LAB 5 :

MICROBIOLOGY

INTRODUCTION
The study of microbiology requires not only an academic understanding of the microscopic
world but also a practical understanding of lab techniques and procedures used to identify,
control, and manipulate microorganisms. The proper identification of a microorganism is not
only important in a microbiology lab but also in the medical, industrial, and pharmaceutical
fields. In this lab report, lab techniques and procedures learned during this course were
performed to assess each students’ practical knowledge in microbiology.

OBJECTIVE
1. To acquire the skill of aseptic technique in the field of Microbiology.
2. To prevent contamination of cultures and media from microbes in the environment.
3. To transfer cultures from one medium by inoculating another medium. This is called
subculturing.

METHODS
(A) Streak plate technique
1. The inoculating loop was sterilize in the Bunsen burner by putting the loop into tha flame
until it was red hot . Allowed it to cool.
2. An isolated colony was picked from the agar plate culture of (a) E.coli and (b) S.aureus
and spread each of them over the first quadrant on separate agar plate .
3. Covered the agar plate with the lid and flame the loop.
4. Turned the plate and lightly streak into the next quadrant without overlapping the previous
streak.
5. Step 3 and 4 was repeated and streak into the third quadrant.
6. Sealed each plate with parafilm.
7. The plates was invert and incubate at 37 ̊ C for 24hours.
(B) Effect of handwashing on bacteria on thumb
1. 4 nutrient agar was obtained and labelled them .
a) control
b) water
c) hand sanitizer
d) soap
2. Each agar plate was divided into 4 section by drawing line using a marker pen on the back
of the petri dish .
3. Aseptic technique was used, gently pressed thumb on the control agar plate.
4. Washed hands (including thumb) with water and step 3 was repeated on the appropriate
agar.
5. step 4 was repeated using hand sanitizer and soap
6. Sealed each plate with parafilm.
7. The plates was invert and incubate at 37 ̊ C for 24 hours .

Gram Staining
1. A sterile inoculating loop was used, add 1 drop of sterile water to the slide . A smear was
prepared :
a) Escherichia coli
b) Staphylococcus aureus
2. Air dry and heat fix
3. The smear was covered with Crystal Violet (primary stain) for 1 min.
4. Wash off gently the slide with water.
5. Gram’s Iodine (mordant) was added for 1 min.
6. Washed with water.
7. Decolorized with 95% ethanol . Stop decolorizing with alcohol as soon as the purple
colour has stopped leaching off the slide. Washed with water immediately.
8. The smear was covered with Safranin for 30 seconds.
9. Both the top & the bottom were washed off the slide with water.
10. The slide was blot.
11. The light microscope was used, viewed the smear up to 100x with immersion oil.
RESULT

Streak plate technique Effect of handwashing on bacteria

on thumb

Gram staining
DISCUSSION
By employing the streaking technique on an agar plate correctly, a single colony can be
obtained. Furthermore, it can be used to separate colonies of mixed culture. Hence, this pure
colony can be picked up and to be grown in large quantity. From the result above, it can be
observed that single colonies of the S.aureus are found. Due to the colour and morphology, it
can be noted that the S.aureus is of a pure culture.
The human skin's surface do carry a large number of microorganism and that by washing
hands, individual can reduce the number of microorganism noticeably. However, even after a
hand wash, microorganisms are still present on the surface.
Different bacteria can live and flourish in certain environments, while others will die or never
grow in the very same environment due to their dissimilar characteristics. These tests help
determine which bacteria can grow in certain situations. For both bacteria, the Gram stain
was a necessity in order to determine if the microbe was Gram Positive or Gram
Negative. Once the Gram Positive bacteria was successfully visible under the microscope, it
was clear that it was cocci. Knowing this information helped to cross two bacteria (both
rods) off the unknown list. The other Gram stained slide was viewed under the microscopes
and pink/reddish rods were clearly visible making it the Gram negative bacteria. This Gram
stain, however, did not eliminate any of the Gram Negative bacteria because they were all
rods.

CONCLUSION
Aseptic technique is a basic laboratory technique that must be employed especially during
Microbiology laboratory session so as to prevent any contamination and affecting the
accuracy of the result. Since microorganism can replicated rapidly, disposal of contaminants
must be done properly so as to protect both the equipments and the health of individuals.
The Gram staining method is a useful tool used in most laboratories as it helps individual to
visualise the bacteria accurately and effectively such as the shape, arrangement and even
whether the culture is a pure or mixed. However, it should be noted that not all bacteria will
give a gram reaction as some of them are gram variable, otherwise known as gram
indeterminate. Therefore, they will give a mix of pink and purple cells after gram staining.
For some of the Gram Positive bacteria, their peptidoglycan breaks easily during cell
division, hence, after staining, they will give pink cells instead of purple. In addition, the
duration of a culture can also affect the gram stain.

REFERENCES
https://www.ukessays.com/essays/biology/a-aseptic-technique.php
http://vlab.amrita.edu/?sub=3&brch=73&sim=212&cnt=1