Recommended Work Practices

1. Pipettes and Pipetting Aids

Pipettes are used for volumetric measurements and transfer of fluids that may contain infectious, toxic, corrosive or radioactive agents. Laboratory associated infections have occurred from oral aspiration of infectious materials, mouth transfer via a contaminated finger and inhalation of aerosols. Exposures to aerosols may occur when liquid from a pipette is dropped onto the work surface, when cultures are mixed by pipetting or when the last drop of an inoculum is blown out. A pipette may become a hazardous piece of equipment if improperly used. The safe pipetting techniques outlined below are required to minimize the potential for exposure to hazardous materials.

  • Never mouth pipette. Always use a pipetting aid.
  • If working with biohazardous or toxic fluid, confine pipetting operations to a biosafety cabinet.
  • Always use cotton-plugged pipettes when pipetting biohazards or toxic materials, even when safety pipetting aids are used.
  • Do not prepare biohazardous materials by bubbling expiratory air through a liquid with a pipette.
  • Do not forcibly expel biohazardous material out of a pipette.
  • Never mix biohazardous or toxic material by suction and expulsion through a pipette.
  • When pipetting, avoid accidental release of infectious droplets. Place a disinfectant soaked towel on the work surface and autoclave the towel after use.
  • Use "to deliver" pipettes rather than those requiring "blowout."
  • Do not discharge material from a pipette at a height. Whenever possible allow the discharge to run down the container wall.
  • Place contaminated, reusable pipettes horizontally in a pan containing enough liquid disinfectant to completely cover them. Do not place pipettes vertically into a cylinder.
  • Discard contaminated disposable pipettes in an appropriate sharps

container. Contact the campus Hazardous Waste Disposal Team at potratzg@uwosh.edu, (x1488) for more information on the disposal of pipettes or sharps.

  • Pans or sharps containers for contaminated pipettes should be placed inside the biosafety cabinet, if possible.

 

2. Syringes and Needles Syringes and hypodermic needles are dangerous instruments. The use of needles and syringes should be restricted to procedures for which there is no alternative. Blunt cannulas should be used as alternatives to needles wherever possible (i.e., procedures such as oral or intranasal animal inoculations). Needles and syringes should never be used as a substitute for pipettes. When needles and syringes must be used, the following procedures are recommended:

  • Use disposable needle locking syringe units whenever possible.
  • When using syringes and needles with biohazardous or potentially infectious agents:
  • Work in a biosafety cabinet whenever possible.
  • Wear gloves, a lab coat, and eye protection.
  • Fill the syringe carefully to minimize air bubbles.
  • Expel air, liquid and bubbles from the syringe vertically into a cotton pledget moistened with disinfectant.
  • Do not use a syringe to mix infectious fluid forcefully.
  • Do not contaminate the needle hub when filling the syringe in order to avoid transfer of infectious material to fingers.
  • Wrap the needle and stopper in a cotton pledget moistened with disinfectant when removing a needle from a rubber-stoppered bottle.
  • Bending, recapping, clipping or removal of needles from syringes is prohibited. If it is essential that a contaminated needle be recapped or removed from a syringe, the use of a mechanical device or the one-handed scoop method must be used. The use of needle nipping devices is prohibited.
  • Use a separate pan of disinfectant for reusable syringes and needles. Do not place them in pans containing pipettes or other glassware to eliminate sorting later.
  • Used disposable needles and syringes must be placed in appropriate   sharps disposal containers and discarded as medical waste. Contact the Hazardous Waste Management Team (potratzg@uwosh.edu, (-1488) for more information on the disposal of sharps.


3. Safe and Effective Use of Biosafety Cabinets

In general:

  • It is recommended that your biological safety cabinet is certified when it is installed or after it is moved, and annually thereafter.  Check the magnahelic gauge regularly for an indication of a problem.
  • Understand how your cabinet works.
  • Do not disrupt the protective airflow pattern of the biological safety cabinet. Such things as rapidly moving your arms in and out of the cabinet, people walking rapidly behind you, and open lab doors may disrupt the airflow pattern and reduce the effectiveness of the biological safety cabinet. Cabinets should be placed in room, such that workflow in area is minimal.
  • Plan your work.
  • Minimize the storage of materials in and around the biological safety cabinet.

 

Operational directions:

  • Before using, wipe work surface with 70% alcohol. Wipe off each item you need for your procedures and place in cabinet.
  • DO NOT place objects over the front air intake grille. DO NOT block the rear exhaust grille.
  • Segregate contaminated and clean items. Work from "clean to dirty."
  • Place a pan with disinfectant and/or a sharps container inside the biological safety cabinet for pipette discard. DO NOT use vertical pipette discard canisters on the floor outside cabinet.
  • It is not necessary to flame items. This creates turbulence in airflow and will compromise sterility; heat buildup may damage the filters.
  • Move arms slowly when removing or introducing new items into the biological safety cabinet.
  • If you use a piece of equipment that creates air turbulence in the biological safety cabinet (such as a centrifuge, blender) place equipment in the back 1/3 of the cabinet; stop other work while equipment is operating.
  • Protect the building vacuum system from biohazards by placing a HEPA cartridge filter or its equivalent between the vacuum trap and the source valve in the cabinet.
  • Clean up all spills in the cabinet immediately. Wait 10 minutes before resuming work.
  • When work is finished, remove all materials and wipe all interior surfaces with 70% alcohol and allow hood to run for 5-10 minutes after cleaning.
  • Remove lab coat and wash hands thoroughly before leaving laboratory.

                       

4. Centrifuge Equipment

Hazards associated with centrifuging include mechanical failure and the creation of aerosols.

To minimize the risk of mechanical failure, centrifuges must be maintained and used according to the manufacturer’s instructions. Users should be properly trained and operating instructions that include safety precautions should be prominently posted on the unit.

Aerosols are created by practices such as filling centrifuge tubes, removing plugs or caps from tubes after centrifugation, removing supernatant, and re-suspending sedimented pellets. The greatest aerosol hazard is created if a tube breaks during centrifugation. To minimize the generation of aerosols when centrifuging biohazardous material, the following procedures should be followed:

  • Avoid overfilling of centrifuge tubes so that closures do not become wet.
  • Add disinfectant to the space between the tube and the bucket to disinfect material in the event of breakage during centrifugation.
  • Always balance buckets, tubes and rotors properly before centrifugation.

High speed/ultra centrifuges pose additional hazards. Precautions should be taken to filter the exhaust air from vacuum lines, to avoid metal fatigue resulting in disintegration of rotors and to use proper cleaning techniques and centrifuge components.

Manufacturers’ recommendations must be meticulously followed to avoid metal fatigue, distortion and corrosion.
Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous materials. Celluloid centrifuge tubes are highly flammable and prone to shrinkage with age. They distort on boiling and can be highly explosive in an autoclave. If celluloid tubes must be used, an appropriate chemical disinfectant must be used to disinfect them.

6. Personal Protective Equipment (PPE)

Personal protective equipment is used to protect personnel from contact with hazardous materials and infectious agents. Appropriate clothing may also protect the experiment from contamination. Personal protective equipment must be provided without cost to personnel.

The following personal protective equipment is recommended for regular use:

  • Face Protection Goggles in combination with masks, or chin length face shields or other splatter guards are required whenever there is the possibility of splashes, sprays or splatters of infectious or other hazardous materials to the face.
  • Laboratory clothing This category includes: laboratory coats, smocks, scrub suits, and gowns. Clean, long sleeved garments should be used to minimize the contamination of skin or street clothes and to reduce shedding of microorganisms from the arms. In circumstances where it is anticipated that splashes may occur, the garment must be resistant to liquid penetration (in order to protect clothing from contamination).
If the garment is not disposable, it must be capable of withstanding sterilization in the event it becomes contaminated. Additional criteria for selecting clothing are: comfort, appearance, closure types and location, anti-static properties and durability. Protective clothing must be removed in non-laboratory areas. Disposables should be available for visitors, maintenance and service workers in the event it is required. All protective clothing should be either discarded in the laboratory or laundered by the facility. Personnel must not launder laboratory clothing at home.
  • Gloves Gloves must be selected based on the hazards involved and the activity to be conducted. Gloves must be worn when working with biohazards, toxic substances and other physically hazardous agents. Temperature resistant gloves must be worn when handling hot material or dry ice. Delicate work requiring a high degree of precision dictates the use of thin walled gloves. Protection from contact with toxic or corrosive chemicals may also be required.
When working with hazardous materials, the lower sleeve and the cuff of the laboratory garment should be overlapped by the glove. A long sleeved glove or disposable arm shield may be worn for further protection of the garment.
In some instances “double gloving” may be appropriate. If a spill occurs, hands will be protected after the contaminated outer gloves are removed. Gloves must be disposed of when contaminated, removed when work with infectious material is completed and not worn outside the laboratory. Disposable gloves must not be washed or reused.
  • Respirators In certain instances additional personal protective equipment may be required. Respirator selection is based on the hazard and the protection factor required. Personnel who require respiratory protection must contact the Campus Safety Office (-4484, schwalbe@uwosh.edu) for information on use of respirators at UW Oshkosh.  Use of respirators requires: a medical examination to ensure no health conditions exist that would be exacerbated by respirator usage; annual fit testing to ensure proper respirator size and type; and training to ensure proper respirator use and maintenance. Under no circumstances shall anyone wear a respirator unless he/she is a participant in the program.
Contact  Campus Safety Office )-4484, schwalbe@uwosh.edu)  for assistance in selection of other personal protective equipment.


7. Blenders, Ultrasonic Disrupters, Grinders and Lyophilizers

The use of any of these devices results in considerable aerosol production. Blenders, grinders and cell disruption equipment should be used in a biological safety cabinet when working with biohazardous materials.

Safety blenders, although expensive, are designed to prevent leakage from the bottom of the blender jar, provide a cooling jacket to avoid biological inactivation and to withstand sterilization by autoclaving. If blender rotors are not leak-proof, they should be tested with sterile saline or dye solution prior to use with biohazardous material. The use of glass blender jars is not recommended because of the breakage potential. If they must be used, glass jars should be covered with a polypropylene jar to prevent spraying of glass and contents in the event the blender jar breaks.

A towel moistened with disinfectant should be placed over the top of the blender during use. Before opening the blender jar allow the unit to rest for at least one minute to allow the aerosol to settle. The device should be decontaminated promptly after use.
Depending on the lyophilizer design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If possible, sample material should be loaded in a biological safety cabinet.

The vacuum pump exhaust should be filtered to remove any hazardous agents or, alternatively, the pump can be vented into a biological safety cabinet. After lyophilization is completed, all surfaces of the unit that have been exposed to the agent should be disinfected. If the lyophilizer is equipped with a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination.

Handling of cultures should be minimized and vapor traps should be used wherever possible.
 Opening an ampoule containing liquid or lyophilized culture material should be performed in a biological safety cabinet to control the aerosol produced. Gloves must be worn. To open, nick the neck of the ampoule with a file, wrap it in disinfectant soaked towel, hold the ampoule upright and snap it open at the nick. Reconstitute the contents of the ampoule by slowly adding liquid to avoid aerosolization of the dried material.

Mix the contents without bubbling and withdraw it into a fresh container. Discard the towel and ampoule top and bottom as infectious waste.
Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye injuries. The use of polypropylene tubes eliminates this hazard. These tubes are available dust free or pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat sealable polypropylene tubes are also available.

8. Loop Sterilizers and Bunsen Burners

Sterilization of inoculation loops or needles in an open flame generates small particle aerosols which may contain viable microorganisms. The use of a shielded electric incinerator minimizes aerosol production during loop sterilization.

Alternatively, disposable plastic loops and needles may be used for culture work where electric incinerators or gas flames are not available. The loops are semi-quantitative and can be used for counting bacteria.
Continuous flame gas burners should not be used in biological safety cabinets.

These burners can produce turbulence which disturbs the protective airflow patterns of the cabinet. Additionally, the heat produced by the continuous flame may damage the HEPA filter. If a gas burner must be used, one with a pilot light should be selected.

9. Housekeeping

Good housekeeping in laboratories is essential to reduce risks and protect the integrity of biological experiments. Routine housekeeping must be relied upon to provide work areas free of significant sources of contamination. Housekeeping procedures should be based on the highest degree of risk to which personnel and experimental integrity may be subjected.


Laboratory personnel are responsible for cleaning laboratory benches, equipment and areas that require specialized technical knowledge. Additional laboratory housekeeping concerns include:

  • Keeping the laboratory neat and free of clutter; surfaces should be clean and free of infrequently used chemicals, glassware and equipment. Access to sinks, eyewashes, emergency showers and fire extinguishers must not be blocked.
  • Proper disposal of chemicals and waste old and unused chemicals should be disposed of promptly and properly. Contact the Hazardous Waste Management Team (ehs@uwosh.edu) for details.
  • Providing a workplace that is free of physical hazards aisles and corridors should be free of tripping hazards. Attention should be paid to electrical safety, especially as it relates to the use of extension cords, proper grounding of equipment, avoidance of overloaded electrical circuits and avoidance of the creation of electrical hazards in wet areas.
  • Removing unnecessary items on floors, under benches or in corners.
  • Properly securing all compressed gas cylinders.
  • Never using fume hoods for storage of chemicals or other materials.


Practical custodial concerns include:

 

  • Dry sweeping and dusting which may lead to the formation of aerosols is not permitted.
  • The usual wet or dry industrial type vacuum cleaner is a potent aerosol generator and, unless equipped with high efficiency particulate air (HEPA) filter, must not be used in the biological research laboratory.

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