Aseptic Technique

This protocol describes basic procedures for aseptic technique for the novice in cell culture technology. One basic concern for successful aseptic technique is personal hygiene. The human skin harbors a naturally occurring and vigorous population of bacterial and fungal inhabitants that shed microscopically and ubiquitously. Most unfortunately for cell culture work, cell culture media, and incubation conditions provide ideal growth environments for these potential microbial contaminants. This procedure outlines steps to prevent introduction of human skin flora during aseptic culture manipulations.

Every item that comes into contact with a culture must be sterile. This includes direct contact (e.g., a pipette used to transfer cells) as well as indirect contact (e.g., flasks or containers used to temporarily hold a sterile reagent prior to aliquoting the solution into sterile media). Single-use, sterile disposable plastic items such as test tubes, culture flasks, filters, and pipettes are widely available and reliable alternatives to the laborious cleaning and sterilization methods needed for recycling equivalent glass items. However, make certain that sterility of plastic items distributed in multiunit packages is not compromised by inadequate storage conditions once the package has been opened.

Ideally, all aseptic work should be conducted in a laminar however, work space preparation is essentially the same for working at the bench. Flame sterilization is used as a direct, localized means of decontamination in aseptic work at the open bench. It is most often used

1. to eliminate potential contaminants from the exposed openings of media bottles, culture flasks, or test tubes during transfers,
2. to sterilize small instruments such as forceps, or
3. to sterilize wire inoculating loops and needles before and after transfers.

Where possible, flame sterilization should be minimized in laminar-flow environments, since the turbulence generated by the flame can significantly disturb the sterile air stream.

Materials

• Antibacterial soap
• 70% ethanol or other appropriate disinfectant
• 95% ethanol
• Clean, cuffed laboratory coats or gowns
• Latex surgical gloves
• Clean, quiet work area
• Shallow discard pans containing disinfectant
• Bunsen burner or pilot-activated burner (e.g., Touch-o-Matic, VWR)

Take Precautions

1. Just prior to aseptic manipulations, tie long hair back behind head. Vigorously scrub hands and arms at least 2 min with an antibacterial soap. Superficial lathering is more prone to loosening than removing flaking skin and microbial contaminants. Loosely adhering skin flora easily dislodge and can potentially fall into sterile containers.
2. Gown appropriately. For nonhazardous sterile-fill applications, wear clean, cuffed laboratory coats and latex gloves. Greater stringencies may be necessary, depending upon laboratory regulatory requirements. Work with potentially hazardous agents certainly mandates additional considerations for safety. Front-closing laboratory coats are not recommended for work with hazardous biological agents. Safety glasses should be worn by laboratory personnel when manipulating biological agents outside the confines of a biosafety cabinet.
3. Frequently disinfect gloved hands with 70% ethanol while doing aseptic work. Although the gloves may initially have been sterile when first worn, they will no doubt have contacted many nonsterile items while in use. Note that 70% ethanol may not be an appropriate agent for latex glove disinfection when working with cultures containing animal viruses, as studies have shown that ethanol increases latex permeability, reducing protection for the wearer in the event of exposure. In this case, quarternary ammonium compounds are more appropriate.
4. Dispose of gloves by autoclaving after use. Do not reuse. Bag and autoclave single-use laboratory coats after use. Bag, autoclave (if necessary), and wash other laboratory coats within the laboratory facility, or send out for cleaning at a laundry certified for handling biologically contaminated linens. Never take laboratory clothing home for washing.
5. Thoroughly wash hands after removing protective gloves.
   Prepare and maintain the work area.
6. Perform all aseptic work in a clean work space, free from contaminating air currents and drafts. For optimal environmental control, work in a laminar-flow cabinet.
7. Clear the work space of all items extraneous to the aseptic operation being performed.
8. Wipe down the work surface before and after use with 70% ethanol or other appropriate disinfectant.
9. Wherever feasible, wipe down items with disinfectant as they are introduced into the clean work space. Arrange necessary items in the work space in a logical pattern from clean to dirty to avoid passing contaminated material (e.g., a pipette used to transfer cultures) over clean items (e.g., flasks of sterile media).
10. Immediately dispose of any small contaminated items into a discard pan.
11. When the aseptic task has been completed, promptly remove any larger contaminated items or other material meant for disposal (e.g., old culture material, spent media, waste containers) from the work space and place in designated bags or pans for autoclaving. Disinfect the work space as in step 8.

Flame-Sterilize the Opening of a Vessel

1. For a right-handed person, hold the vessel in the left hand at an ~45 ° angle (or as much as possible without spilling contents) and gently remove its closure. Do not permit any part of the closure that directly comes in contact with the contents of the vessel to touch any contaminating object (e.g., hands or work bench). Ideally, and with practice, one should be able to hold the closure in the crook of the little finger of the right hand while still being able to manipulate an inoculating loop or pipettor with the other fingers of the hand. Holding the vessel off the vertical while opening will prevent any airborne particulates from entering the container.
2. Slowly pass the opening of the vessel over the top of (rather than through) a Bunsen burner flame to burn off any contaminating matter. Be careful when flaming containers of infectious material. Any liquid lodged in the threads of a screw cap container will spatter as it is heated. Aerosols thus formed may actually disseminate entrapped biological agents before the heat of the flame is hot enough to inactivate them.
3. While still holding the vessel at a slant, use a sterile pipette and pipettor to slowly add or remove aliquots to avoid aerosol formation.
4. Flame-sterilize again as in step 13, allow the container to cool slightly, and carefully recap the vessel. Flame-sterilize small hand instruments.
5. Dip critical areas of the instrument (i.e., those that come into contact with the material of concern) in 95% ethanol. Make certain that the alcohol is in a container heavy enough to support the instrument without tipping over. Caution: 95% ethanol is flammable; keep the container at a safe distance
   from any open flame.
6. Remove the instrument from the alcohol, being careful not to touch the disinfected parts of the instrument. Allow excess ethanol to drain off into the container.
7. Pass the alcohol-treated part of the instrument through the flame of a Bunsen burner and allow residual alcohol to burn off.
8. Do not let the sterilized portion of the instrument contact any nonsterile material before use. Let the heated part of the instrument cool for ~10 sec before use.
9. After use, return the instrument to the alcohol disinfectant until needed again. Flame-sterilize inoculating loops and needles.
10. Hold the inoculating wire by its handle and begin in the center of the wir to slowly heat the wire with the flame of a Bunsen burner. Proceed back and forth across the wire’s full length until it glows orange.
11. While still holding the handle, allow the inoculating wire to cool back to room temperature (~10 sec) before attempting any transfer of material. If transfers are made while the inoculating wire is hot, cells will be killed by the hot wire, and aerosols created from spattering material can disperse biological material throughout the work space.
12. After the transfer is made, reheat the inoculating wire as in step 21 to destroy any remaining biological material. Let it cool to room temperature before putting it aside for next use.