In this cell culture contamination aseptic technique post we have briefly explained about aseptic tissue culture techniques, elements of aseptic tissue culture techniques, sterile handling, and vertical laminar flow.
Aseptic Culture Techniques
Microorganism contamination is still a big issue in tissue culture. Bacteria, mycoplasma, yeast, and fungal spores can enter the system through the operator, the environment, work surfaces, solutions, and a variety of other sources. By establishing a tight code of practise and ensuring that everyone who uses the facility follows it, aseptic tissue culture techniques tries to prevent infection.
Why Aseptic Technique?
Maintaining sterility correct aseptic tissue culture techniques technique should provide a barrier between microorganisms in the environment outside the culture and the pure, uncontaminated culture within its flask or dish. Hence all materials that will come into direct contact with the culture must be sterile, and manipulations must be designed such that there is no direct link between the culture and its nonsterile surroundings.
Although laboratory conditions have improved in some ways (with air conditioning and filtration, laminar-flow facilities, and so on), they are typically more crowded, and accommodations may have to be shared.
Cell Culture Contamination Aseptic Technique
Elements of Aseptic Technique
Antibiotics, laminar-flow cabinets, and filtered air-conditioning have all improved the conditions for producing a clean space for cell growth throughout the years. Antibiotics should not be used indefinitely because clean room air combined with laminar flow has made achieving and maintaining an aseptic tissue culture techniques makes cell culture easier and more dependable.
A constant, consistent flow of filtered air passing over the work surface protects the work space from dust and pollution, which is a primary benefit of working in a laminar-flow hood.
There are two types of flow: (1) horizontal, where airflow blows from the side facing you, parallel to the work surface, and is not recirculated, and (2) vertical, where air blows down from the top of the hood onto the work surface, is drawn through the work surface, and is either recirculated or vented.
In the absence of a laminar-flow cabinet, sterile work should be performed in a separate sterile room. If this isn’t possible, choose a quiet spot of the lab with little or no traffic and no other activity. Make sure the location is clear of draughts from doors, windows, and other sources. There should be no through traffic and no equipment that produces air currents (e.g., centrifuges, refrigerators, and freezers); air conditioners and exhaust manifolds should be placed so that effluent air does not interfere with the hood’s operation.
Swab the surface liberally with 70% alcohol to get a fully clear surface. Bring only the objects you need for a certain procedure onto the surface, and remove everything that isn’t needed between processes and swab the surface clean.
Arrange your work area so that you have (a) easy access to all products without having to reach over one to get at another and (b) a broad, clear surface in the centre of the bench to work on. Allowing your hands or any other nonsterile materials (even the outside of a flask) to pass over an open flask or dish is not recommended.
Immediately mop up any spills and wipe the surface with 70% alcohol. When you’re done, take everything off the work surface and swab it clean. Make sure the area beneath the work surface is wiped up at least once a week.
There has been much discussion about whether hand washing encourages or reduces the bacterial count on the skin. Regardless of this debate, washing will moisten the hands and remove dry skin that would otherwise be likely to blow onto your culture. Washing will also reduce loosely adherent microorganisms, which are the greatest risk to your culture.
Caps, gowns, and face masks are required under good manufacturing practice (GMP) [Food and Drug Administration, 2007; Rules and Guidance for Pharmaceutical Manufacturers and Distributors, 2007] conditions but are not necessary under normal conditions, particularly when working with laminar flow.
Commercially procured reagents and media have previously been subjected to stringent quality control to guarantee that they are sterile, but the exterior surface of the bottle in which they are packaged is not.
Some manufacturers provide bottles that are wrapped with polyethylene, which keeps them clean and allows them to be warmed or thawed in a water bath. Outside the hood, the wrapping should be removed. When unwrapped bottles are removed from the refrigerator or a water bath, they should be swabbed in 70% alcohol.
Cultures brought in from another lab run the danger of being contaminated at the point of origin or while in transit. Imported cell lines should always be quarantined, which means they should be handled separately from the rest of your stocks and maintained antibiotic-free until proven uncontaminated. They may then be incorporated into your main stock. Antibiotics should not be used routinely as they may suppress, but not eliminate some contaminations and encourage poor technique.
Swab the work surface with 70% alcohol before and during work, especially after any spills, and then swab it clean when you’re done. Before utilising any bottles, especially those that have come from cold storage or a water bath, swab them, as well as any flasks or boxes from the incubator. Isopropyl alcohol can be used instead of ethanol or methanol and is available as a proprietary spray or as prepacked swabs.
Stoppers are recommended over deep screw caps, but when washing caps, make sure all detergent is drained out from behind rubber liners. As a result, disposables or wadless polypropylene caps should be used. Although the introduction of deep polypropylene closures (e.g., Duran) has made foil shrouding less required, the screw cap should be covered with aluminium foil to protect the neck of the bottle from sedimentary dust.
When working on an open bench, flame glass pipettes and the necks of bottles and screw caps before and after opening and closing a bottle, work close to the flame where there is an updraft due to convection, and do not leave bottles open. Screw caps should be placed with the open side down on a clean surface and flamed before being replaced on the bottle. Alternatively, screw caps may be held in the hand during pipetting, avoiding the need to flame them or lay them down.
Flaming is not advisable when you are working in a laminar-flow hood, as it disrupts the laminar flow, which in turn compromises both the sterility of the hood and its containment of any biohazardous material.
When working on an open bench, you should not keep bottles vertical when open; instead, keep them at an angle as shallow as possible without risking spillage. A bottle rack can be used to keep the bottles or flasks tilted. Culture flasks should be laid down horizontally when open and, like bottles, held at an angle during manipulations.
When you are working in laminar flow, bottles can be left open and vertical, but do not let your hands or any other items come between an open vessel or sterile pipette and the HEPA filter. Flasks with angled necks facilitate pipetting when the flask is lying flat.
Standard glass or disposable plastic pipettes are still the easiest way to manipulate liquids. Syringes are sometimes used but should be discouraged as regular needles are too short to reach into most bottles. Syringing can also produce high shearing forces when you are dispensing cells, and the practice also increases the risk of self-inoculation. Wide-bore cannulae are preferable to needles but still not as rapid to use, except with multiple-stepping or repeating dispensers.
Do not pour from one sterile container into another, unless the bottle you are pouring from is to be used once only to deliver all its contents (premeasured) in one single delivery. The major risk in pouring lies in the generation of a bridge of liquid between the outside of the bottle and the inside, permitting contamination to enter the bottle during storage or incubation.
Vertical Laminar Flow
- Swab down the work surface and all other inside surfaces of laminar-flow hood, including inside of the front screen, with alcohol and a lint-free swab or tissue.
- Bring medium and reagents from cold store, water bath, or otherwise thawed from freezer; swab bottles with alcohol, and place those that you will need first in the hood.
- Collect pipettes, and place at one side of the back of work surface in an accessible position.
- Open pipette cans and place lids on top or alongside, with the open side down, or stack individually wrapped pipettes, sorted by size, on a rack or in cans.
- Collect any other glassware, plastics, and instruments that you will need, and place them close by.
- Slacken, but do not remove, caps of all bottles about to be used.
- Remove the cap of the flask into which you are about to pipette, and the bottles that you wish to pipette from, and place the caps open side uppermost on the work surface, at the back of the hood and behind the bottle, so that your hand will not pass over them.
- The pipette in the bulb or pipette controller will now be at right angles to your arm. Take care that the tip of the pipette does not touch the outside of a bottle or the inner surface of the hood, always be aware of where the pipette is.
- Tilt the medium bottle toward the pipette so that your hand does not come over the open neck, withdraw 5 mL of medium, and transfer it a flask, also tilted.
- Discard the pipette into the pipette cylinder containing disinfectant. Plastic pipettes can be discarded into double-thickness autoclavable biohazard bags.
- Recap the flask.
- Replace the cap on the medium bottle and flasks. Bottles may be left open while you complete a particular manoeuvre, but should always be closed if you leave the hood for any reason.
- On completion of the operation, tighten all caps, and place flasks in incubator.
- Remove all solutions and materials no longer required from the work surface, and swab down.
- Pauly’s Diazo Test for Amino Acids
- Sakaguchi Test for Arginine
- Xanthoproteic Test for Amino Acids
- Acid Hematin Method of Hemoglobin Determination
- Red Blood Cell Count Manual Method
- Anthrone Test for Carbohydrates
- Barfoed’s Test for Monosaccharides
- Benedict’s Reagent Test for Reducing Sugars
- Bial’s test for Carbohydrates
- Estimation of Reducing Sugar by DNS Method
- Aseptic Tissue Culture Techniques | Thermo Fisher Scientific – IN
- Aseptic Tissue Culture Techniques.pdf (cpp.edu)
- Aseptic Tissue Culture Techniques for cell culture – PubMed (nih.gov)
- Aseptic Tissue Culture Techniques| Thermo Fisher Scientific – IN
- Aseptic Tissue Culture Techniques guidelines | protocol | Abcam