LAB 10A – ASEPTIC TECHNIQUE

When working in a microbiology laboratory it is important to always remember that

microorganisms are ubiquitous. You cannot see them, but they are there by the billions  in the air,
on the bench top, on your skin, etc. When working with a culture of bacteria, you must take
precautions so as not to allow the culture to become contaminated with organisms from the
surrounding environment. It is equally important not to contaminate yourself or your surroundings
with the culture.

To avoid contamination of bacterial cultures and the surrounding environment we must use what
is called aseptic technique (also called sterile technique). Everything you use in lab will be sterilized
before touching microbial colonies, and everything should be sterilized (or properly disposed of)
after touching the microbes to avoid contaminating other surfaces or samples. At first the necessary
procedures seem awkward, but after practice, they will become simple and automatic. Mastering
good aseptic technique is very important because it will ensure reliable experimental results by
preventing your cultures from becoming contaminated, as well as ensuring your safety, and the safety
of those around you from the bacteria you are working with. This will also be an important practice
to keep you and your patients safe from infectious disease. The object of this lab exercises is to
practice aseptic technique by using a sterile loop to streak a sterile agar plate without introducing
any contaminating organisms onto your plate.

As a reminder: You must pass the aseptic technique skills check.  Our nursing program has
told us that they DEPEND on you to have learned aseptic technique in microbiology.  It has always
been an essential skill for healthcare, and with COVID-19, it is even more so!  Therefore, even if you
are getting a 4.0 in the class, if you do not pass the aseptic technique skills check, you will receive a
0.0 in the class.

Reference: Brown, A.E. Benson’s Microbiological Applications, Laboratory Manual in General

Microbiology, Short Version, 14th Edition, McGraw Hill, 2017.
 LAB EXERCISE 8, Aseptic Technique

IMPORTANT: The Benson Manual shows techniques involving Bunsen burners and flames to
sterilize reusable metal loops. We are using sterile individually wrapped disposable plastic
loops this quarter, which should NEVER be passed over a flame. You will never re-use the
disposable plastic loops, because they cannot be reliably sterilized after they have been used.
Once they are contaminated, they should be disposed of immediately in the small biohazard bag.

Prepare a pre-lab for Periods 1 and 2 (all procedures).

PERIOD 1 (DO THIS BY Thursday, May 14th)

Materials per person:

Squirt bottle of diluted disinfectant 3 sterile loops (individually wrapped)

Paper towels Ziploc bag
Candle (and something to light it) Home kit incubator
Permanent marker Small biohazard bag
1 TSA plate

Procedure:

1. Before starting, disinfect your work area and wash your hands with soap and water and get all of
your materials above ready.
Note: Always check your agar plates for contamination before you start!
1
2. Light your candle: this creates an area of warm air to reduce chances of contamination.

3. Label the bottom of the plate around the edges with permanent marker clearly with:
 your name (initials)  type of media (for this experiment, the media is TSA)
 the date  incubation temperature (room temperature or RT)
 a name of the sample (aseptic technique)

Note: You may need to dry your plate off to label it. Every plate that you use in this course must
be labeled with these 5 pieces of information!

4. Draw a “T” with a permanent marker on the bottom of the plate:

1. A
3. 2. C
B

5. Turn your plate over, keeping the lid on.

6. Open the end of one sterile loop (the side with the handle), by peeling the paper from the plastic.
Do this immediately before you are ready to streak your plate.

7. Remove the sterile loop from the packaging, without touching the loop end to any surface other
than the surface of the agar.

8. Use the first sterile loop to streak the surface shown in region “A” in the figure below.
Note: You may want to practice drawing this streak pattern, exactly as show in the figure below,
using a pencil and paper, since we have a limited number of TSA plates!

A. Tilt the lid open with the other hand, while

holding the plate steady with that same hand.
A Gently glide the sterile loop across the surface of
the agar. Dispose of loop immediately in the
small biohazard bag and close the lid.
B = use new sterile loop.
C B. Use new sterile loop to streak through section A into section B
several times (2-3 times). Continue streaking in section B without
touching section A. Dispose of loop immediately in the small
biohazard bag.
C. Use new sterile loop to streak through section B into section C
several times (2-3 times). Continue streaking in section C without
touching section A. Incubate plate. Dispose of loop immediately in
the small biohazard bag.
9. When finished, place your TSA plate (inverted, as previously shown) into a ziploc bag and into
your incubator in a warm place (like on top of your refrigerator).

2
10. Disinfect your work area and then wash your hands with soap and water.

PERIOD 2 – 5-7 days later (Turn in Lab report Thursday, May 21 st)

1. Disinfect your work area and then wash your hands with soap and water.

2. DO NOT OPEN YOUR PLATE IF THERE ARE MOLD COLONIES ON IT!!!! You do not need your
candle today.

3. Make drawings and notes about your observations on a piece of paper. You must ALWAYS make
notes and colored drawings in your notebook for ALL observations of labs for our
microbiology course!

4. If your plate has bacterial or mold colonies growing on it, repeat the procedure with a new plate.
 If your plate has growth, try to think of an idea for what you might have done wrong, and
write this down ON YOUR NOTES. You will also need to re-do the Period 1 procedure with a
new TSA plate, and then check the plate the following lab period to see if you have mastered
aseptic technique. If so, you can then get checked off by your instructor!

5. After incubation for 5-7 days, take three photos to include in your lab report:
1) your face and your finished agar plate clearly seen in one picture together.
2) a close-up of your plate from the bottom showing your initials clearly on the plate.
3) a close-up of your plate from the top (with the lid still on).

6. When you are finished:

1 place your TSA plate back into the ziploc bag,
2 put the ziploc bag into the small biohazard bag,
3 put the small biohazard bag into the large biohazard bag,
4 use the twist-tie to close the large bag,
5 store the bag out of reach from children/pets (for example, in the garage, or in a
bucket with lid).

7. Disinfect your work area and then wash your hands with soap and water.

3
 Name: __________________________________________

Section: ________________________________________

Lab 10A – Aseptic Technique Post Lab Report

What you will submit in ONE document uploaded to Canvas:
 Lab notes (such as any protocol changes or mistakes that were made)
 Results and observations, including:
 Drawings of your plates in color
 Written notes (i.e. colony counts, amount of growth, interpretation of results)
o 3 required Photos after 5-7 day incubation
 Laboratory Report (below) – not the one from the Benson manual!
 The usual 3 questions for your Lab Journal.

1. List 3 three reasons why the use of aseptic technique is essential when handling microbial cultures in
the laboratory.

1:

2:

3:

2. (a) Define the word inoculate as it relates to this experiment:

(b) use the word inoculate in a sentence that relates to microbiology lab:

3. When you are transferring microbes from one plate to another, why should you not set the lid of the
Petri dish down on the counter?

4. Where should a label be written on an agar plate? WHY?

5. How should agar plates be incubated? WHY?

6. Discussion (on a separate piece of paper): Consulting the results in your lab notes from this
exercise, did you find contamination anywhere? If so, in your discussion/conclusion describe what
you think caused the contamination (when did the contamination happen during your procedure).

4
 Explain the importance of aseptic technique and why/how you will use aseptic technique to culture
organisms in this class, and why it’s important for handling patient samples.