successful cell culture depends heavily onkeeping the cells free from microorganism contamination. this can be accomplished withproper knowledge of sterile environments, working slowly and deliberately, and followingall of the guidelines for sterile technique. always wear basic personal protective equipment,or ppe, when working in the laboratory. talk to the safety team at your institute for yourrequired ppe. also, remember to review the msds information before working with any mediaor reagents. wear closed toe shoes, and clothes that coveryour legs. washing your hands before handling cells orperforming culture work removes bacteria and microscopic dead skin particles. dead skincan be a potential source of contamination.
a 70% ethanol wash kills microorganisms thatcould contaminate the cultures. cleanliness is one of the most important parts of steriletechnique. always clean the hood before and after use. spray alcohol should not be usedin any area where a flame is being used, due to the fire hazard.the outside of containers carry dust and contaminants. remember to clean each item placed in thehood with ethanol. you may choose to spray the item before placing it in the hood, orimmediately after as demonstrated. as you clean the item and place into the cell culturehood, put it in the correct position. enter the hood with your hands in a forward motion,try not to sweep across the front and disrupt the air flow.proper set-up of the cell culture hood includes
not over-crowding the work surface. not onlydoes this increase the risk of contamination through accidental touching, but it also interruptsthe air flow through the chamber, which will not maintain the sterile field. for this samereason, it is important to keep the front sash in the lower position when working.the basic idea is to keep your media and reagents sterile by only touching them with sterileobjects. using sterile media, reagents and supplies is a big step in keeping your culturesfree from contamination. notice how the cell culture hood is set up to make the followingactions easier. the pipette aid is on the right side so you can easily control the pipettingin your right hand. reagents are in the center back so you can easily open the bottles andpipette from them. it is very important to
have the set up in this manner, so you arenot crossing your hands or supplies over top of sterile items. any movement over the topof a sterile item can result in contamination. we’ll now demonstrate the basic techniqueof handling a pipette and a bottle of medium while aseptically supplementing dmem withglutamax. when opening the pipette package, be surethe pipette does not touch anything non-sterile. grasp the pipette high on the neck, insertthe pipette into the pipette aid, turn the desired measurement marks toward you and thendiscard the wrapper. it may take some practice, but it is a very important first step. discardany pipette you accidentally contaminate. only open your media, reagents, and suppliesin the cell culture hood. opening these items
outside the sterile field will result in contamination.when holding the cap, it is important not to touch the inside edge or you could contaminateit. replace the cap as soon as possible, in order to decrease your risk of contamination.if you must set the cap down in order to free your hand, set it down with the interior surfacefacing down. when pipetting, try not to touch the pipetteto anything non-sterile, particularly the outside of containers, or contamination couldresult. each pipette should be used one time and discarded (or washed if using glass pipettes).reusing pipettes by dipping directly into another media bottle, or by leaving it standingin a media bottle, increases your chances of spreading contamination.gently mix the contents after supplementation.
labeling the bottle after supplementationis good laboratory practice. sometimes you will need to transfer largevolumes of liquid from one container to another. the best way to transfer is always to usea sterile pipette, but with practice, it is possible to aseptically pour instead. youshould pour quickly and deliberately, using the front corner of the bottle to channelthe liquid and improve the speed and accuracy of your pour. any spills should be wiped withethanol immediately. when passaging cells or changing their medium,the basics of aseptic technique are the same: only open the containers in the sterile field,do not cross your arms or other items over an open flask, don’t rush (but work at agood pace and with deliberate motions), do
not re-use pipettes, and do not use itemsthat were inadvertently contaminated. when finished, make sure everything is closedtightly before removing from the cell culture hood. anything opened outside the sterilefield will now be non-sterile and should not be used for cell culture work. wipe down thework surface with ethanol again and straighten up the hood before you leave.
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