Good sterile technique is the first and most important
step in insuring consistent results when employing
recombinant DNA and protein expression techniques.
Sterile technique refers to procedures by which cultures
may be manipulated without infecting the worker or
contaminating the cultures or the laboratory environment.
Because contaminating bacteria are ubiquitous and are
found on fingertips, bench tops, etc., it is important
to minimize contact with these contaminating surfaces.
When students are working with the inoculation loops
and agar plates, you should stress that the round circle
at the end of the loop, the tip of the pipetter, and
the surface of the agar plate should not be touched
or placed onto contaminating surfaces.
The flaming of lips of tubes and flasks must ALWAYS
be done whenever culture liquid is to be poured from
a container (e.g., pouring plates). Flaming should
be routinely done when caps are removed from tubes
during transfer of cultures. The purpose of flaming
is not to sterilize, but to warm the tube and create
warm air convection currents up and away from the opening.
This "umbrella" of warm, rising air will
help to prevent the entrance of dust particles upon
which contaminating bacteria reside.
Petri dish lids prevent dust from falling directly onto
plates but allow diffusion of air around the edges.
There are no direct air currents into the plate, and
to enter, dust particles would have to rise vertically
more than a centimeter. This does not often occur because
of the density of the particles. Whenever the lid is
removed, it should be held over the plate as a shield.
Do not place the lid on the bench top. Do not leave
plates uncovered. Do not walk around the room with
an open plate.
When working with cultures in testtubes, work as rapidly
as is consistent with careful technique. Keep the tubes
open a minimum amount of time. While the tubes are
open, hold them at a 45 degree angle so that dust cannot
fall into the open tube. Hold the tubes away from your
face while transferring.
Testtubes are handled in the following manner:
Label all cultures with the name or number of the organism,
and your name.
- The testtube is held in the left hand (for a right-handed
- 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 tube is lightly flamed and the instrument
is manipulated appropriately, and withdrawn.
- The cap is replaced on the testtube and the testtube
is put back into the rack.
Always clean all work areas (your bench, balance area,
sink area, gel area, etc.) thoroughly before leaving
the laboratory! The last step before leaving the lab
is to wash your hands thoroughly.
These are guidelines. You may find a set of techniques
that best suite your working style. This is fine as
long as you adhere to the basic concepts of good sterile