TCC techniques-1: Basic Aseptic Rules CEAC514 Assoc. Prof. Dr. Yasemin G. İşgör

Contamination may ocur, it can be minor and confined to one or two cultures Such a minor Contamination, however, can spread among several cultures and compromise a whole experiment, or can be widespread and wipe out your (or even the whole laboratory’s) entire stock. Correct aseptic technique should provide a barrier between microorganisms in the environment outside the culture and the pure, uncontaminated culture within its flask or dish. 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 respects (with air-conditioning and filtration, laminar-flow facilities, etc.), the modern laboratory is often more crowded, and facilities may have to be shared.

ELEMENTS OF ASEPTIC ENVIRONMENT Quiet Area The laminar flow cabinet or sterile room is required. The quiet area should be less crowded, be easily cleaned, apart from waste material storage, and should contain special air filters (HEPA) to block pathogens. The laminar flow cabinet (TCC hood) must be free from air currents from doors, windows, etc.; the area should also have no through traffic (except a few researchers, no other people should move around) no equipment that generates air currents (e.g., centrifuges, refrigerators, and freezers; air conditioners should be positioned so that air currents do not compromise the functioning of the hood Nonsterile activities, such as sample processing, staining, or extractions, should be carried out elsewhere

While workin at TCC Lab ALWAYS Be Sterile! Always wash your hands before and after your work Never talk through sterile material Use 70% ethanol to Spray on surfaces and wipe (just soft touch with tissue paper) Spray on containers (bags, glass jars, etc.) containing sterile material before bringing into sterile hoods and leave there till alcohol evaporates or wipe in the hood softly Autoclave distilled water to yield sterile water Use sterile filter units to sterilize complete media and drugs extracts etc (to be in contact with cells) Refer to Carolina’s “Plant Tissue Culture Definitions and Techniques” packet

Protective equipments: protects you and your work Always wear gloves, before entering the hood spray with 70% alcohol Always wear lab coat, wach your sleeves that do not touch surfaces Preferentially elastic band wrist lab coats are safer than the regular ones For long hairs make pony tail, preferentially wear sterile hair cap For beards be aware your beard can collect dusts and pathogens from air, for protecting sterility in hood and lab, please wear mask, For scarfs, the edges should be under the lab coat, tha sides should not close your visiual range Please dress such that your skirts and pants do not touch to ground while bending, walking or sitting. If availabe, bring clean clothes for tissue culture lab for sterile room use.

Work Surface Start with a completely clear surface. Swab the surface liberally with 70% alcohol Bring onto the surface only those items you require for a particular procedure. Remove everything that is not required, and swab the surface down between procedures. Arrange your work area so that you have easy Access to all items without having to reach over one to get at another a wide, clear space in the center of the bench (not just the front edge!) to work on. Never close the air flow line with labwares, note paper or pen etc.

Basic application rules Wipe down equipment and lab areas with 70% ethanol Minimize airflow- keep door closed Turn on sterile hoods about 20 minutes before experiment and clean with 70% ETOH Wash hands and arms, but don’t scrub too hard- do not irritate your skin. Spray outside of gloves and packages to enter the sterile hoods with 70% ETOH

Humidified incubators are a major source of contamination. They should be cleaned out at regular intervals (weekly or monthly, depending on the level of atmospheric contamination and frequency of access) For cleaning, remove all the contents, including all the racks or trays, and spray down or wipe down the interior surfaces and wash the the racks or shelves with a nontoxic detergent such as dilute soft soap. Traces of detergent should then be removed with 70% alcohol, then allow to evaporate completely before replacingthe shelves and cultures.

Waste Bins are also major source of contamination. They should be emptied regularly Wastebins should be cleaned with sofsoap and rinse with 70% ethanol Wastebins should contain biohazard trash bags, red in color, and while disposing to specific biohazard waste collection sites, the bag should be closed and mouth will be tightly closed and wrapped with appropriate bag strings.