Standard Operating Procedures for departmental equipment are found on the AFNS safety drive. Using equipment with biohazardous materials requires some special considerations. Below are some special considerations for the most commonly used equipment in Level 1 and 2 labs.
Biological Safety Cabinets (BSC)
Biological safety cabinets should be used when working with pathogens, tissue culture and human samples.
- Turn on fluorescent lights
- Check air grilles for obstructions, switch on blower
- Allow air to purge workspace for 5 minutes
- Spray or swab all interior surfaces with appropriate disinfectant (germicide)
- Preplan and have all supplies ready for the entire procedure
- Introduce only material required to perform procedure (do not block air intake or exhaust grill by over filling the cabinet)
- Place material such that clean and contaminated items do not meet
- Place contaminated material container at right rear
- Allow air purge period with no activity inside (leave blower on!)
- Record the static pressure on the appropriate paper.
- Don protective clothing, gloves, mask, etc. as appropriate
- Introduce hands into work space, work carefully and methodically (i.e. from clean to work area to discard).
- DO NOT remove hands from work space until procedures are complete and all critical material is secured.
- Remove gloves into contaminated material container
- Allow air purge period with no activity inside (leave blower on!).
- Use fresh gloves, remove materials to incubator, to biohazard bag, autoclave as appropriate.
- Spray or swab all interior surfaces with appropriate disinfectant.
- Replenish communal supplies
- Refill the bottle of disinfectant, put a new plastic bag in the waste bucket.
Other helpful hints with biosafety cabinets
- Keep activity in the area surrounding the hood to a minimum to reduce the amount of air disruption in the hood.
- Place everything at least 4” inside the work area to reduce chance of contamination.
- If a spill occurs clean it up immediately before continuing work.
- Do not use an open flame in a biosafety cabinet.
- Every time the cabinet is used, record the static pressure on the chart provided by EHS.
What to do in case of a cabinet failure while working
- Seal containers, surface decontaminate and remove any biohazardous material.
- Decontaminate the interior of the BSC.
- Switch off the power if the motor is making noise.
- Place a sign on the cabinet to indicate that it is broken and must not be used.
- Contact Heather Vandertol-Vanier (firstname.lastname@example.org).
- If personnel may have been exposed to infectious material due to cabinet failure, then the supervisor must be promptly notified and an incident report completed and the appropriate first aid and medical follow-up action taken.
Clean air benches
Clean air benches are not Biological safety cabinets and should never be used with pathogens or other materials that are potential aerosol hazards. These benches only protect the product not the user.
Autoclaves are used to sterilize items potentially exposed to biohazards. Autoclaves operate with steam and pressure. Both of these can be dangerous, so autoclaves should be used with caution. Before using any autoclave, you must receive training from Urmila Basu, Kelley Dunfield, Heather Vandertol-Vanier or Jackson Mah.
When loading an autoclave, do not overload by stacking or overflowing containers. Some general guidelines as follows:
- Containers should not be any more than 2/3 full.
- Put in an autoclave safe container.
- Be careful when removing to prevent bumping (liquid boiling over).
- Waste bags should be open so that steam can penetrate to sterilize all items.
- Each cycle should be about 1 hour.
Important Safety Practices for autoclaves
- Load the autoclave properly per the manufacturer recommendations.
- Before loading containers of liquids into the autoclave, the caps must be loosened.
- Use an autoclave safe tray with a solid bottom and walls to contain the bottles and catch spills.
- Don't load plastic materials that are not compatible with the autoclave.
- Individual glassware pieces should be within a heat resistant plastic tray on a shelf or rack and never placed directly on the autoclave bottom.
- Make sure the door of the autoclave is fully closed and the correct cycle has been selected before starting the cycle.
- Wear heat-resistant gloves when cracking the autoclave door open after a run.
- Before removing autoclaved items, wait 5 minutes for loads containing only dry glassware, and 10 minutes for autoclaved liquid loads.
- When removing items from the autoclave use heat resistant mitts.
- Be alert for autoclaved liquid bottles still bubbling. Let liquid loads stand in an out of-the-way place for a full hour before touching with ungloved hands. Hot glassware and scalding liquids will cause burns and serious harm.
- Do not autoclave items containing bleach or phenol.
- PPE including long pants, closed toe shoes, lab coat, safety glasses and heat resistant gloves should be worn when loading and unloading an autoclave.
- An SOP for using an autoclave is available on the AFNS safety drive.
High-speed spins generated by centrifuges can create large amounts of aerosol if a spill, leak or tube breakage occurs. An SOP for using centrifuges is available on the AFNS safety drive.
To avoid contaminating your centrifuge:
- Check glass and plastic centrifuge tubes for stress lines, hairline cracks and chipped rims before use. Use unbreakable tubes whenever possible.
- Avoid filling tubes to the rim.
- Use caps or stoppers on centrifuge tubes.
- Use sealed centrifuge buckets or rotors which can be loaded and unloaded in a biological safety cabinet. Decontaminate the outside of the cups or buckets before and after centrifugation. Inspect o-rings regularly and replace if cracked or dry.
- Ensure that the centrifuge is properly balanced.
- Do not open the lid during or immediately after operation, attempt to stop a spinning rotor by hand or with an object or interfere with the interlock safety device.
- Decant supernatants carefully and avoid vigorous shaking when resuspending packed cells.
- Clean spills promptly.
Aerosols may be produced during operation of a freeze drier and when material is being removed from the chamber. When lyophilizing biohazardous materials:
- Load samples in a biological safety cabinet.
- Check glass vacuum containers for nicks and scratches.
- Use only glassware that was designed for high vacuum use.
- Use a disinfectant-containing trap for the vacuum pump exhaust.
- After completion of the run, decontaminate all accessible surfaces.
Glass vacuum vessels may rupture and shower laboratory personnel with glass fragments and flask contents. To reduce these risks:
- Use metal flasks and vacuum traps whenever possible.
- Tape glass containers with duct or adhesive tape to contain glass shards in case of rupture, or use a secondary metal container that is at least as tall as the vacuum flask.
To prevent exposure of lab personnel or maintenance employees who may be required to repair the central vacuum system, vacuum line connections that draw biohazardous aerosols or fluids should be fitted with:
- a HEPA filter in the line leading into the vacuum line: cartridge-type in-line filters provide an effective barrier to escape of aerosols into vacuum systems, and are commercially available for this purpose (discard used filters as biomedical waste).
- an overflow flask in case of accidental aspiration of liquids out of the collection vessel. This flask should be of sufficient capacity, and be placed between the collection flask and the air filter.
Filtration of biohazardous materials must be done in a biosafety cabinet.
Needles and Syringes
Needles and syringes present hazards of spill, autoinoculation and aerosol generation, and should be used only when absolutely necessary. When working with syringes and needles, the following precautions are recommended:
- Perform all operations with infectious material in a biological safety cabinet.
- Keep syringes and sharps pointed away from the worker and others.
- Fill syringes carefully; avoid frothing or introduction of air bubbles.
- Use luer-lock needles and syringes or units in which needles are integral to syringes. Do not bend, shear by hand, or recap needles.
- Do not disassemble a needle/syringe assembly.
- Place used needles and syringes in puncture-resistant containers and dispose of the container using Chematix.
- Incidents with needles must be reported to your PI as soon as possible and an incident report must be filled out and sent to EHS.
Selection of a Mechanical Pipetting Aid
Improper handling of pipettes can lead to contamination of the user and/or to generation of hazardous aerosols. Mechanical pipetting aids should be used for all pipetting procedures. Never pipette by mouth.
Selection of a pipetting device should be based upon:
- intended use
- ease of handling
- delivery accuracy
- user preference
- quality of seal formed with pipettes to be used; liquid should not leak from the pipette tip
- whether the pipetting aid can be sterilized
Safe Use of Pipettes
If infectious aerosols are likely to be generated, pipette in a biological safety cabinet. Handling pipettes as described below will reduce splashing and aerosolization:
- Plug pipettes with cotton.
- Check pipettes before using; cracked or chipped suction ends may damage the seals of the pipetting aid.
- Keep pipettes upright while in use and between steps of a procedure to prevent contamination of the mechanical aid.
- Gently expel contents close to the surface of a liquid or allow to flow down the side of the container.
- Avoid mixing fluids by alternate suction and blowing, or by bubbling air from the pipette.
- Avoid forceful ejection of the contents; use TD (short for "to deliver", also referred to as "mark-to-mark") rather than TC ("to contain") pipettes, as the last drop of fluid does not have to be expelled with TD pipettes.
- Use easier-to-handle shorter pipettes when working inside a biological safety cabinet.
- Submerge used non-disposable pipettes horizontally in disinfectant solution; dropping them in vertically may force out any liquid remaining in the pipette.
- Disinfect the stage, eyepieces, knobs and any other contaminated parts.
- Select a disinfectant that will be effective on the pathogens and non-corrosive to the microscope.
- Clean regularly; add disinfectant, such as a phenolic detergent, to the water. Avoid using sodium azide to prevent growth of microorganisms because sodium azide forms explosive compounds with some metals.
- Raise the temperature to 90 oC or higher for 30 minutes once a week for decontamination purposes.
- To prevent electrical shocks, unplug the unit before filling or emptying and have the continuity-to-ground checked on a regular basis.
- Allow aerosols to settler prior to opening vessels.
- Use these in a biological safety cabinet when working with biohazardous material.
- Wear gloves and hearing protection.
Microbiological transfer loops
To eliminate the spattering and aerosolization associated with flaming of loops, char the material before fully inserting the loop into the flame: i.e., before flaming, hold the loop close to (but not into) the flame. Alternatively, use disposable loops or a microincinerator.