Lab Module 2: Aseptic Transfers
INTRODUCTION
In our local environments, microorganisms are ubiquitous. They are in the air, on our skin, on the
lab benches, and on every other surface. This is problematic when working in a 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 making
aseptic transfers to minimize the risk of culture contamination (and the risk of making oneself
sick). In today’s lab we will learn the steps used to make an aseptic transfer.
The steps used in making an aseptic transfer:
When culturing microbes, we must assume that everything (except the inside of sterile culture
media) is contaminated. In order to inoculate (transfer cells to) new sterile media, we must make
sure that we sterilize everything that can potentially contaminate the sterile media.
Step 1. Necessary equipment is shown in the picture below. Note: a bacterial incinerator may be
used instead of a Bunsen burner. Ours requires approximately 10 minutes to warm up, and
should appear red hot inside. The loop should be inserted into the incinerator for about
10-15 seconds (if you keep it in too long, the loop will break).
Stock culture = already contains cells. It is used as a source of cells from which to inoculate new
sterile media.
CAUTION:
Lids on test tubes are loose. Always firmly grasp the glass test tube (not the lid)
when moving the test tubes. TRANSPORT TEST TUBES IN A TUBE RACK, NOT INDIVIDUALLY.
Stock Culture
(contains cells)
Blue Cone = hottest
part of flame
Test Tube Rack
Sterile Medium Bunsen Burner
Bacticinerator
Plastic or rubber portion of handle
Metal portion of handle
Wire loop
(imagesource
www.amazon.com)
Inoculating Loop
1
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. BUNSEN: 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).
BACTICINERATOR: insert the inoculating loop all the way into the hole and leave for 1015 sec.
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 the loop.
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 bench;
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).
2
Step 5.
BUNSEN: Flame the mouths of the test tubes.
BACTICINERATOR: wave the test tube mouths immediately above the hole for 2-3 sec.
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 by submerging it 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. Repeat step 5. Then put the caps back on.
Step 9. Re-flame/re-incinerate the inoculating loop.
DAY ONE ACTIVITIES
Today, EACH PERSON will complete the following activities.
Materials needed:
Test tube rack
Bacterial incinerator
Inoculating loop
Two empty test tubes
One set per pair of students
Stock culture of Serratia marcescens (labeled S. marcescens) [Shared among the whole class].
One sterile Nutrient Broth with Yeast Extract (NBYE)
One sterile NAYE slant
One set FOR EACH STUDENT
3
Activity 1: Dry run with empty test tubes.
1. Using two empty test tubes, run through the nine steps listed above.
a. Pretend that one empty test tube is the stock culture and the other tube is the
sterile medium.
b. Your partner will critique your technique.
Activity 2: Transfer from stock culture to a sterile broth.
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. Remember, the
label should have:
a. Your name.
b. The date.
c. The type of medium (NBYE in this case).
d. The incubation temperature (30C in this case).
e. The inoculum (S. marcescens in this case).
2. Stick the label on the NBYE test tube (ensure it does not cover the liquid).
3. Perform the aseptic transfer of cells from the S. marcescens stock to the sterile NBYE tube.
Activity 3: Transfer from stock culture to a sterile agar slant.
Agar slant cultures are useful because high cell densities can be grown (as in a broth culture).
Contamination can be more easily detected than in a broth. Agar slants are better than agar
plates for maintaining long-term cultures because the small mouth of the test tube minimizes risk
of airborne contamination.
1. Use a small piece of labeling tape to make a label for your agar slant culture.
Remember, the label should have:
a. Your name.
b. The date.
c. The type of medium (NAYE in this case).
d. The incubation temperature (30C in this case).
e. The inoculum (S. marcescens in this case).
2. Stick the label on the NAYE test tube (ensure it does not cover the liquid).
3. Perform the aseptic transfer of cells from the S. marcescens stock to the sterile
NAYE slant. The entire slanted area should be inoculated from bottom to top in
a zigzag motion.
Place both your newly inoculated broth and slant cultures in the class test tube rack in the front
of the lab.
4
DAY TWO 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.
d. You should NOT see:
i. a pellicle (a solid cap of cells on top of the broth);
ii. flocculent material (floaties).
2. Observe your S. marcescens agar slant culture.
a. If your aseptic technique was successful, you should see:
i. uniform pink growth on the slanted surface (perhaps with cream-colored
edges);
ii. growth over the majority of the slanted surface (good coverage).
b. You should NOT see other colors of growth (except a possible cream coloration on
the edges of the growth).
QUESTIONS
1. There’s an old saying, “slow and steady wins the race.” Why is this not necessarily true when
performing aseptic transfers?
2. What is a stock culture?
3. What is a pure culture? A mixed culture?
4. Give two reasons for using aseptic technique.
5. List possible sources of contamination that you should be concerned about while transferring
bacteria from a stock culture to a sterile medium.
5