This protocol describes basic procedures for aseptic technique for the
culture technology. One basic concern for successful aseptic technique
personal hygiene. The human skin harbors a naturally occurring and
population of bacterial and fungal inhabitants that shed microscopically
ubiquitously. Most unfortunately for cell culture work, cell culture
incubation conditions provide ideal growth environments for these
microbial contaminants. This procedure outlines steps to prevent
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
(e.g., flasks or containers used to temporarily hold a sterile reagent
aliquoting the solution into sterile media). Single-use, sterile
such as test tubes, culture flasks, filters, and pipettes are widely
reliable alternatives to the laborious cleaning and sterilization
for recycling equivalent glass items. However, make certain that
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
preparation is essentially the same for working at the bench. Flame
sterilization is used as a direct, localized means of decontamination in
the open bench. It is most often used
- to eliminate potential
contaminants from the exposed openings of media bottles, culture flasks, or test
tubes during transfers,
- to sterilize small instruments such as forceps, or
- to sterilize wire inoculating loops and needles before and after transfers.
possible, flame sterilization should be minimized in laminar-flow
the turbulence generated by the flame can significantly disturb the
- 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)
Flame-Sterilize the Opening of a Vessel
- Just prior to aseptic manipulations, tie long hair back behind head.
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
can potentially fall into sterile containers.
- Gown appropriately. For nonhazardous sterile-fill applications, wear
cuffed laboratory coats and latex gloves.
Greater stringencies may be necessary, depending upon laboratory
requirements. Work with potentially hazardous agents certainly mandates
additional considerations for safety. Front-closing laboratory coats are
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.
- Frequently disinfect gloved hands with 70% ethanol while doing aseptic
work. Although the gloves may initially have been sterile when first
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,
ammonium compounds are more appropriate.
- Dispose of gloves by autoclaving after use. Do not reuse. Bag and
single-use laboratory coats after use. Bag, autoclave (if necessary),
wash other laboratory coats within the laboratory facility, or send out
cleaning at a laundry certified for handling biologically contaminated
Never take laboratory clothing home for washing.
- Thoroughly wash hands after removing protective gloves.
Prepare and maintain the work area.
all aseptic work in a clean work space, free from contaminating
currents and drafts. For optimal environmental control, work in a
- Clear the work space of all items extraneous to the aseptic operation being
- Wipe down the work surface before and after use with 70% ethanol or
other appropriate disinfectant.
- Wherever feasible, wipe down items with disinfectant as they are
into the clean work space. Arrange necessary items in the work space in
logical pattern from clean to dirty to avoid passing contaminated
(e.g., a pipette used to transfer cultures) over clean items (e.g.,
- Immediately dispose of any small contaminated items into a discard pan.
- When the aseptic task has been completed, promptly remove any larger
contaminated items or other material meant for disposal (e.g., old
material, spent media, waste containers) from the work space and place
designated bags or pans for autoclaving. Disinfect the work space as in
- 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
closure. Do not permit any part of the closure that directly comes in
contact with the contents of the vessel to touch any contaminating
(e.g., hands or work bench).
Ideally, and with practice, one should be able to hold the closure in
crook of the little finger of the right hand while still being able to
inoculating loop or pipettor with the other fingers of the hand.
Holding the vessel off the vertical while opening will prevent any
particulates from entering the container.
- Slowly pass the opening of the vessel over the top of (rather than
Bunsen burner flame to burn off any contaminating matter.
careful when flaming containers of infectious material. Any liquid
the threads of a screw cap container will spatter as it is heated.
thus formed may actually disseminate entrapped biological agents before
heat of the flame is hot enough to inactivate them.
- While still holding the vessel at a slant, use a sterile pipette and
slowly add or remove aliquots to avoid aerosol formation.
- Flame-sterilize again as in step 13, allow the container to cool slightly, and
carefully recap the vessel.
Flame-sterilize small hand instruments.
- Dip critical areas of the instrument (i.e., those that come into contact
material of concern) in 95% ethanol.
Make certain that the alcohol is in a container heavy enough to support
instrument without tipping over.
Caution: 95% ethanol is flammable; keep the container at a safe distance
from any open flame.
- Remove the instrument from the alcohol, being careful not to touch the
disinfected parts of the instrument. Allow excess ethanol to drain off
- Pass the alcohol-treated part of the instrument through the flame of a
Bunsen burner and allow residual alcohol to burn off.
- Do not let the sterilized portion of the instrument contact any
material before use. Let the heated part of the instrument cool for ~10
- After use, return the instrument to the alcohol disinfectant until needed
again. Flame-sterilize inoculating loops and needles.
- Hold the inoculating wire by its handle and begin in the center of the
slowly heat the wire with the flame of a Bunsen burner. Proceed back
forth across the wire’s full length until it glows orange.
- While still holding the handle, allow the inoculating wire to cool back
room temperature (~10 sec) before attempting any transfer of material.
transfers are made while the inoculating wire is hot, cells will be
the hot wire, and aerosols created from spattering material can disperse
biological material throughout the work space.
- After the transfer is made, reheat the inoculating wire as in step 21 to
destroy any remaining biological material. Let it cool to room
before putting it aside for next use.