Sterile Technique
Good sterile technique is the firstand most important step in insuring consistent results whenemploying recombinant DNA and protein expression techniques.Sterile technique refers to procedures by which cultures may bemanipulated without infecting the worker or contaminating thecultures or the laboratory environment.
Because contaminatingbacteria are ubiquitous and are found on fingertips, bench tops,etc., it is important to minimize contact with these contaminatingsurfaces. When students are working with the inoculation loops andagar plates, you should stress that the round circle at the end ofthe loop, the tip of the pipetter, and the surface of the agarplate should not be touched or placed onto contaminatingsurfaces.
The flaming of lips of tubes and flasks must ALWAYS be donewhenever culture liquid is to be poured from a container (e.g.,pouring plates). Flaming should be routinely done when caps areremoved from tubes during transfer of cultures. The purpose offlaming is not to sterilize, but to warm the tube and create warmair convection currents up and away from the opening. This"umbrella" of warm, rising air will help to prevent the entrance ofdust particles upon which contaminating bacteria reside.
Petri dish lids prevent dust from falling directly onto plates butallow diffusion of air around the edges. There are no direct aircurrents into the plate, and to enter, dust particles would have torise vertically more than a centimeter. This does not often occurbecause of the density of the particles. Whenever the lid isremoved, it should be held over the plate as a shield. Do not placethe lid on the bench top. Do not leave plates uncovered. Do notwalk around the room with an open plate.
When working with cultures in testtubes, work as rapidly as isconsistent with careful technique. Keep the tubes open a minimumamount of time. While the tubes are open, hold them at a 45 degreeangle so that dust cannot fall into the open tube. Hold the tubesaway from your face while transferring.
Testtubes are handled in the following manner:
The testtube is held in the left hand (for a right-handed person).
The instrument (loop, pipet, or needle) is held in the right hand.
The testtube cap is grasped by the little finger of the right hand,and removed.
While continuing to hold the cap with the little finger, the tubeis lightly flamed and the instrument is manipulated appropriately,and withdrawn.
The cap is replaced on the testtube and the testtube is put backinto the rack.
Label all cultures with the name or number of the organism, andyour name.
Always clean all work areas (your bench, balance area, sink area,gel area, etc.) thoroughly before leaving the laboratory! The laststep before leaving the lab is to wash your hands thoroughly.
These are guidelines. You may find a set of techniques that bestsuite your working style. This is fine as long as you adhere to thebasic concepts of good sterile technique.