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SBL1023

TECHNIQUES IN BIOLOGICAL AND


BIOCHEMISTRY LABORATORY

MICROBIOLOGY

NAME: NUR IZZATIE BINTI KHAIZAAR


MATRICS NUMBER: E20161013729
GROUP: A
LECTURERS NAME: ASSC. PROF DR. SHAKINAZ BINTI DESA
MICROBIOLOGY

INTRODUCTION
Microbiology is the study of living things that could not be seen by naked eyes.
The living things is also includes the bacteria. The study of bacteria nowadays is very
important since the bacteria is half is very good for us while other is very dangerous
to us. There many ways to handle all of these bacteria by using these methods. For
instance, aseptic techniques with streak plate method and gram staining. Streak plate
method is about the qualitative isolation method of bacteria rapidly meanwhile gram
staining is the common method to differentiate between the positive gram staining and
negative gram staining.

METHODOLOGY
A) Streak plate techniques

1. The inoculating loop is sterilized with ethanol (70%) and then burned loop
in the Bunsen burner until it is red hot. Allowed it to cool.
2. An isolated colony from the agar plate culture of E. coli is picked up by the
loop and spread each of them on separate agar plate as the first quadrant.
3. The agar plate is covered with lid and the loop is flamed the loop again.
4. The plate is turned and lightly streak into the next quadrant without
overlapping the previous streak.
5. The step 3 and 4 are repeated into the third and fourth quadrant.
6. The plate is sealed with parafilm.
7. The plate is inverted and incubated at 37C for 24 hours.

B) Effect of hand washing on bacteria on finger.

1. The plate that contain of agar is labelled and divided into four:
a) control
b) water
c) hand sanitizer
d) soap

2. Using aseptic technique, the thumb is gently pressed on the control agar
plate.
3. One of other fingers is washed with water and step 2 is repeated on the
appropriate agar.
4. Step 3 is repeated by using hand sanitizer and soap.
5. The plate is then sealed with parafilm.
6. The plate is inverted and incubated at 37C for 24 hours.
C) Gram staining

1. 1 drop of sterile water is added on the centre of slide. By using the sterile
inoculating loop, a smear of:
a) Escherichia coli and b) Staphylococcus aureus
2. The bacteria is touched by using the loop then swiped it on the centre of
slide.
3. The slide is heated for a while horizontally at centre of it.
4. The smear is covered with Crystal Violet (primary stain) for 1 min.
5. The slide is washed off gently with water.
6. Grams Iodine (mordant) is added for 1 min. Then, washed it with water.
7. Decolourized the slide with 95% ethanol until the colour is leached off. Then,
immediately washed with water.
8. The smear is then covered with Safranin for 30 seconds.
9. The slide is washed with at the top and bottom of it.
10. The slide is blotted.
11. The light microscope is used to view smear up to 100x with immersion oil.
RESULTS
A) Streak plate techniques

The diagram above shows that dilution of the bacteria by using the streaking
technique. The plate is numbered with green marker. 1 is the first quadrant. 2 is
the second quadrant. 3 is third quadrant. 4 is the fourth quadrant.
B) Effect of hand washing on bacteria on finger

The diagram above shows that the growth of bacteria for each situation.

C) Gram staining

The diagram above shows that the result gram staining before we put under
microscope. Staphylococcus aureus (left) and Escherichia coli (right).
Bacteria Observation Gram bacteria

Escherichia coli Negative.


Because the colour
that we saw
through
microscope is pink.

Staphylococcus Positive.
aureus Because the colour
that we saw
through
microscope is blue.
Escherichia coli

Staphylococcus aureus
DISCUSSION
Based on this experiment, for section A, we used to apply aseptic technique in
streak plate method. The aseptic techniques is fundamental laboratory skill that we
need to apply in order to avoid contamination of a sample. All the precaution steps
that we did apply before start the experiment, including sterile work area, personal
hygiene, apparatus, reagent and handling. Sterile the work area, e.g. in the laminar
flow, we have to spray the wall and base of the laminar flow by using ethanol 70% and
switch on the UV light. We supposed to wear glove and mask. Mask is used so that
no dust or bacteria or pathogens will contaminate the work area. We also have to keep
silence while carrying out the experiment at the laminar flow. The streak plate method
is used to dilute the bacteria from colonies to individual so that we could determine
that present in the agar plate. To ensure that the streak plate method worked
successfully, we used to apply aseptic techniques so that the number of colony
bacteria more accurate since there is the absence of contamination.
For section B, we used our fingers to determine the level of cleanliness for each
situation. The result shows that control is the most growth of bacteria. This is because
in control section, we did not wash our finger, which means that our finger contain a
lot of bacteria. This is not good if we want to eat something without washing our hands.
The section that free of bacteria is by using hand sanitizer. It means that by using hand
sanitizer, the cleanliness of our hands could be guaranteed. However, this experiment,
the most section that free from bacteria should be the washed hand. This is because
of we did washed our hand with antibacterial soap. The growth of bacteria at this
section because we washed our hand with antibacterial soap improperly.
For section C, gram staining is about classification of bacteria. The result shows that
Escherichia coli gives the result negative gram. Negative gram means that the thin
peptidoglycan layer is surrounded by the plasma membrane so that it will not stain
with crystal violet. Meanwhile, gives result positive gram. Positive gram means that
the thick of peptidoglycan layer surrounded the plasma membrane so that it will stain
with the crustal violet.

REFERENCES
1. Archived on 20 December 2017
https://microbiologysociety.org/about/what-is-microbiology-.html

2. Archived on 20 December 2017


https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-
examples-and-animation/

3. Archived on 20 December 2017


https://bandbdental.com/flashcard/difference-gram-positive-gram-negative-
cell-walls/

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