EXPERIMENT OF MICROBIOLOGY

INTRODUCTION

PART 1: ASEPTIC TECHNIQUE

Aseptic technique is a fundamental and important laboratory skill in the

field of microbiology. Microbiologists use aseptic technique for a variety of
procedures such as transferring cultures, inoculating media, isolation of pure
cultures, and for performing microbiological tests. Proper aseptic technique
prevents contamination of cultures from foreign bacteria inherent in the
environment. Using proper aseptic technique can greatly minimize or even
eliminate the risk of contamination. Aseptic technique is also essential for isolation
of a single species of microorganism from a mixed culture to obtain a pure culture.
Furthermore, proper aseptic technique prevents microbes used in the laboratory
from accidentally being released into the environment and/ or infecting people
working in the laboratory. This is especially relevant when pathogens are being
handled.

Handwashing is thought to be effective for the prevention of transmission of

diarrhoea pathogens. However it is not conclusive that handwashing with soap is
more effective at reducing contamination with bacteria associated with diarrhoea
than using water only. This experiment is carry out by (1) handwashing with
water, (2) handwashing with soap, (3) no handwashing and (4) handwashing with
sanitizer.

PART 2: GRAM STAINING

Gram staining is a common technique used to differentiate two large groups

of bacteria based on their different cell wall constituents. The gram stain procedure
distinguishes between Gram positive and Gram negative groups by coloring these
cells red or violet. Gram positive bacteria stain violet due to the presence of a thick
layer of peptidoglycan in their cell walls, which retains the crystal violet these cells
are stained with. Alternatively, Gram negative bacteria stain red, which is
attributed to a thinner peptidoglycan wall, which does not retain the crystal violet
during the decoloring process.

METHODOLOGY

PART 1: ASEPTIC TECHNIQUE

A) STREAK PLATE TECHNIQUE

1) The placed that involved in carry out a streak plate is sterilized. The
inoculating loop in the Bunsen burner is sterilized by putting the loop
into the flame until it is red hot. Then, allowed it to cool.
 2) After that, an isolated colony is 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) The agar plate is covered with the lid and the loop is flamed.
4) The plate is turned and lightly streak into the next quadrant without
overlapping the previous streak.
5) Step 3 and 4 are repeated and streaked into the third quadrant.
6) Each plate is sealed with parafilm.
7) The plates are inverted and incubate at 37oC for 24 hours.

B) EFFECT OF HANDWASHING ON BACTERIA ON THUMB

1) In 1 nutrient agar, it is labelled with:
a) control
b) water
c) soap
d) hand sanitizer
2) The agar plate is divided into 4 sections by drawing line using a marker pen
on the back of the petri dish.
3) Using aseptic technique, gently pressed thumb on the control agar plate.
4) Then, hands (including thumb) is washed with water and step 3 is repeated
on the appropriate agar.
5) Next, step 4 is repeated using hand sanitizer and soap.
6) The plate is sealed with parafilm.
7) The plate is inverted and incubated at 37oC for 24 hours.

PART 2: GRAM STAINING

1. By using a sterile inoculating loop, 1 drop of sterile water is added to the

slide. Then, prepared a smear of:
(a) Escherichia coli
(b) Staphylococcus aureus
2. Next, dried with air and fixed the heat.
3. The smear is covered with crystal violet (primary stain) for 1 min.
4. Washed off the slide gently with the water.
5. Gram’s Iodine (mordant) is added for 1 min.
6. Washed with water.
7. Decolorized it with 95% ethanol. This is the "tricky" step. Stop decolorizing
with alcohol as soon as the purple color has stopped leaching off the slide
(time will vary depending on thickness of smear). Immediately wash with
water. Be sure to dispose of all ethanol waste in the appropriately labelled
waste container.
8. Then, the smear is covered with Safranin for 30 seconds.
9. Both the top & the bottom of the slide are washed with water.
 10. Blotted the slide.
11. Using the light microscope, viewed the smear up to 100x with immersion oil.

RESULTS

A) STREAK PLATE TECHNIQUE

Figure 1: streak plate result by using bacteria S. aureus

Figure 2: streak plate result by using bacteria E. coli

B) EFFECT OF HANDWASHING ON BACTERIA ON THUMB

Figure 3: thumb print result for control, water, soap and sanitizer

C) GRAM STAINING

Figure 4: Gram staining result Figure 5: Gram staining result

for Escherichia coli (gram-negative) for Staphylococcus aureus
(gram-positive)
DISCUSSIONS

From this experiment, we have done three experiment of microbiology which

are aseptic technique that have part of streak plate technique and effect of
handwashing on bacteria on thumb and part of gram staining.

Firstly, we want to discuss for the part A which is streak plate technique. In
this experiment, we are using two bacteria which are E. coli and S. aureus. These
experiments show that the appearanced of the colonies in the agar after we are
streak it by different of bacteria. What we can say from the experiment, the colonies
in the agar plate for the both bacteria show the different characteritics on the agar
plate. For the E.coli bacteria, the colonies are small in size and the pigmentation of
the colonies is colourless. For the S.aureus bacteria, the colonies are big in size
and the pigmentation of the colonies is yellow. So, the growth of the colonies for the
both of bacteria is different from their characteristics for both bacteria.

Next, we want to discuss for the part B which is effect of handwashing on

bacteria on thumb. In this experiment, for the part of thumb by using sanitizer, the
contaminat is almost similar to the part of soap, control and water. As we know,
sanitizer will reduced the contaminat on the finger but in this case, the sanitizer is
giving higher contaminat on the finger. It is because the content in the sanitizer
maybe same like content in the soap. That’s why, the contaminat that produced in
the part of sanitizer same like part in soap. What we can see from this experiment,
water is more effective method in handwashing to reduce the bacteria from the
fingers.

Then, for the part C, two bacteria are used to know which bacteria have
gram-positive and gram-negative. In this experiment, the result show that E.coli is
a gram-negative which is pink in colour that lose the crystal violet stain and are
instead stained by the safranin in the final staining process. For the S.aureus, the
result shows that it is gram-positive which is purple in colour that has a thicker
peptidoglycan layer which remains crystal violet stain during the decolorization
process.

CONCLUSIONS

In conclusion, for the experiment A, the growth of colonies in the agar plate
is rapidly for both bacteria after incubated for 48 hours. Then, for the experiment B
that carried out, handwashing with water only is much more effective in removing
bacteria from hands than handwashing with sanitizer and soap. For the
information, the sanitizer is much more effective in removing bacteria on the hand
than water only but in this experiment, the sanitizer not removed the bacteria on
the hand but it is giving more bacteria on the hand. After that, for the experiment
C, the result show that E.coli is a gram-negative while S. aureus is a gram-positive
when we looked at the view 100x of microscope.
REFERENCES

http://vlab.amrita.edu/?sub=3&brch=73&sim=213&cnt=1

https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.ht
ml