General Aseptic Techniques in Microbiology Laboratory

- Aseptic technique is a set of routine measures that are taken to prevent cultures, sterile media stocks, and other solutions from being contaminated by unwanted microorganisms (i.e., sepsis).

- While such actions are sometimes called “sterile technique,” that terminology is appropriate only in reference to preventing the introduction of any organisms to the laboratory or medical equipment and reagents (e.g., during surgery).

- Since the goal of a biologist is to grow microorganisms or eukaryotic cells without the introduction of extraneous organisms, aseptic techniques are crucial for accurate and meaningful experimentation.

- One should always remember that a completely sterile working environment does not exist.

- However, there are a number of simple, common sense procedures that will reduce the risk of culture contaminations.

- The aseptic techniques control the opportunities for contamination of cultures by microorganisms from the environment, or contamination of the environment by the microorganisms being handled.

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Examples of aseptic technique are:

- Cleaning and disinfecting lab surfaces prior to use

- limiting the duration that cultures or media are uncapped and exposed to the air

- keeping petri dishes closed whenever possible

- effectively sterilizing inoculating loops and other equipment that comes into contact with cultures or media, and

- avoiding breathing on cultures or sterile instruments.

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There are some general rules to follow for any aseptic technique.

- Close windows and doors to reduce draughts and prevent sudden movements which might disturb the air.

- Make transfers over a disinfected surface. Ethanol disinfection is recommended because of its rapid action. If the bench surface is difficult to clean, cover the bench with a sheet of tough material which is more easily disinfected.

- Start the operations only when all apparatus and materials are within immediate reach.

- Complete all operations as quickly as possible, but without any hurry.

- Vessels must be open for the minimum amount of time possible.

- While vessels are open, all work must be done close to a Bunsen burner flame where air currents are drawn upwards.

- On opening a test tube or bottle, the neck must be immediately warmed by flaming (see below) with the vessel held as near to horizontal as possible and so that any movement of air is outwards from the vessel.

- During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to contamination from the air.

- The parts of sterile pipettes which will be put into cultures or sterile vessels must not be touched or allowed to come into contact with other non-sterile surfaces, such as clothing, the surface of the working area, or the outside of bottles/ test tubes.

- All items which come into contact with microorganisms must be sterilised before and after each such exposure. This could be either by the technical team preparing for and clearing up after a piece of practical work (for example, in the case of glassware to be used), or by the worker during the course of the practical (for example, in flaming a wire loop).

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Sterile Handling

- Always wipe your hands and work area with 70% ethanol.

- It is recommended to wear gloves. This will prevent any foreign contaminants from coming in contact with the customers and sample during testing. If gloves are not used, it is necessary to wash hands before and after testing.

- Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood.

- Avoid pouring media and reagents directly from bottles or flasks.

- Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be used. Keep pipettes at the work area.

- Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resalable bags to prevent microorganisms and airborne contaminants from gaining entry.

- Never uncover a sterile flask, bottle, Petri dish, etc. until the instant you are ready to use it and never leave it open to the environment. Return the cover as soon as you are finished.

- If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down.

- Use only sterile glassware and other equipment.

- Be careful not to talk, sing, or whistle while performing sterile procedures.

- Perform experiments as rapidly as possible to minimize contamination.

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Inoculating agar plates, slopes and cultures

- Carry out the transfer of cultures as quickly as possible, with tubes and plates open to the air for the minimum length of time.

- Normal practice is to open agar plates away from the body and without removing the lid completely from the base.

- In instances when the lid of the Petri dish may be removed for longer periods than normal, work very close to the Bunsen burner flame to reduce the chances of contamination.

- If you experience frequent contamination of plates with fungal spores, reduce the chance of draughts further, and consider inoculating plates from below with the agar surface facing downwards. In this way there is perhaps less chance of spores settling onto the plate from the air.

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Using a wire loop

- While using a wire loop, hold the handle of the wire loop close to the top, as you would hold a pen, at an angle that is almost vertical. This leaves the little finger free to take hold of the screw cap/ cotton wool plug of the bottle/ test tube. It also ensures that any liquid culture on the loop will run down into the flame.

- Sterilise a wire loop by heating to red hot in a roaring blue Bunsen burner flame before and after use. This ensures that contaminating bacterial spores are destroyed.

- The flaming procedure should heat the tip of the loop gradually. This is because after use it will contain culture, which may splutter on rapid heating and possibly release small particles of culture, forming an aerosol.

- Position the handle end of the wire in the light blue cone of the flame. This is the coolest area of the flame.

- Draw the rest of the wire upwards slowly into the hottest region of the flame – immediately above the blue cone.

- Hold there until it is red hot.

- Ensure the full length of the wire receives adequate heating.

- Allow to cool for a few seconds in the air, then use immediately.

- Do not put the loop down, or wave it around.

- Re-sterilise the loop immediately after use.

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Using a pipette

- Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and sterile solutions.

- Remove the pipette from its container/ wrapper by the end containing a cotton wool plug, taking care to touch as little of the pipette as you need to take a firm hold.

- Fit the teat. It is sometimes helpful to dip the teat first in sterile liquid to lubricate it.

- Hold the pipette barrel as you would a pen, but do not grasp the teat. This leaves your little finger free to take hold of the cap/ cotton wool plug of a bottle/ test tube and your thumb free to control the teat.

- Depress the teat cautiously and take up an amount of fluid which is adequate for the amount required, but does not reach and wet the cotton wool plug.

- Squeezing the teat with the pipette tip beneath the liquid surface introduces air bubbles which may cause ‘spitting’ and, consequently, aerosol formation. Avoid this by squeezing the teat before placing the tip into the liquid.

- Then gently release the pressure until the required amount of liquid is drawn up, and lift the pipette tip out of the liquid.

- 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.

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Flaming the neck of bottles and test tubes

This ensures that no microorganisms enter the mouth of the vessel to contaminate the culture or the medium. Passing the mouth of the bottle through a flame produces a convection current away from the opening, and helps to prevent contamination. The hot part of the flame is above the inner bright blue ‘cone’ and the vessel needs to be moved through the flame, not held in place.

- Loosen the cap of the bottle so that it can be removed easily.

- Lift the bottle/ test tube with your left hand.

- Remove the cap/ cotton wool plug of the bottle/ test tube with the little finger curled towards the palm of your right hand. (Turn the bottle, not the cap.)

- Do not put down the cap/ cotton wool plug.

- Flame the neck of the bottle/ test tube by passing the neck forwards and back through a hot Bunsen burner flame.

- After carrying out the procedure required, for example, withdrawing culture, replace the cap/ cotton wool plug on the bottle/ test tube using your little finger. Take care! The bottle will be hot. (Turn the bottle, not the cap.)

- If cotton wool plugs have partly lost their shape, they can be more easily guided back into the neck of the vessel by slowly twisting the mouth of the vessel as the plug is pushed down.

*Disinfecting surfaces*

- For technicians, ethanol disinfection is recommended because of its rapid action (around 5 minutes). Technicians will be more experienced and able to deal with the associated fire hazards of working with ethanol.

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The Bunsen Burner

- Probably the easiest way to create a relatively sterile environment on the laboratory bench is by using a simple gaspowered burner.

- This common piece of equipment burns a continuous stream of a flammable gas—usually natural gas (methane)—based upon a design made almost 150 years ago by the German chemist Robert Wilhelm Bunsen (1811-1899).

- A major purpose of the open flame in aseptic technique is to create a cone of hot air above and around the laboratory bench to reduce the viability of organisms on suspended dust particles.

- The ability of the Bunsen burner flame to heat things very quickly also makes it an ideal choice for sterilizing inoculating loops, warming glass bottle necks, or igniting alcohol on culture spreaders.

- A Bunsen burner is not practical in some situations, e.g., within a laminar flow unit where the heat will disrupt airflow.

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The Laminar Flow Unit

- A laminar flow unit (or hood) is a sophisticated appliance that can further help prevent contamination of reagents and biological cultures.

- Used correctly, it provides the work space with clean, ultrafiltered air.

- It also keeps room air from entering the work area and both suspends and removes airborne contaminants introduced into the work area by personnel.

- The most important part of a laminar flow hood is a high-efficiency bacteria-retentive filter, i.e., the HEPA (high-efficiency particulate air) filter.

- A certified HEPA filter must capture a minimum of 99.97% of dust, pollen, mold, bacteria, and any airborne particles with a size of >0.3 μm at 85 liters/min.

- The first HEPA filters were developed in the 1940s by the U.S.A.Atomic Energy Commission as part of the Manhattan Project (the development of the atomic bomb) to provide an efficient, effective way to filter radioactive particulate contaminants.

- HEPA filter technology was declassified after World War II, allowing extensive research and commercial use.

- Laminar flow hoods are essential components of many biosafety level (BSL)-2 laboratories, where they help prevent spread of viruses and some bacteria.

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References

1) pbf.unizg.hr/content/download/24827/96881/version/…/Aseptic+Technique.pdf 2)http://www.nuffieldfoundation.org/practical-biology/aseptic-techniques 3)https://support.hach.com/app/answers/answer_view/a_id/1020204/~/microbiology-guide%3A-introduction-to-aseptic-technique- 4)https://www.thermofisher.com/np/en/home/references/gibco-cell-culture-basics/aseptic-technique.html 5)http://www.austincc.edu/microbugz/aseptic_technique.php