Aseptic technique refers to a set of routine practices used to prevent contamination of cultures, sterile media stocks, and other laboratory solutions by unwanted microorganisms (sepsis).
The term “sterile technique” is sometimes used interchangeably, but it is more accurately applied to situations requiring the complete prevention of any organism introduction, such as during surgical procedures.
The main goal in biological work is to cultivate microorganisms or eukaryotic cells without introducing foreign organisms, making aseptic techniques essential for producing accurate and meaningful experimental results.
It is important to remember that achieving a completely sterile working environment is impossible.
However, several straightforward, practical procedures can significantly reduce the risk of culture contamination.
Aseptic techniques aim to minimize the chances of contaminating cultures with environmental microorganisms or contaminating the environment with microorganisms being handled.
Common examples of aseptic techniques include:
Cleaning and disinfecting laboratory surfaces before use.
Limiting the time that cultures or media are uncapped and exposed to the air.
Keeping Petri dishes closed whenever possible.
Sterilizing inoculating loops and other equipment that come into contact with cultures or media effectively.
Avoiding breathing directly onto cultures or sterile instruments.
General rules for maintaining aseptic conditions include:
Keeping windows and doors closed to reduce drafts and prevent sudden air movements that could disturb the working area.
Performing all transfers over a disinfected surface; ethanol is recommended for disinfection because of its rapid and effective action.
If the bench surface is difficult to clean, covering it with a tough, easily disinfected material is advised.
Starting any operation only after ensuring all necessary apparatus and materials are within immediate reach.
Completing all procedures as quickly as possible while maintaining precision and avoiding unnecessary haste.
Minimizing the time vessels remain open to reduce exposure to airborne contaminants.
Conducting all open-vessel work close to a Bunsen burner flame to take advantage of the upward airflow, which helps draw air currents away from the working area.
When opening a test tube or bottle, immediately flame the neck of the vessel while holding it nearly horizontal to ensure air movement flows outward, preventing contaminants from entering.
During manipulations involving Petri dishes, limit exposure of sterile inner surfaces to the surrounding air.
Avoid touching or allowing sterile parts of pipettes—those that will enter cultures or sterile vessels—to come into contact with non-sterile surfaces such as clothing, the working bench, or the exterior of bottles and test tubes.
Ensure that all items coming into contact with microorganisms are sterilized both before and after each exposure.
Sterilization can be performed either by the technical team during preparation and cleanup (for instance, sterilizing glassware before use) or by the worker during the experiment itself (for example, flaming a wire loop before and after inoculation).
Specific Aseptic Techniques
Sterile Handling
Always wipe your hands and work area with 70% ethanol before starting any procedure.
Wearing gloves is recommended to prevent foreign contaminants from coming into contact with samples or personnel during testing.
If gloves are not used, hands must be washed thoroughly before and after testing.
Wipe the outside surfaces of containers, flasks, plates, and dishes with 70% ethanol before placing them inside the cell culture hood.
Avoid pouring media or reagents directly from bottles or flasks.
Use sterile glass or disposable plastic pipettes with a pipettor for liquid handling.
Each pipette should be used only once to prevent cross-contamination.
Do not unwrap sterile pipettes until they are ready to be used, and keep them within the immediate work area.
Always cap bottles and flasks after use.
Seal multi-well plates with tape or place them in resealable bags to prevent microbial or airborne contamination.
Never uncover sterile items (flasks, bottles, Petri dishes, etc.) until the moment they are needed, and never leave them open to the environment.
Replace the cover immediately after use.
If a cap or cover must be placed on the work surface, position it with the opening facing downward.
Use only sterile glassware and sterile equipment during all procedures.
Avoid talking, singing, or whistling while performing sterile tasks.
Carry out all experiments rapidly but carefully to reduce exposure time and minimize contamination risk.
Inoculating agar plates, slopes and cultures
Perform culture transfers as quickly as possible, keeping tubes and plates open for the minimum time.
Open agar plates away from your body without completely removing the lid from the base.
When the Petri dish lid must be removed for a longer time, work very close to a Bunsen burner flame to minimize contamination risk.
If contamination by fungal spores is frequent, reduce drafts further and consider inoculating plates from below, with the agar surface facing downwards, to limit exposure to airborne spores.
Using a wire loop
Hold the wire loop handle close to the top, similar to holding a pen, at an almost vertical angle.
Keep the little finger free to handle the screw cap or cotton wool plug of a bottle or test tube.
Sterilize the wire loop by heating it to red hot in a roaring blue Bunsen burner flame before and after each use.
This sterilization step destroys bacterial spores and prevents contamination.
Heat the loop gradually, as rapid heating of liquid culture residues can cause splattering or aerosol formation.
Position the handle end of the loop in the light blue cone of the flame (the coolest part).
Slowly move the wire upward into the hottest region just above the blue cone until it becomes red hot.
Ensure the entire wire is properly sterilized by heating along its length.
Allow the loop to cool in the air for a few seconds before use—do not wave it or place it down.
After use, re-sterilize the loop immediately by reheating it to red hot.
Using a pipette
Use sterile graduated or Pasteur pipettes for transferring cultures, sterile media, or sterile solutions.
Remove the pipette from its container or wrapper by the plugged end, touching only what is necessary for a firm grip.
Attach the teat to the pipette; dipping it into sterile liquid first can help lubricate it.
Hold the pipette like a pen, keeping the little finger free for handling caps or plugs and the thumb for controlling suction.
Press the teat gently to draw up the required amount of liquid, ensuring it does not reach or wet the cotton wool plug.
Avoid forming air bubbles by squeezing the teat before immersing the tip into the liquid and then releasing pressure gently to draw up liquid.
Dispense excess liquid gently to prevent splashing.
After use, immediately place the contaminated pipette into a discard pot containing disinfectant.
Remove the teat only once the pipette is inside the disinfectant pot to prevent contamination of the workspace.
Return any excess gently.
Immediately after use put the contaminated pipette into a nearby discard pot of disinfectant.
Remove the teat only once the pipette is within the discard pot otherwise drops of culture will contaminate the working surface.
Flaming the neck of bottles and test tubes
Flaming the neck prevents microorganisms from entering and contaminating the culture or medium by creating an upward convection current.
The hot zone of the flame lies just above the inner bright blue cone; the vessel should be passed through this area, not held stationary.
Loosen the cap of the bottle before starting, making it easy to remove.
Hold the bottle or test tube with the left hand.
Use the little finger of your right hand to remove the cap or cotton wool plug by turning the bottle, not the cap.
Do not place the cap or plug down during the procedure.
Pass the mouth of the vessel quickly back and forth through the flame.
After completing the required task (e.g., withdrawing or adding culture), replace the cap or plug using your little finger.
Be cautious as the neck of the bottle will be hot.
If cotton plugs have lost shape, guide them gently into the neck by twisting the vessel mouth as you insert the plug.
Disinfecting surfaces
Ethanol disinfection is recommended due to its rapid action, typically requiring around 5 minutes.
Technicians should handle ethanol with care, as it is flammable.
Experienced personnel can safely manage ethanol use while ensuring effective disinfection and maintaining aseptic conditions.
Tools Used for Maintaining Aseptic Conditions
The Bunsen Burner
One of the simplest and most effective tools for creating a relatively sterile environment on a laboratory bench is the gas-powered Bunsen burner.
This common laboratory instrument burns a continuous stream of flammable gas, usually natural gas (methane), and is based on a design created nearly 150 years ago by the German chemist Robert Wilhelm Bunsen (1811–1899).
The open flame serves a key role in aseptic technique by generating a cone of hot air above and around the workspace.
This rising air current reduces the number of viable microorganisms carried by suspended dust particles, helping maintain a cleaner working environment.
The intense heat of the Bunsen burner also makes it ideal for:
Rapidly sterilizing inoculating loops and needles.
Flaming and warming the necks of glass bottles and tubes.
Igniting ethanol or other alcohol-based disinfectants on spreaders used for culturing microorganisms.
However, a Bunsen burner is not suitable for all laboratory settings.
It should not be used inside a laminar flow unit, as the flame’s heat can disrupt the airflow pattern that maintains sterility.
In such cases, a microincinerator can serve as an alternative to the Bunsen burner.
A microincinerator consists of a circular or tubular heating element; inserting an inoculating loop or needle into this ring quickly heats and sterilizes it.
While a microincinerator effectively sterilizes tools, it does not provide the additional aseptic benefits (such as upward airflow and dust reduction) that an open Bunsen burner flame offers.
The Laminar Flow Unit
A laminar flow unit (or laminar flow hood) is a highly advanced apparatus designed to prevent contamination of biological cultures and reagents.
When operated correctly, it provides a controlled work environment with clean, ultrafiltered air, protecting both the sample and the user from airborne contaminants.
The hood functions by ensuring that room air does not enter the work area, while also suspending and removing airborne particles introduced by personnel during work.
The most critical component of a laminar flow hood is the HEPA (High-Efficiency Particulate Air) filter, which is a high-efficiency, bacteria-retentive filter.
A certified HEPA filter must capture at least 99.97% of dust, pollen, mold, bacteria, and other airborne particles larger than 0.3 μm at a flow rate of 85 liters per minute.
HEPA filter technology originated in the 1940s, developed by the U.S. Atomic Energy Commission as part of the Manhattan Project (the development of the atomic bomb).
These filters were initially designed to trap and remove radioactive particulate contaminants.
After World War II, the technology was declassified, leading to widespread research and commercial applications across various scientific fields.
Laminar flow hoods are now essential in biosafety level 2 (BSL-2) laboratories, where they play a vital role in preventing the spread of viruses and certain pathogenic bacteria, ensuring safe and aseptic handling of biological materials.
References
Faculty of Food Technology and Biotechnology, University of Zagreb. Aseptic Technique. Available at: pbf.unizg.hr
Nuffield Foundation. Aseptic Techniques – Practical Biology. Available at: www.nuffieldfoundation.org/practical-biology/aseptic-techniques
Hach Company. Microbiology Guide: Introduction to Aseptic Technique. Available at: support.hach.com
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