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In our local environments, microorganisms are ubiquitous. They are in the air, on our skin, on the benchtop, and on every other surface.
This is problematic when working in microbiology lab because we generally want to work with pure cultures (cultures that contain one
and only one species of microorganism). In the mid-1800s, Louis Pasteur developed the techniques of aseptic transfer to minimize the
risk of culture contamination (and the risk of making oneself sick). In today’s lab we will learn the steps in aseptic transfer.
CAUTION: Lids on test tubes are loose. Always hold the glass test tube (not the
lid) when carrying them.
Stock Culture
(contains cells)
Sterile Medium
Bunsen Burner
Blue Cone = hottest
part of flame
Inoculating Loop
Plastic or rubber portion of handle Wire loop Metal portion of handle
Test Tube Rack
Step 2. Grasp both the stock culture and the sterile medium in one hand and the
inoculating loop in the other.
a. Loosen, but don’t remove the caps on the tubes.
b. Hold the inoculating loop at the end of the rubber/plastic portion of the
handle.
Step 3. Stick the loop into the flame until it becomes RED HOT. Flame the entire wire
loop and about half of the metal portion of the handle (because part of the metal
portion will extend into the test tubes).
Step 4. Remove the caps and hold them in the same hand as the loop.
a. To avoid contamination of the caps:
i. don’t set them down on the benchtop;
ii. don’t touch the bottom (opening) of the caps;
iii. try to keep the caps pointed downwards (so that airborne
contaminants don’t fall into them).
The loop should be put into the flame along the edge of the blue cone. This uses the hottest part of the flame and sterilizes.
Step 5. Flame the mouths of the test tubes.
The mouths may be contaminated. We don’t want to drag the loop across the mouth of a tube and contaminate our culture.
Step 6. Cool the loop by tapping it on the sterile agar or in the sterile broth. Cooling is
instantaneous.
Step 7. Insert the loop into the stock culture to obtain [a small amount of] cells. You
don’t need to see cells on the loop to have enough. Transfer cells to the sterile
medium. This should be very quick.
Step 8. Re-flame the mouths of the tubes. Then put the caps back on.
DAY 1 ACTIVITIES
Broth cultures are useful because very high cell densities can be achieved. Also, some
characteristics, like oxygen requirements, can be observed. Broth cultures are
problematic because it is difficult to determine if contamination has occurred (because all
the cells mix together in a broth).
1. Use a small piece of labeling tape to make a label for your broth culture.
Place both your newly inoculated broth and slant cultures in the class test tube rack in the front of the lab (near the overhead
projector).
Day 2 Observations
1. Observe your S. marcescens broth culture. If your aseptic transfer technique was
successful you should see:
a. a faint pink or orange coloration (especially in the meniscus of the broth);
b. light turbidity (cloudiness) throughout the broth;
c. a pink sediment at the bottom of the test tube.
2. Observe your S. marcescens agar slant culture. If your aseptic technique was
successful, you should see:
a. uniform pink growth on the slanted surface;
b. growth over the majority of the slanted surface (good coverage).
*****inoculatingloop- for streaking bacteria onto plates, and can be used to pick colonies to start
liquid cultures.