UNIVERSITI SAINS MALAYSIA

SCHOOL OF BIOLOGICAL SCIENCES

BOI207/4 GENERAL MICROBIOLOGY
LAB PRACTICAL 3: ASEPTIC TECHNIQUE

NAME: NOOR EZETY NAJWA BINTI TERMIZI

MATRIC NUMBER: 141954
DATE SUBMITTED: NOVEMBER 13TH 2019
LECTURER: DR DARAH IBRAHIM
INTRODUCTION
Microorganisms are very diverse and include all bacteria, fungi, yeast and algae. They can be
found everywhere, in the air, in water, and on every kind of surface imaginable. Their
widespread presence means that microbiologists have to take certain precautions when working
with the microorganisms to avoid contamination.
Aseptic techniques (also called sterile techniques) are defined as the processes required for
transferring a culture from one vessel to another without introducing any additional organisms
to the culture or contaminating the environment with the culture.

OBJECTIVE
To learn and apply aseptic techniques for working with bacterial cultures.

MATERIALS
1. Test tube rack
2. Nutrient broth culture of bacteria (Escherichia coli)
3. Nutrient agar slant culture of bacteria (E. coli)
4. 10 ml tubes of sterile nutrient broth
5. Nutrient agar plate
6. Inoculating loop
PROCEDURES
Transfer of bacteria from broth to broth
1. Plates and tubes are labelled. The BOTTOM of the plate is labelled rather than the lid.
2. Bunsen burner is lighted up.
3. The loop is sterilized by holding it in the flame of the Bunsen burner until it glows read.
The loop is help slanted so that 1 to ½’ of the loop is in the flame. Heating the loop
handle is avoided.

4. The loop is let to cool thoroughly. You may set the loop down on the bench, but make
sure the sterilized end does not touch anything. Failure to let the loop cool thoroughly
can kill the bacteria that you are trying to transfer with the loop. You know it’s too hot
if the broth sizzles when you put the loop into it, or if the agar melts when you touch the
loop to an agar plate.
5. With the loop in dominant hand, the broth culture is picked up in the other hand and
gently shook to make sure it has been mixed.
6. With the little finger of the hand holding the the loop, the cap of the tube is grasped and
pulled off.
7. The mouth of the tube is flamed immediately. The tube is removed from the flame.
8. The loop is inserted into the culture.
9. The loop is removed, the mouth of the culture is flamed and the cap is replaced. The
tube is set down in the rack.
10. The new tube of the sterile broth is picked up, the cap is removed as before and the
mouth of the tube is flamed.
11. Without flaming the loop, the loop containing some of the original culture into the new
tube. The loop is removed and the mouth of the tube is flamed before replacing the cap.
12. To sterilize it the loop is flamed.
Transfer of bacteria from agar slant to broth
1. With a loop sterilized and cooled as described in one hand, the agar slanted culture of
bacteria is picked up in the other. The cap is removed as before, and the mouth of the
tube is flamed.
2. The loop is inserted and a small amount of bacterial growth is picked up. The loop is
removed, the mouth of the tube is flamed and the cap is replaced. The culture is put
back in the rack.
3. A fresh tube of nutrient broth is picked up, the cap is removed and the mouth of the
tube is flamed.
4. The loop containing the bacteria from the slanted agar culture is inserted and gently
stirred to dislodge the bacteria into the broth.
5. The loop is removed, the mouth of the tube is flamed, the cap is replaced and set on
the rack.
6. The loop is sterilized.
Transfer of bacteria from broth to agar plate
1. With a loop sterilized and cooled as described, the broth culture of bacteria is picked
up. The cap is removed, and the mouth of the tube is flamed.
2. The loop is inserted to get a drop of the culture. The loop is removed, the mouth of
the tube is flamed, and the cap is replaced. The culture is put back to the rack.
3. Carefully lift the lid of your agar plate with the hand that is not holding the loop. Do
NOT set the lid down anywhere at any time and try to avoid breathing on the plate
while the lid is open.
4. The loop is carefully streaked back and forth over the surface of the plate so that the
plate look like this:

5. The lid is put back down and the loop is flamed.

6. All the inoculated broth and plate cultures are incubated at 37 °C for 48 hours. Then,
the growth of the bacteria in the broth and the plate cultures are observed.
 RESULTS AND QUESTIONS

The pictures shown below are the original nutrient broth and agar plate and the incubated broth
and agar plate that transferred with bacterial culture using aseptic technique.

From broth culture to broth

Sterile nutrient broth After incubated

From agar slant culture to broth

Sterile nutrient broth After incubated

 From broth culture to agar plate

Sterile agar plate After incubated

1. Did something grow in all of your transfers? If not, what could have been the
problem?

- Yes, something grew in the agar. Both the broths become turbid or cloudy and there
are white lines and spots grown on the agar, these are the proof that microorganism
had grown in the medium provided.

2. Do any of your agar plates appear to be contaminated?

- No. Because the color of the broth and the agar plate is same with the broth and agar
plate culture we used to take up bacteria.

3. How would you know if your broth cultures were contaminated with a microbe other
than the one you wanted to grow?

- The cultures that grow should all look the same, they should have the same cultural
characteristics like color and shape. So when they are contaminated, the culture turns
into other colors or become more cloudy than the original broth culture.
CONCLUSION
In this experiment, we learned about the aseptic technique. We are also exposed to bacterial
culture preparation. Aseptic technique is very important to prepare a good microbial culture
which is not contaminated and to prevent the original bacterial culture to be contaminated. A
good practice of aseptic technique also ensure that we do not contaminate our working
environment with bacteria. The proper practical and procedure is learned and precautions are
taken to make sure that the aseptic technique is nicely done. We are also exposed to bacterial
culture preparation.

References
1. kashort0609. (2013). Lab Report Questions. Retrieved November 3, 2019, from Quizlet:
https://quizlet.com/38657163/lab-report-questions-pg-25-26-flash-cards/#