SBL1023

TECHNIQUES IN BIOLOGICAL AND

BIOCHEMISTRY LABORATORY

LAB 5
MICROBIOLOGY

NAME: FARAH WAHIDAH BINTI JEPPRI

MATRICS NUMBER: E20161013726
GROUP: A
LECTURERS NAME: ASSOCIATE PROF DR. SHAKINAZ BINTI DESA
 LAB 5 MICROBIOLOGY

INTRODUCTION
Microbiology is the study of microorganisms that are too small to be seen by unaided eyes. In
microbiology, there are several techniques that can be carried out to study about this living organisms
which is microorganisms or microbes that include bacteria, fungi, protozoa, microscopic algae and
viruses. The streak plate method is a rapid qualitative technique used to isolate a pure strain from a
single species of microorganism, Escherichia coli (E.coli) bacteria. This method consists of dilution
technique that involves spreading a loopful of culture over the surface of agar plate. The resulting
colonies can be taken as samples and a microbiological culture can be grown on a new plate so that the
resulting diminution can be observed, identified, studied and tested.

In daily life, we use hand to do variety activities. This part of human is the part that easily to
transfer different microbes to other peoples or objects as it was easily to come in contact with many
things. Different types of hand washer or other cleaning products have different efficiency to kill the
germ. Sometimes, by washing hand using water only is more effective to reduce bacteria contamination.
The experiment will prove the real facts about the effectiveness of the hand washer or cleaning products.

Gram staining is the method used in microbiology laboratory that distinguish bacteria on the
basis of their cell wall structure that differentiate between gram positive and gram negative group. This
method resulting the colouring changes of the cells which are red or violet. Based on the composition
of bacteria cell wall, the bacteria could be differentiate as gram positive bacteria as it stain violet due to
the presence of thick layer of peptidoglycan in their cell walls, which retains the crystal violet these
cells are stained with. The gram negative bacteria usually stained with red colour.

OBJECTIVES

To apply aseptic technique in isolation bacteria to produce colony without contamination.

To prove the effectiveness of hand washer with soap and other cleaning products in reducing
bacteria contamination.
To differentiate between the gram positive and negative bacteria based on the change colour
of the stain observed.

METHADOLOGY
PART 1: ASEPTIC TECHNIQUE

a) Streak plate technique

1. The inoculating loop in the Bunsen burner was sterilized by putting the loop in the flame
until it is red hot. The inoculating loop was allowed to cool.
2. An isolated colony was picked from the agar plate culture of E. coli and the bacteria was
spread over the first quadrant on separate agar plate.
3. The agar plate was covered with the lid and loop was flamed.
4. The plate was turned and streaked lightly into the next quadrant without overlapping the
previous streak.
5. Step 3 and step 4 was repeated and the streaking into the third quadrant was done.
6. The plate was sealed with parafilm.
7. The plates was inverted and incubated at 37C for 24 hours.
 b) Effect of hand washing on bacteria 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 sections and the line was drawn using a marker pen on the
back of the petri dish.
3. Using aseptic technique, the control agar plate was gently pressed with thumb.
4. Other hands (including thumb) was washed with water and step 3 was repeated on the
appropriate agar.
5. Step 4 was repeated using hand sanitizer and soap on the other hand.
6. The plate was seal with parafilm.
7. The plates was inverted and incubated at 37C for 24 hours.

PART 2: GRAM STAINING

1. 1 drop of water was added to the slide using a sterile inoculating loop. A smear of
Escherichia coli and Staphylococcus aureus was prepared.
2. The process of air dry and heat fix was done.
3. The smear was covered with Crystal Violet (primary stain) for 1 minute.
4. The slide was gently washed off with water.
5. Grams Iodine (mordant) was added for 1 minute.
6. The slide with iodine was washed with water.
7. The slide was decolorized with 95% ethanol. The decolorizing process was stopped as soon as
the purple colour has stopped leaching off the slide. The slide was immediately washed with
water. All ethanol waste was disposed in the appropriately labelled waste container.
8. The smear was covered with Safranin for 30 seconds.
9. Both top and bottom of the slide was washed with water.
10. The slide was blotted.
11. The smear was viewed up to 100x by using light microscope with immersion oil.
RESULTS
PART 1: ASEPTIC TECHNIQUE

a) Streak plate technique

Figure 2
Figure 2 above shows that the result. The right side of the plate shows that the present of
bacterial colonies which is called as heavy confluent growth. The left and right side of the
plate is called heavy growth and there is discrete colonies at the upper of the plate.
a) Effect of hand washing on bacteria thumb

Figure 3

The figure shows the effectiveness of hand washer, cleaning product, water and control towards
bacteria thumbprint contamination.
PART 2: GRAM STAINING

Figure 4

Image of Bacteria positive gram of Staphylococcus aureus in microscope based on the colour stain
observed.

Figure 5

Image of bacteria negative gram of Escherichia coli in microscope based on the colour stain observed.
DISCUSSION

This experiment involve two parts which are aseptic technique before gram staining
process. Aseptic technique need high attention and careful to prevent contamination of cultures
and media from microbes in the environment. Streak plate method is the important step to
isolate a microorganism from a mixed culture to obtain a pure culture. All steps in this method
needed to take serious to prevent contamination. The principle of this method which are
flaming the loop, flaming the mouth of the test tube and cleaning the work place with 95%
ethanol is very significant step in this method to kill the bacteria surrounding. Based on
experiment that I had carried out, from figure 2, the result shows that there was the presence of
bacterial cells in the plate indicate the microbial growth in the pure culture surface. The
bacterial cells form fourstreak way. The right side of the plate shows that the present of
bacterial colonies which is called as heavy confluent growth. The left and right side of the plate
is called heavy growth and there is discrete colonies at the upper of the plate. This experiment
was successfully carried out as no contamination occur in the culture as all the steps as followed
well.

Different soap or hand cleaning product have different effectiveness towards bacteria
contamination for our hand. Many products claim that their product can kill until 99.99% germ
and make our hand clean and sterile from the transfer of bacteria. This experiment will use
thumbprint for the testing with the products and the effect of thumbprint will be observed on
the agar plate after incubation of 24 hours. However, this experiment prove us that by washing
thumbprint using water only is more effective in killing bacteria from the thumbprint than
washing thumbprint with soap. This X brand of soap does not effective in killing bacteria as it
left more colony of bacteria on the agar plate. By using hand cleaning product like hand
sanitizer, all the bacteria are killed as no bacteria left on the thumbprint section. The
effectiveness are made based on the bacteria left observed on the agar plate compared with the
control.

Gram staining is method to distinguish between positive and negative bacteria. The
result in coloured stain observed will make us know the type of bacteria when it was observed
under the microscope. Based on figure 4, the purple colour of stain indicate that the
Staphylococcus aureus bacteria is positive gram cells. Based on figure 5, Bacteria Escherichia coli stain
pink with the gram stain indicate the gram-negative cells.
REFERENCES
1. Archived on 20 December 2017 from
http://www.academia.edu/9655093/LAB_REPORT_OF_MICROBIOLOGY_3

2. Archived on 20 December 2017 from

http://vlab.amrita.edu/?sub=3&brch=73&sim=212&cnt=1
3. Lecture note, Microbiology.
4. Laboratory Manual SBL 1023.