LABORATORY EQUIPMENT

Autoclaves are used to sterilize equipment, products or culture media prior to use in an experiment. They can also be used to render items non-infectious prior to disposal. Autoclaves sterilize materials using saturated steam under pressure ("moist heat"). Due to the use of pressure, steam and high temperatures, there is a significant risk for injury. Individuals that intend to use an autoclave must be properly trained on operational procedures and use appropriate personal protective equipment (PPE). 

General guidelines

• Autoclaves should be inspected and serviced on a regular basis.
• Individuals using autoclaves must be properly trained and familiar with the autoclave specifications and related SOPs prior to working with them.
• Autoclaves should be tested on a regular basis with a commercial biological indicator (i.e. Geobacillus stearothermophilus) to ensure that it is functioning properly.
• Tape indicators (autoclave tape) with heat sensitive, chemical indicators should be used in every load. Autoclave tape will only verify that the autoclave has reached normal operating temperatures. NOTE: It does not indicate proof that organisms were actually killed during an autoclave run.
• High density wastes or materials that insulate agents from heat and steam penetration are not suitable for steam sterilization. Items covered with dirt or film require additional retention times.
• All autoclaved infectious waste must be placed into red biohazard bags for disposal.
• Upon completion of a cycle wait until the autoclaved has cooled prior to opening the door (most autoclaves have safety interlocks that prevent the door from opening when the temperature inside is greater than 80°C). Do not stand in front of the autoclave when opening the door, a puff of steam may be ejected immediately after the cycle has been completed.
• Containers with liquids should be allowed to cool for at least 20 minutes before transport to prevent sudden eruption from the containment vessel.

Appropriate Personal Protective Equipment (PPE)
The following PPE should be used during loading and unloading of the autoclave:

• Standard laboratory clothing including long pants and closed-toed shoes
• Eye/face protection
• Heat resistant gloves
• Laboratory coat

When autoclaving dry materials

• Only use approved autoclave bags and ensure that they are not filled beyond 75% of their holding capacity.
• Steam resistant autoclaveable bags must be left open (or have holes punched into the top of them to ensure proper steam penetration.

When autoclaving liquid materials

• Bottle caps and stoppers must be loosened after placement into the autoclave chamber. This will allow proper steam penetration to sterilize and prevent the bottles from shattering or bursting from the pressurization and extreme heat during operation.
• Containers should not be filled beyond 25-50% of their capacity to prevent spill and boil over.
• Bottles/flasks/containers can be placed in an autoclave pan with about 5-10 inches of water for even heating, ensure that are no air bubbles under the containers.

Incompatible materials
Not all materials are autoclaveable as they can generate toxic vapors or create explosive environments. The following materials should not be autoclaved:

• Materials containing solvents, volatile, corrosive or flammable chemicals
• Materials contaminated with chemotherapeutic agents or cytotoxic drugs
• Materials containing bleach
• Carcinogens or mutagens
• Phenol and Trizol
• Polystyrene, polyethylene, low-density polyethylene and high-density polyethylene plastics and polyurethane tubing
• Household glassware
• Radioactive materials 

Biosafety Cabinet (BSC) - is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level (BSL). The primary purpose of a BSC is to serve as a means to protect the laboratory worker and the surrrounding environment from pathogens.  All air is HEPA-filtered as it exits the BSC, removing harmful bacteria and viruses. Many (but not all) types of BSCs are also designed to maintain the sterility of the materials being worked with.

Note: Chemical fume hoods are not designed nor intended to be used as BSCs. Chemical fume hoods fail to provide the environmental protection that HEPA filtration in a BSC would provide. Likewise, a laminar flow clean bench cannot be used as a BSC. A laminar flow clean bench blows unfiltered exhaust air towards the user and is not safe for work with pathogenic agents.

For a thorough description of BSCs, including selection, installation, and use please see Appendix A of the Biosafety in Microbiological and Biomedical Laboratories Guidelines (6th ed.).

If work is being performed in a BSL-2 laboratory, all work in which infectious aerosols may be produced must be conducted in a BSC or other type of physical containment. If work is being performed in a BSL-3 laboratory, all work must be performed in a BSC or other type of physical containment.

Work practices and procedures

1. Cabinet blowers should be operated at least 3-5 minutes before beginning work to allow the BSC to "purge" particulates.
2. Turn off BSC Ultraviolet (UV) light (if present) before beginning work.
3. Appropriate Personal Protective Equipment (PPE) must be worn. Lab coats must be buttoned. Gloves should be pulled over the knitted wrists of the lab coat, not worn inside the coat.
4. Place only the necessary materials in the BSC before beginning work.
5. Extra supplies (gloves, plates, media, etc.) should be stored outside BSC: material placed inside BSC may cause disruption to the airflow.
6. Move arms in and out of BSC slowly, perpendicular to the face opening to reduce disruption of air curtain. Manipulation of materials should be delayed momentarily to allow air circulation to stabilize.
7. Do not block front grille with papers or materials or rest arms on front grille. This allows room air to flow directly into the work area rather than being drawn through the front grille. Work with arms raised slightly.
8. All operations should be performed at least 4 inches from the front grille on the work surface.
9. Interior walls, interior surface of the window, and the surfaces of all materials to be placed in the BSC should be wiped with 70% ethanol or other appropriate disinfectant before and after each use. If bleach is used, make sure that it is followed by sterile water or 70% ethanol.  Bleach will eventually ruin the stainless steel surfaces of the BSC.
10. Plastic backed absorbent toweling can be placed on the work surface (but not on the front grille) to aid in cleanup and spill containment.
11. Active work should flow from the clean to contaminated area across the work surface.
12. To minimize frequent in/out arm movement and maintain air barrier, do not tape autoclavable biohazard collections bags to the outside of the BSC and upright pipette collection containers should not be used in the BSC nor placed on the floor outside the BSC (use horizontal discard trays containing an appropriate chemical disinfectant within the BSC).
13. Potentially contaminated materials should not be brought out of the BSC until they have been surface decontaminated.
14. Use techniques to reduce splatter and aerosol generation: Open bottles or tubes should not be held in a vertical position, hold the lid above open sterile surfaces to minimize direct impact of downward air, open flames create turbulence which disrupts the pattern of air supplied to the work surface and should not be used. If necessary, touch plate microburners which provide a flame on demand or electric furnaces are available. All flames must be off before disinfectants are used.
15. Aspirator bottles or suction flasks should be connected to an overflow collection plastic flask containing an appropriate disinfectant, and to an in-line HEPA filter.
16. If spilled liquid enters through the front or rear grilles, close the drain valves and pour decontaminating solution into the drain pans. After 20-30 minutes, collect the spilled liquid and disinfectant with paper towels.

Use of vacuum lines
All vacuum lines used to aspirate supernatant, tissue culture media, and other liquids that may contain microorganisms should be protected from contamination by the use of a collection flask and overflow flask. In addition, a hydrophobic vacuum line filter or HEPA filter should be used. This will prevent fluid and aerosol contamination of central vacuum systems or vacuum pumps. Full strength chemical disinfectant should be added to collection flasks and allow the aspirated fluids to complete the dilution (for example: start with 100 ml of undiluted bleach, aspirate 900 ml of fluids, and discard for a 10% final bleach concentration).  Flasks containing aspirated fluids and disinfectants should be changed often and on a regular basis.

Biosafety Cabinet Certifications (BSCs)

• All BSCs should be certified annually. If Biosafety Level 2 or higher materials are used in the BSC, the cabinet must be certified annually.
• If the BSC is relocated, it must be re-certified prior to use.

Reference source
United States Department of Health and Human Services. Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets. Washington, D.C: U.S. Government. Printing Office, 1995.

Three factors that govern a safe life for any rotor are:

1. Design and manufacture
2. Proper care and handling during use
3. Retirement, when damage or fatigue make continued use unsafe

Proper care and handling

• Record the purchase date of each rotor, along with manufacturing date and serial number.
• Read the manuals for the rotors and tubes before using the equipment. Follow all operational specifications published in each rotor manual.
• Rotors must be used with the correct centrifuge (Beckman rotors in Beckman centrifuges). Proper rotor and centrifuge combinations will meet laboratory equipment standards and regulations of UL.
• Maximum speed and sample density ratings designated by the manufacturer for each rotor are intended to prevent stress failures and should always be observed.
• Speed reductions required for running high-density solutions, plastic adapters, or stainless-steel tubes should always be observed.
• Sample loads must be balanced and swinging bucket rotors must not be run with missing buckets.
• Before running an ultracentrifuge, check the classification decal on the ultracentrifuge and make sure it matches the classification decal on the rotor.
• The correct overspeed disk must be used with ultracentrifuges. The disk must be on the bottom of the rotor and the disk must be in good condition.
• A speed-derating disk must be installed if and when the warranty conditions require it.
• A well-kept rotor log is essential for continued safe operation of an ultracentrifuge. Include date, user, rotor used, and any problems encountered.
• Set the proper run speed on each time to prevent overspeeding.
• Use a titanium rotor if corrosive salt solutions will be used frequently.
• Do not scratch or otherwise damage the aluminum oxide layer that protects the underlying metal.
• Rotor cavities and buckets must never be cleaned with an ordinary bottle brush with sharp wire ends. Use special plastic-coated brushes.
• Do not use alkaline detergents or cleaning solutions that may remove the anodized coating. Most commercially available solutions for radioactive decontamination are highly alkaline.
• If corrosive materials have been run or spilled on the rotor, wash it immediately.
• Clean all spills or breakage involving radiological, toxic, pathogenic or biological material immediately. Refer to appropriate safety guides for information.
• Only wash the buckets of a swinging bucket rotor. The body of the rotor should never be immersed: the hanger mechanisms are hard to dry and can rust.
• Air dry the rotor after it has been cleaned and thoroughly rinsed with water.
• Store all fixed angle vertical tube and near-vertical tube rotors upside down, with the lids or plugs removed.
• Swinging bucket rotors should be stored with the bucket caps removed.
• Store all rotors in a dry environment, not in the centrifuge.
• Lubricate O-rings and threads as recommended by the manufacturer.
• Observe warranty period and retirement recommendations for each class of rotor.
• Consideration should be given to retiring the rotor when the warranty period has expired.
• Do not use a rotor after the expiration date permanently marked (on some models) on the rotor or rotor accessories. The components must be taken out of service.
• If using centrifuges with Biosafety Level 2 or higher material, rotors must have aerosol containment ("O-rings") or be used in a biosafety cabinet. Rotors must be loaded and unloaded in a biosafety cabinet.
• If using centrifuges with radioactive material, keep centrifuge behind an appropriate shield.
• Rotors and accessories must be made non-radioactive, non-pathogenic, non-toxic and otherwise safe prior to maintenance or repair. A signed statement must be included with the equipment.

Reference source
Beckman and Sorvall technical guides

Work practices and procedures

• Before using a fume hood, make sure your work area is clean and uncluttered.
• Never use the fume hood to store chemicals and equipment between procedures.
• Verify the date on the inspection sticker on the fume hood. The fume hood should be inspected annually. Contact Environmental Health & Safety for inspection.
• The fume hood average face velocity should be between 80-150 feet per minute.
• If the hood is not equipped with an air measuring device, verify adequate inward airflow by using smoke tubes or tissue paper.
• Do not use the fume hood if it is not working properly. Contact Building Manager / Lab Director / Physical Plant at (631) 632-6400 or (631) 444-2400 if the fume hood is not working. Contact Environmental Health & Safety to verify that it is working properly.
• Inspect the bypass area, airfoil, sash and access opening to verify that no air passages are blocked.
• Never put your head inside a fume hood.
• Electrical extension cords are not safe to use in a fume hood due to the danger of an explosion or fire.
• Large equipment must be elevated on solid blocks to maintain an airflow space of 1-2 inches above the work surface.
• Make sure equipment does not block the baffles at the rear of the hood.
• Keep all apparatus at least 6 inches inside the fume hood. The best way to maintain this distance is to mark a safety line with tape.
• Avoid opening and closing the sash rapidly and avoid swift arm and body movements in front of or inside the hood. These actions may increase turbulence and reduce the effectiveness of the fume hood.
• Position the sash so that it acts as a shield. Keep the sash as low as possible. The inspection sticker will indicate the maximum height. Always look through the sash, not under it.
• If you observe defective or overheating equipment, shut off the equipment, disconnect it, close the sash, and report the problem to your supervisor.
• Keep chemical containers closed at all times. Use condensers, traps, or scrubbers to contain and collect waste solvents, vapors or dusts.
• Clean all spills immediately. Do not allow spilled liquid chemicals to evaporate.
• If a fire occurs inside the fume hood, immediately close the sash and activate the fire alarm, exit the room, close the door and from a safe area, contact University Police to report a chemical fire.
• Keep fume hood exhaust on at all times.
• Keep the sash closed completely when the fume hood is not in use.

Close the sash for safety
The sash on a fume hood serves many purposes, but the most important one is to protect persons working in the laboratory. When the sash is closed it prevents any "leakage" of chemical fumes from the hood. A closed sash also protects you from "escapes" caused by accidents. Shattered glass, chemical spills, and vapors are contained in the hood if the sash is closed and an "event" occurs. Closing the sash improves overall hood performance for other hoods in the lab and within the building. Also, in case of a power outage or hood ventilation failure, chemical vapors will not back up into the laboratory. Closing the Sash for Safety is a very healthy habit to develop!

Reference materials

• • Fume hood work practices and procedures
  • EH&S Policy 4.5 laboratory chemical fume hood safety program
  • National Research Council prudent practices in the laboratory: handling and management of chemical hazards

Gloveboxes can be used to protect the items inside, the people outside, or both. It is important to monitor whether the box is leaking and to keep records so you can spot trends. There can never be too many checks! Check with the glovebox manufacturer for specific checklists and operating requirements.

Gloves information

• Glove inspections - A visual inspection and dexterity check must be performed before gloves are used.
• Glove changes - Changing of a glove must be documented (date, manufacturer and model of glove and person performing change).
• Glove date lifetime limits - The maximum life for a glove is 10 years from the date of manufacture (stamped on the inside surface of each glove).
• Glove protection - Disposable nitrile gloves should be used over the glovebox gloves to protect from contamination and wear and tear.
• Inactive glove ports - Plugging ports that are never or infrequently used is allowed and encouraged. A properly plugged port should have a stub glove and a glove port cap installed.
• Glove breaches and failure - Document who was working, the material they were working with and potentially contaminated with and the tasks that may have contributed to the failure.
• Pressure check - The glovebox pressure must be checked every day, before use and immediately after gloves are changed. The pressure check must be documented. There are several ways the pressure can be checked:
  • • Visual: If the glovebox is kept under positive pressure, the gloves should be extended outside of the box.
    • Gauges: There may be built in oxygen, water and pressure sensors.
    • Hand-held air flow meter used on all the seams.
    • Soap tests the gloves, seals and fittings for a qualitative test.
    • Pull a vacuum on the airlock each night. If the seal on the airlock was compromised, the vacuum will be gone.
    • Monitor gas consumption: Gas consumed much faster than expected is an indication of a leak.
    • Smoke test: use dry ice or liquid smoke inside the box, purge the box with inert gas and once under pressure watch to see if any of the smoke escapes from the box.
    • Be sure soft connections for articles attached to the box (e.g. drums, gloves, HEPA filters), are completely sealed. Even small leaks allow contaminants to escape or enter. Test whether the gas tanks were leaking.

Work practices
Individuals must be trained in the use of the glovebox prior to using it. They must understand the design features and limitations of a glovebox before using it, including:

• Physical limitations of components, gloves and support systems (e.g. barriers that maintain contamination control).
• Ventilation and vacuum controls that maintain a pressure differential between the glovebox and outside.
• Atmospheric controls (e.g., controlling oxygen concentrations and moisture).
• Features that prevent over-pressurizations, flooding and fire. Over-pressurization can cause a breach in the glovebox, endangering the user, damaging other equipment, spreading hazardous contaminants, and letting air with oxygen and moisture into the box. This design criterion especially applies to existing systems when they are connected to a pressurized support system for the first time. Pressure relief systems should be retrofitted into existing systems that do not have adequate pressure relief.
  • • All personal protective equipment (PPE) needed for the hazardous material (e.g. protective eyewear, gloves and lab coat) must be worn when using the glovebox.
    • Avoid abruptly extending gloves in the glovebox. This "pumping" can cause a pressure pulse that will result in contamination. Multiple users need to coordinate their movement to prevent their actions from having a compounding effect.
    • Respond to off-normal indications or alarms with established procedures. Promptly convey problems and abnormal conditions to your supervisor. Stop work until the cause and consequence of an alarm have been identified and safe working conditions have been restored.
    • Keep sharps in an approved container. Use tools with points on sharps ground down if appropriate.
    • Do not work in the glovebox unless the lighting is working.
    • Follow all safe work practices for using and handling compressed gas that may be associated with working in the glovebox. Review Compressed Gas Safety information.
    • Remove waste in a timely manner. Do not allow it to accumulate. Hazardous waste containers in the glovebox must be appropriately labeled at all times.

Documentation
Document the following:

• Condition of gloves and glove seals
• Daily pressure differential (gauge readings)
• Condition and configuration of other systems installed on the box (e.g. valves and readings on pressure and flow gauges fall within acceptable ranges)
• Condition of box and fittings (e.g., rust or other conditions)
• Compressed gas cylinder: record date changed, tank serial number (if available), and daily pressure reading.

Responsible individuals

• Periodically assess glove boxes to determine if ventilation design and monitoring requirements are adequate for authorized operations. Record the results of these assessments using a logbook (or other durable, easily retrievable record) for each glovebox. The record should include:
  • • A characterization of the type and intended use of the glove box
    • Ventilation monitoring and design information
    • Glovebox use history
    • Applicable operating procedures, alarm response procedures, and safety plans.
• Conspicuously label gloveboxes with the following:
  • • Identifying information about the glove box and authorized types of work activities
    • Appropriate hazard warning labels
    • The names and phone numbers of the responsible individual and other knowledgeable people to contact about the glovebox
• Ensure documentation listed above is completed

Reference materials

• American Glovebox Society 

Always place an appropriate trap between experimental apparatus and the vacuum source. The vacuum trap protects the pump, pump oil and piping from the potentially damaging effects of the material; protects people who must work on the vacuum lines or system, and; prevents vapors and related odors from being emitted back into the laboratory or system exhaust.

Improper trapping can allow vapor to be emitted from the exhaust of the vacuum system, resulting in either reentry into the laboratory and building or potential exposure to maintenance workers. Proper traps are important for both local pumps and building systems.

Proper trapping techniques
To prevent contamination, all lines leading from experimental apparatus to the vacuum source must be equipped with filtration or other trapping as appropriate.

• Particulates: use filtration capable of efficiently trapping the particles in the size range being generated.
• Biological Material: use a High Efficiency Particulate Air (HEPA) filter. Liquid disinfectant (e.g. bleach or other appropriate material) traps may also be required.
• Aqueous or non-volatile liquids: a filter flask at room temperature is adequate to prevent liquids from getting to the vacuum source.
• Solvents and other volatile liquids: use a cold trap of sufficient size and cold enough to condense vapors generated, followed by a filter flask capable of collecting fluid that could be aspirated out of the cold trap.
• Highly reactive, corrosive or toxic gases: use a sorbent canister or scrubbing device capable of trapping the gas.

Cold traps
For most volatile liquids, a cold trap using a slush of dry ice and either isopropanol or ethanol is sufficient (to -78 deg. C). Avoid using acetone. Ethanol and isopropanol are cheaper and less likely to foam.

Liquid nitrogen can only be used with sealed or evacuated equipment. If the system is opened while the cooling bath is still in contact with the trap, oxygen may condense from the atmosphere and react vigorously with any organic material present.

Water aspirators
Water aspirators also provide a source of vacuum. While convenient, the use of water aspirators is discouraged for several reasons:

• They waste water, since you have to run the water at full blast to create a good vacuum.
• Volatile solvents are carried out from your flask and through the aspirator to the water that is running down the sink - this means you are contaminating the waste water with organic solvents.
• Aspirators often back water up into your glassware, whether it is a filtering flask or an evaporating flask.

Only use this set-up if your solvent is non-volatile and non-hazardous, such as water. Methylene chloride and similar solvents will be drawn out of the flask and end up in the water being flushed down the drain through the water aspirator.

Biological material traps
A suction flask is used to collect the contaminated fluids into a suitable decontamination solution. A second flask serves as a fluid overflow collection vessel and an in-line HEPA filter is used to protect the vacuum system from aerosolized microorganisms.

Reference materials

• View a PDF file of this information