DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE AND MATHEMATICS

SULTAN IDRIS EDUCATION UNIVERSITY

SBL 1023
TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY

EXPERIMENT 5: MICROBIOLOGY
BY

NAME NUR NATRAH NATASHA BT

MUSTAPA AYUB
MATRIC NUMBER E 20161015941
GROUP B
LECTURE’R NAME PROFESSOR MADYA DR.
SHAKINAZ BT DESA

Date and time of practical class

28th November 2017 2.00 p.m. – 5.00 p.m.

TITLE: MICROBIOLOGY
PART 1: ASEPTIC TECHNIQUE

INTRODUCTION

Microbiology, micro as we know is something extremely small. In this world, an organism

that was extremely is recognised as microorganisms. In order to observe them clearly, human
needs to occupy themselves with a microscope. We cannot simply rely on naked eyes to
observe them as microorganism is very small and microscope can help us to detect their
shape and behaviour.

In this experiment, the first experiment that we do is streak plate technique. The streak plate
technique is a rapid isolation method. The techniques commonly used for isolation of discrete
colonies initially require the number of organism in the inoculums be reduced. It is
essentially a dilution technique that involves spreading a loopful of culture over the surface
agar plate. The bacteria that used to isolate the colony are Escherichia coli and
Staphylococcus aureus.

Figure 1: Four quadrant streak on agar plate

Then, we continue with the second experiment, that is effect of hand washing on bacteria on
thumb. Hands are parts of human body that are contact with the all things in this world.
People use their hand for variety of all activities days. It is extremely easy to come in contact
with different microbes and to transfer them to other object even people. Hand washing is
thought be effective for prevention of transmission of pathogen. There are many products in
the market that able to kill almost half of the microbes. However it is not conclusive that hand
washing with soup or other type of cleansing is more effective at reducing the bacteria
contamination than only using the tap water.

OBJECTIVE

1. To apply aseptic techniques in microbial cultivation.

2. To isolate pure colony by streak plate method.
3. To transfer bacteria by several methods.
4. To observe the bacteria in a different nutrient agar.

APPARATUS

Bunsen burner Water

Inoculating loop Hand sanitizer
Pure bacterial culture on nutrient agar Soap
Agar plate Parafilm

METHODOLOGY

A. Streak plate technique

1. The inoculating loop was sterilized in the Bunsen burner by putting the loop into the
flame until it is red hot. Allow it to cool.
2. An isolated colony from the agar plate culture (Escherichia coli)
3. The colony was spread over the first quadrant on a agar plate
4. The agar plate was cover with the lid.
5. The next quadrant was lightly streak without overlapping the previous streak.
6. Step 4 and 5 was repeated into the third quadrant.
7. The plate was seal with parafilm.
8. The plate was inverted and incubates at 37°C for 24 hours.

B. Effect of hand washing on bacteria on thumb

1. 4 nutrient agars were labelled as control, water, hand sanitizer and soap.
2. Each agar plate was divided into 4 sections by drawing the line using a marker pen on
the back of the petri dish.
 3. Press thumb on the control agar plate gently by using aseptic technique.
4. Step 3 was repeated on the appropriate agar by washing hand (including thumb) with
water.
5. Step 4 was repeated by using a hand sanitizer and soap.
6. The plate was seal with parafilm.
7. The plate was inverted and incubates at 37°C for 24 hours.

RESULT

(PART A) (PART B)

DISCUSSION

Based on our result in part A, it’s shown a heavy growth of colony. This is because we used a
wrong technique while streaking the loop and we are forgetting the position of the quadrant.

In part B, we observed the effect of hand washing on thumb with water, soap, hand
sanitizer and negative control. Based on theory, hand washing with soap and hand sanitizer
are more effective for reduce the bacteria. From the result, hand washing with sanitizer is
more effective while it shows the less colony growth of bacteria in a plate agar. However,
from the result soup shown more contamination. So based on this experiment, we recommend
people to use hand sanitizer to wash our hand before eat and doing any activity to protect our
body from any infection.
CONCLUSION

In this experiment we learned how to transfer bacteria from one container to another while
maintaining the purity of the culture. Application of aseptic techniques would ensure pure
cultures could be maintained and dissemination of the microbes to the environment can be
avoided. Aseptic techniques work by avoiding contacts between contaminants and pure
cultures.

REFERENCE

Mescopio Aisyah. Aseptic Technique Lab Report. Retrieved from

http://www.academia.edu/6573320/Aseptic_Technique_Lab_Report
PART 2: GRAM STAINING
INTRODUCTION

Gram staining is a different stain commonly used in microbiology that different bacteria
structure depends of their cell wall structure. Most bacteria can be divided into two based of
composition in the cell wall such as gram-positive and gram-negative.

Gram positive cell walls have a thick peptidoglycan layer beyond the plasma membrane. The
characteristics of the polymer is called teichoic and lipoteichoic acids stick out above the
peptidoglycan and it is because of their negative charge that the cell is overall negative. The
acids are important in the body ability to recognize foreign bacteria. Gram-positive cell walls
stain purple with the Gram stain.

Gram-negative cell walls are more complex. It has a thin peptidoglycan layer and an outer
membrane beyond the plasma membrane. The space between the plasma membrane and outer
membrane is called the periplasmic space. The outer leaflet of the outer membrane is
composed largely of a molecule that called lipopolysaccharide (LPS). Lipopolysaccharide is
an endotoxin that is important in triggering the body immune response and contributing to the
overall negative charge of the cell. Spanning the outer membrane is porin protein that enables
the small particle passage on it. LPS join the outer membrane and the thin peptidoglycan
layer. Gram-negative cells will satin pink with the Gram stain.

OBJECTIVE

1. To differentiate between the two major categories of bacteria, Gram positive and Gram
negative.

APPARATUS

Inoculating loop Gram iodine

Sterile water Decolorize with 95% ethanol
Slide Safranin
Bacteria (Escherichia coli and Microscope
Staphylococcus aures)
Crystal Violet Penunu Bunsen
METHODOLOGY

1. 1 drop of sterile water was added to the slide by using a sterile inoculating loop. A smear
of Escherichia coli and Staphylococcus aures was prepared.
2. Air dry and heat fix.
3. The smear was covered with Crystal violet for 1 minute.
4. The slide was washed with water gently.
5. Grams Iodine was added for 1 minute.
6. The solution was washed with water.
7. Then, the slide was decolorizing with 95% ethanol. *stop decolorizing with alcohol as
soon as the purple colour has stopped leaching off the slide.
8. The smear was covered with Safrain for 30 seconds.
9. The slide was washed both top and bottom with water.
10. The smear was viewed up to 100x with emulsion oil by using light microscope.

RESULT

Escherichia coli Staphylococcus aures

DISCUSSION

The purpose of the Gram stain is to separate bacteria into two different categories, Gram-
positive and Gram-negative based on chemical and physical properties of their cell wall. The
result of Gram stain reveals whether the organism is positive or negative based on the colour
the organism appears after staining. Gram-positive organisms will not be easily decolorized
and retain the purple stain of crystal violet Gram-negative organisms will be decolorized by
the alcohol and are subsequently stained by the safranin and appear pink.

Firstly, we prepared smears of two microorganisms which are Escherichia coli and
Staphylococcus aures. After preparing the smears of the microorganisms, the smears were air
dried and heat fixed. Then we stain both smears using crystal violet dyes. Crystal violet dyes
are basic dyes that have positively charged particle that helps them to bind to negatively
charged molecule like teichoic acid at the cell wall of bacteria. This crystal violet dye can
dissociate into cv+ and cv- ions. These ions can penetrate deeps into the cell wall of bacteria
and interacts with the negatively component on the bacterial cell wall. After one minutes, the
crystal violet was washed with tap water.

The next step is to add iodine. Iodine was being added as a mordent to form crystal
violet-iodine complex, CVI complex. This complex enables the dyes to not be easily being
removed. Next, we washed the iodine with tap water and dried off the excess water. After
that, 95% of ethanol was being added to acts as a decolorizing agent. It interacts with the
lipid membrane of both positive and negative bacteria, and this would cause the gram-
negative bacteria to lost their outer membrane and exposing the peptidoglycan. The CVI
complex are being washed from the outer membrane of gram-negative bacteria and cause the
purple color to decolorize. Meanwhile for gram-positive bacteria, the addition of alcohol
dehydrated the layer of peptidoglycan which in turn would trap the CVI complex. This cause
the gram positive bacteria appeared to be purple colour as the CVI complex are being
retained. The addition of alcohol more than 15 seconds would break the cell wall of the
bacteria so no stain to be observed.

In the next step, safranin was used to counterstain both smears. This is to enables the
gram negative bacteria to be visualized easily as it can be stain in pink colour. The gram-
positive bacteria does not being stained pink when safranin was being introduced because the
peptidoglycan layer already have CVI complex. Then, the slides were washed using tap water
and dried off. Finally, we observed our specimen using microscope.
CONCLUSION

Based on this experiment, we can conclude that gram staining is the method of distinguishing
between gram positive and gram negative bacteria. In our result, we stained Staphylococcus
aures epidermis which appeared to be Gram-positive and Escherichia coli appeared to be
Gram-negative.

REFERENCE

Amrita (2011). Gram Satin Technique. Retrieved from

http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1
Akbar Haqi. Lab report of microbiology 3. Retrieved from
http://www.academia.edu/9655093/LAB_REPORT_OF_MICROBIOLOGY_3