DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE AND MATHEMATICS

SULTAN IDRIS EDUCATION UNIVERSITY

SBL 1023

TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY

EXPERIMENT 5 : MICROBIOLOGY

NAME: NOR HAIZATUL BINTI SABIDI

NO MATRIC: E20161015640

GROUP : B

LECTURE NAME : PROFESOR MADYA DR. SHAKINAZ BINTI DESA

Title

Experiment 5: Microbiology

Introduction

Part 1: Aseptic Technique

The streak plate method is a rapid qualitative isolation method. The techniques
commonly used for isolation of discrete colonies initially require that the number of
organisms in the inoculums be reduced. It is essentially a dilution technique that involves
spreading a loopful of culture over the surface of an agar plate. The resulting diminution of
the population size ensures that, following inoculation, individual cells will be sufficiently far
apart on the surface of the agar medium to effect a separation of the different species present.
Although many type of procedures are performed, the four ways or quadrant streak is mostly
done.

The hands are the parts of the human body that are in most contact with the outside
world. People use their hands for a variety of activities everyday. It it extremely easy to come
in contact with different microbes and to transfer them to other objects and mybe even
people. Handwashing is thought to be effective for the prevention of transmission of
pathogens. There are many products in the market claim to be able to kill the germs. However
it is not conclusive that handwashing with soap or other cleaning products is more effective at
reducing bacteria contamination than using water only.

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 peptidogly
can wall, which does not retain the crystal violet during the decoloring process.

Objective

 To isolate a microorganism from a mixed culture to obtain a pure culture.

 To compare the effect of handwashing of bacteria on thumb.
 To determine the gram-postive and gram-negative of gram stain.
 To compare the differentiates bacteria of gram stain.
Materials

Part 1: Aseptic Technique

 A stock culture of E.coli

 Inoculation loop
 A striker
 Bunsen burner
 Ethanol
 Agar plates
 Paper towel
 Water
 Hand sanitizer
 Soap

Part 2: Gram Staining

 Slit
 Tube holder
 Bunsen burner
 Microscope under 100x magnification
 Inoculating loop
 Slide
 Distilled water
 Crystal violet
 Gram iodine
 Ethanol
 safranin

Methods

A. Streak plate technique

1. The inoculating loop was sterilized with Bunsen burner by putting the loop into
the flame until it is red hot. Allow it to cool.
2. An isolated colony was picked from the agar plate culture of (a) E.coli and (b) S.
Aureus and each of them was spread over the first quadrant on separate agar plate.
3. The agar plate was covered with the lid and the loop was flamed.
4. The plate was turned 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. Each plate was sealed with parafilm.
7. The plate was inverted and incubate at 37˚C for 24 hours.
B. Effect of handwashing on bacteria on thumb
1. 4 nutrient agar was obtained and label them:
a) Control
b) Water
c) Hand sanitizer
d) Soap
2. Each agar plate was divided into 4 sections by drawing line using a marker pen on
the back of the petri dish.
3. Aseptic technique was using by press thumb on the control agar plate
4. Hands (including thumb) was washed with water and repeat step 3 on the
appropriate agar.
5. Step 4 was repeated using hand sanitizer and soap.
6. Each plate was sealed with parafilm.
7. The plates was inverted and incubate at 37˚C for 24 hours.

C. Gram staining
1. The inoculating loop was used and 1 drop of sterile water was added to the slide.
Prepare a smear of:
a) Escherichia coli and b) staphylococcus aures
2. Air dry and heat fix.
3. The smear was covered with Crystal Violet (primary stain) for 1 min
4. The slide was washed with water.
5. Grams’Iodine (mordant) was added for 1 min.
6. Then wash with water.
7. Decolorize with 95% ethanol. This is the “tricky” step. Stop decolorizing with
alcohol as soon as the purple color has stopped leaching off the slide then
immediately wash with water.
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 bloted.
11. The smear was observed using the light microscope. The smear was viewed up to
100x immersion oil.
 Results

A. Streak plate technique B. Effect of handwashing on C. Gram staining

bacteria on thumb

a) Escherichia coli

b) Staphylococcus aureus

Discussion

Part 1: Aseptic Technique

Streak plate technique is used to grow bacteria on a growth media surface so that
individual bacterial colonies are isolated and sampled. Isolated colonies indicate a clone of
cells, being derived from a single precursor cell. When the selected culture media is
inoculated using a single isolated colony, the resulting culture grows from that selected single
clone. The modern streak plate method has evolved from the efforts by Robert Koch and
other microbiologists to obtain pure culture of bacteria in order to study them. The dilution or
isolation by streaking procedure was originally developed by Loeffler and Gaffky in Koch's
laboratory, which involves the dilution of bacteria by systematically streaking them over the
surface of the agar in a petri dish to obtain isolated colonies which will subsequently grow
into mass of cells, or isolated colonies. If the agar surface grows microorganisms which are
all the genetically same, the culture is then considered as a pure culture..

In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated

microbial culture. The process is called "picking colonies" when it is done from an agar plate
with isolated colonies and is transferred to a new agar or gelatin plate using a sterile loop or
needle. The inoculating loop or needle is then streaked over an agar surface. On the initial
region of the streak, many microorganisms are deposited resulting in confluent growth or the
growth of culture over the entire surface of the streaked area. The loop is sterilized by heating
the loop in the blue flame of the Bunsen burner, between streaking different sections, or
zones and thus lesser microorganisms are deposited as the streaking progresses. The
streaking process will dilutes out the sample that was placed in the initial region of the agar
surface. There are two most commonly used streak patterns, a three sector "T streak " and a
four quadrant streak methods.

Streaked plate are incubated at 37°C for 24 hours. The colonies grown in the plate
was examined carefully. Based ob my result, the water and hand sanitizer do not show any
effect of bacteria on thumb. In generally, hand sanitizer is the least effect of bacteria on
thumb. Control, soap and water section were effected by colonies of bacteria. So, the most
effective handwashing is sanitizer.

Part 2: Gram Staining

Gram-positive bacteria have a thick mesh-like cell wall which is made up of

peptidoglycan (50-90% of cell wall), which stains purple. Peptidoglycan is mainly a
polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic
acid. As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of
peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell
wall. The thick peptidoglycan layer of Gram-positive organisms allows these organisms to
retain the crystal violet-iodine complex and stains the cells as purple. Lipoteichoic acid
(LTA) is another major constituent of the cell wall of Gram-positive bacteria which is
embedded in the peptidoglycan layer. It consists of teichoic acids which are long chains of
ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as regulator of autolytic
wall enzymes (muramidases: Bacterial enzymes located in the cell wall that cause
disintegration of the cell following injury or death.)

Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall)
and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but
retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional
outer membrane which contains lipids, which is separated from the cell wall by means of
periplasmic space.Based on this experiment, E coli show the gram-negative bacteria while
staphylococcus aureusis is gram-positive bacteria.
Conclusion

In the conclusion, sanitizer is a good effective handwashing because it contain least

colonies of bacteria. In gram staining experiment, we conclude that the E coli is gram-
negative bacteria while staphylococcus aureusis is gram-positive bacteria.

Reference

 Microbial life. What is gram stainin?. 3 November 2016. From

https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html

 amrita. Gram stain technique. 20 January 2018. Form

http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1

 Wikipedia. Streaking(microbiology). 1 December 2017. From

https://en.wikipedia.org/wiki/Streaking_(microbiology)

Reflection

This experiment, I have been done from the previous semester. So I have an
experience in handling this experiment. The worst part is we need to careful while handling e
coli because e coli is bacteria that can harmful human. After making this experiment, I have
observed that thumb print without washing with water has more bacteria compare to thumb
that has been wash with sanitizer. So the lesson learn is we must wash hand before eat to
prevent food poising.