-culture x

tubes-micro-centrifuge x

vials- cryo-storage x

vials-hypodermic x

weigh boats x


MICROBIOLOGICAL PRACTICES FOR BIOSAFETY LEVEL 2 LABS

1. The biological and pathogenic properties of organisms should be reviewed by all workers before they are used. Review in advance all safety procedures for handling each organism. Work surfaces are decontaminated before and after working with an infectious or toxigenic agent. Any spills are decontaminated with an appropriate agent at once (see page 9).

2. All infectious liquid or solid waste must be marked as such and is to be decontaminated in the autoclave before being disposed.

3. Mechanical pipetting devices are used. Mouth pipetting is prohibited.

4. Eating, drinking, applying cosmetics, wearing contact lenses and smoking are not permitted in the lab. The wearing of shorts and sandals is prohibited in the labs.

5. Laboratory personnel are to wash their hands before leaving the lab after handling infectious materials.

6. Aerosols are suspensions of fine liquid particles in gas, such as air. Aerosols should be kept to a minimum. All procedures that can create aerosols are to be performed under a class II biological hood.

7. Contaminated plates are to be placed in a durable, leakproof autoclave bag that is to be left loosely sealed with autoclave tape. All bags must be transported in a plastic autoclave pan and be placed on a cart.

8. When an infectious or toxigenic agent (such as Clostridium botulinum) is being worked with in a lab, biohazard signs will be posted on the entrances to warn susceptible persons. Access to the laboratory may be limited to authorized personnel.

9. Spills or accidents that result in exposure of personnel to infectious agents are to be reported to the responsible supervisor at once.

10. Disposable lab coats and latex gloves must be worn at all times when working with infectious or toxigenic agents, and are not to be worn outside of the second floor MFS unit. All gloves and lab coats worn in the laboratory are to be left in the laboratory when going to the cafeteria, library, or other non-laboratory areas. Gloves and lab coats must be autoclaved before disposal.

11. Eye protection must be readily available at all times and always worn when working with strong acids, bases or other corrosive or caustic materials.

12. Keep personal and communal spaces clean, safe and free from clutter. Frequently clean out cabinets and drawers of unwanted items, and discard them.

13. The use of animals for experimentation is permitted under specific, controlled conditions. Protocols for animal work must be submitted and approved by a licensed veterinarian and the chair of the Animal Care Committee. Contact Dr. Vijay K. Juneja, ext. 6500 (committee chairperson) in advance of the work commencing to submit your protocol. Additionally, approved protocols require annual updates. All animals used for research, their bedding and any material that comes in contact with them must be autoclaved and disposed of with the infectious waste. Contact Jeff Call (ext. 6789) with any questions regarding animal waste disposal.

14.Radio labeled compounds can be used at the Center under specific, controlled conditions in designated locations. Handling, waste disposal, and inventory of isotopes is controlled by ARS/NRC guidelines. For further details, contact Dr. Harry Farrell (ext. 6462) or the current chair of the Radiological Safety Committee.

15. Food products that have not been used with microbial cultures may be disposed of as ordinary trash but not in the laboratory trash cans.


CLEAN-UP PROCEDURES FOR SPILLS OF INFECTIOUS OR TOXIGENIC ORGANISMS

ORGANISM CLEAN-UP

1. Staphylococcus Quatsyl 256 (1:256 dil.) for 5 minutes (Quaternary ammonium disinfectant)

2. Listeiia Quatsyl 256 detergent (1:256 dil.) for 5 minutes

3. E. coli Quatsyl 256 detergent (1:256 dil.) for 5 minutes (and other gram negative bacteria)

4. Closttidium 0.5 % bleach for a small spill, and B. cereus and undiluted bleach for a large spill for 10 minutes

Alert everyone in the vicinity that a spill of a pathogenic culture has occurred. Always wear gloves and a disposable lab coat for clean-up. After saturation with appropriate solution, drop paper towels onto spill and allow them to absorb liquid. Use the absorbent pads in the spill control station if spill is extensive. Dispose of towels or absorbent pads carefully in autoclave bags that are to be sealed at the spill site. If the spill was extensive, the contaminated area may have to be retreated. Everything that came in contact with the spill must be autoclaved before disposal.


TRANSPORT OF HAZARDOUS MATERIALS IN THE HALLWAYS

ALL BIOHAZARDOUS MATERIALS SHOULD BE LABELED AS SUCH. All materials should be transported in non-breakable, leakproof containers, which can be decontaminated if necessary. Biohazardous liquids should be enclosed in leakproof containers prior to transport. Transport of all materials by a cart with a barrier that prevents objects from sliding off is recommended. Carrying biohazardous materials in the common-use hallways may result in a spill.


GENERAL OPERATING PROCEDURES FOR WORKING WITH BACTERIAL CULTURES

1. DO NOT MOUTH PIPETTE. Use a pipetting device. Avoid generation of aerosols.

2. NO EATING, DRINKING, OR SMOKING IN THE LAB. No food, dishes, or beverages are to be stored in the lab.

3. ALL MATERIAL THAT HAS COME IN CONTACT WITH A CULTURE MUST BE AUTOCLAVED BEFORE WASHING OR DISPOSAL. This includes culture plates, bottles, tubes, flasks, beakers, food packaging material, etc.

4. CONTACT LENSES, SHORTS AND SANDALS MUST NOT BE WORN IN THE LABORATORY.

5. ALL TRANSFERS OF BACTERIAL CULTURES MUST BE DONE UNDER A CERTIFIED CLASS II BIOLOGICAL HOOD. Sanitize all work surfaces before and after operations with Quatsyl 256 (cleaner/disinfectant) diluted 1:256. This may be followed by 70% ethanol.

6. DISPOSABLE GLOVES SHOULD BE WORN. Be especially careful when working with open cuts, and always wear a Band-Aid to cover cuts.

7. LABEL INOCULATED CULTURE FLASKS, PLATES, FOODS, ETC. CAREFULLY. Clearly state the organism, the date of the initial inoculation and the name of the responsible individual when such items are being held in common use incubators, cold rooms, refrigerators, etc. Any unlabeled material will be immediately autoclaved and discarded.

8. DISINFECT ANY SPILLED CULTURE BROTH WITH A CHEMICAL DISINFECTANT, BLEACH, OR ETHANOL. Warn others of the spill, tell your immediate supervisor, wear disposable gloves and a disposable lab coat, and autoclave all materials that have come in contact with the spilled culture. In the event that a culture container is broken, disinfect as above and carefully autoclave the container prior to discarding it. (See page six for specific clean-up procedures.)

9. USE OF THE SPIRAL PLATER. Be careful to create as little aerosol as possible. Cover the dispensing arm of the spiral plater with a petri dish cover during sampling operations to avoid splashes. Use 0.5% bleach or 70% ethanol followed by sterile water before starting to run samples. Use 70% ethanol and sterile water between samples. When finished, use either 0.5% bleach or 70% ethanol followed by sterile water.

10. ONLY AUTHORIZED AND IMMUNIZED INDIVIDUALS MAY WORK WITH C. BOTULINUM OR BE PRESENT IN THE LABORATORY (rooms 2125 and 2127) WHEN C. BOTULINUM OR ITS TOXINS ARE PRESENT.

11. Avoid generation of aerosols when vortexing, using the spiral plate, throwing out plates etc.

12. USE OF LABORATORY ANIMALS. Only properly trained individuals may work with or care for laboratory animals. Contact [deleted].


Last Modified: 4/29/2005
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