DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE & MATHEMATICS

UNIVERSITI PENDIDIKAN SULTAN IDRIS

SBL 1023

LABORATORY TECHNIQUE

IN BIOCHEMISTRY

STUDENT’S NAME NAZIRA BINTI NASLAMROS

LECTURER’S NAME ASSOC.PROF SHAKINAZ DESA
MATRIC NO E20161013757
LABORATORY MP 13
Introduction

Bacteria, yeast and fungi, protozoa and viruses are known as microorganism.

These are most commonly found microorganisms in the surrounding, capable of

settling and living in various environments such as soil, water, plant and animal

tissues also human body. Some simple methods include cultivation and microscopic

techniques could be applied to identify and isolate most of microbiological culture.

When working in a microbiology laboratory, aseptic technique is fundamental

to the safety and success of an experiment. Generally, aseptic techniques performed

by scientist under sterile conditions to ensure that microbial contaminants not doing

harm to the colleagues, supply or experimental cultures. One of the aseptic

technique is streak plate. The streak plate method is a rapid qualitative isolation

method commonly used for isolation of discrete colonies that require the number of

organisms in the inoculums be reduced. Inoculation is performed from a sector in

order to isolate a pure culture.

Also in this lab, we are going to determine the effect of handwashing on

bacteria on thumb. People used their hands for variety of activities everyday so it is

extremely easy to come in contact with microbes. Handwashing is thought to be

effective for the prevention of pathogens transmission. However, there is no

conclusion that handwashing with soap is more effective at reducing bacteria

contamination than using water only.

The gram stain is a differential stain commonly used to differentiates bacteria

on the basis of their cell wall structure.

 The gram stain is key procedure in the identification of bacteria based on

staining characteristics, and enable the bacteria to be examined using light

microscope. Most bacteria can be divided into 2 groups based on the cell wall

structure.

Gram staining positive cell walls have single membrane enclosed by thick,

cross link peptidoglycan. The thick peptidoglycan takes up dye and appears in

purple. Alcohol cannot wash out the dye-mordant complex because it firmly secured

in the thick peptidoglycan layer. Saturated with crystal violet dye. The cell counter

stain has no little to effect. Cell wall ranges in colour from mid to dark purple.

Gram staining negative cell wall have thin layer of peptidoglycan in the

periplasmic space within its inner and outer lipid membrane. Cell wall takes up dye. It

appears in purple. Dye and mordant complex, but does not adhere to the thin layer

of peptidoglycan. It easily removed by alcohol and turn to colourless. It easy to take

the counterstain. It colour with safranin and turn to pink.

Objective :

 To produce isolated colonies of an organisms ( bacteria ) on an agar plate

using streak plate technique

 To determine the effect of handwashing using water, hand soap and hand

sanitizer on bacteria.

 To differentiate between the 2 major categories of bacteria which is gram

positive and gram negative.

 To understand how gram stain affects the gram (+) and gram (-) based on the

structural differences of their cell wall

Result :

1. Streak plate technique.

2. Effect of handwashing using water, hand soap and hand sanitizer on

bacteria.

3. Gram staining
Discussion :
Aseptic techniques are performed to prevent contamination of the growth
medium. Bacteria often occur in mixed populations and very rare to find a single
species of bacteria. So, it is vital to divide the bacteria from the other species that
originate in the environment so that we can study the cultural, physiological and
morphological characteristic of the individual species. The streak plate method is
commonly used as isolation method to grow bacteria on a media surface (agar plate)
so that individual bacterial colonies are isolated and can be sampled.
When the selected culture media is inoculate using a single isolated colony,
the resulting culture will grow from the selected single colony. This techniques is
useful when we need to separate an organism in a mixed culture. It is also useful
when we need to study the colony morphology of an organism because biochemical
test are only valid when performed on pure culture. In this lab, we must use sterile
tool because the crucial aspects is to maintain a sterile work area surrounding us.
Before we start the experiment, it is important to disinfect all of our work
surface using 70% ethanol or any other disinfectant reagent. Sterile technique is
absolutely necessary in many laboratory practical. It is a method that prevents
foreign cells from getting into our plates and cultures. Generally, the key to
successful experiment is to work quickly and efficiently otherwise, we will get a
contaminant resulting culture.
To differentiate between the two major categories of bacteria which is gram
positive and gram negative. S. aures are choose as experimental species. S.aures is
a facultative anaerobic that can grow without need of oxygen and coccal (round)
bacterium.
S. aures is not always pathogenic, it is a common cause of skin and
respiratory infections also food poisoning. After we examined the bacteria under light
microscope, we identifying the bacteria to be gram- positive bacteria as it stains
purple in colour. Gram-positive differ from gram-negative through their cell walls
structure and composition. Gram-positive bacteria have a thick cell walls made up of
peptidoglycan layer beyond the plasma membrane which is cross linked by short
chains of peptides. It is then resulting in the shape and rigidity of the cell wall. The
thick layer of peptidoglycan allows to retain the dye-mordant complex (crystal violet
and iodine). The alcohol cannot wash out the dye-mordant complex because it firmly
secured in the thick layer of peptidoglycan. As a result the cells are stain as purple
Conclusion:

Staining is a technique used to enhance the clarify of the microscopic image. Gram

stain is a very important preliminary step in the classification and characterization of

bacteria. Gram stain procedure enables bacteria to retain the colour of stains based

on the differences in the composition of their cell walls. From this experiment, we can

differentiate between the two major categories (gram-positive and gram-negative).

We can obtain the individual colonies from a bacterial culture using streak

plate method. This is ideal for experimentation because the colonies are genetically

identical. When doing a microbiological laboratory, there are many aspects that we

need to pay attentions. Firstly, it is very crucial to maintain a sterile work area

surrounding us. This requires common sense and a continuous practices.

Reference :

 https://microbeonline.com/streak-plate-method-principle-purpose-procedure

 http://www.austincc.edu/microbugz/streak_isolation.php 3.

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