WEEK 3 BIO 253L Drs. Hengartner & Vega
Finish Last Week's Exercise (Simple Stains: Positive and Negative Stains & Differential and
Special Stains)
If you ran out of time last week, examine the stained slides you prepared and record observations
about cell shape, arrangement, and color.
Pure Culture and Aseptic Technique
The two goals of aseptic (or sterile) techniques:
1) Safety: prevent the microbes you are working with from contaminating you, the
environment, or others.
2) The experiment: prevent contamination of your culture with unwanted organisms from
the environment
Note: The meaning of the words aseptic and sterile are slightly different, but both words are used
to describe the techniques you are learning today. See word definitions below.
Loop Inoculation
Loop inoculation and tube handling basics: Fundamental microbiology lab skills you need to
master.
1. Hold loop like pencil, flame loop starting at base and slowly move toward the end (make
sure all heated parts
become a uniform red-
orange). Allow wire to
cool for a few seconds
2. Remove tube cap with the
little finger of the hand
holding loop
3. Briefly flame tube lip by
passing through flames
4. Harvest sample, take care
loop does not touch lip of
the tube on way in or out
(a bit of broth should
remain trapped in loop)
5. Flame tube lip again, place
cap, set tube down
6. You can now inoculate a
fresh media (transfer the
inoculum to fresh media to
start a culture)
a. If inoculating a
liquid media or agar slant in a tube, open tube as described above, flame lip, and
insert loop without touching the lip of the tube, flame lip again, cap tube. After
inoculating broth tubes, touch the loop tip to side of glass to remove liquid
trapped in loop (this prevents aerosol effect when you flame the loop afterwards).
Avoid touching the loop to the lip of the tube.
b. If inoculating an agar plate, tilt lid of plate open, using it as a shield to minimize
airborne contaminants falling in. Use zig-zag pattern to spread bacteria from
loop onto plate.
7. Always flame loop after inoculation to avoid contamination of the work space.
