INTRODUCTION

Microbial staining method is a technique to enhance contrast in samples.

This is because most of microbial cytoplasm is usually transparent and it is
necessary to stain microorganism before they can be viewed with the light
microscope.Staining microorganisms enables to see greater contrast between
microorganisms, differentiate various micromorphological and observe certain
structures such as flagella, capsules and endospores.The aim of this
experiment is to learn more on the microbial staining method. There are three
types of microbial staining method which is simple staining , differential
staining and structure staining .This staining brings different function towards
the microorganisms.

Simple staining can be used to determine the cell shape, size and
arrangement. This method is a very simple procedure where it is directly
staining the bacterial cell with a positively dye in order to see the bacterial
details in contrast to negative staining where the bacteria remains unstained.
Gram staining is a common technique which is used to differentiate two large
groups of bacteria based on their different cell wall constituents which is gram
positive and gram negative. This is done by colouring the cells with 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 peptidoglycan wall, which does not retain the crystal
violet during the decoloring process. As for endospore staining is type of
staining which are used to visualize bacterial endospore which are usually
visible on bacterial such as Bacillus.

References

Fereshteh Azari, Michael Radermacher, Keith P. Mintz and Teresa Ruiz, Interactions between

the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional
Electron Tomography, Journal of Bacteriology, 10.1128/JB.00297-19, 201, 16, (2019).

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

https://www.austincc.edu/microbugz/endospore_stain.php
MATERIALS AND METHODS

APPARATUS

Stain solutions (Methylene blue, Crystal violet, Safranin red, ethyl alcohol,
iodine solution and malachite green solutions) , Bacterial cultures ( Bacillus
cereus , Staphylococcus aereus, Escherichia coli ( E. coli) ) , Blotting paper ,
clean glass slides, Immersion oil , Microscope

PROCEDURES

Simple staining

A smear on agar plate is prepared by using fixing method. The simple stains
is added by drop towards the bacterial cultures which is crystal violet for about
10 sec. Then , the excess of the stain is rinsed off from the slide and blot by
using the blotting paper. The smear is allowed to dry by air. The slide is being
observed using the oil immersions lens and the observation is recorded.

Gram staining

Smear is prepared on a slide. Then the slide is flooded with crystal violet
stain and leaved for about 1 minute. Then, the excess is being wash by water
and gram iodine solution is added for 1 minute. Then, the excess is being bot
by the blotting paper. The slide is being held slant and flooded with 95%
alcohol to remove the excess crystal violet. As the crystal violet dye stops
leached , the slide is washed with water and stopped the decolourisation
methods. The slide is counterstain with safranin red for about 1 minute. As the
excess of the stain is rinsed off the slide is being blot and allowed to air dry.
Endospore staining

The smear is being prepared. Then, the slide is being saturated with
malachite green solution and leaved for 1 minute. The slide is held by using
forceps and heated the slide carefully over the flame until the stain began to
steamed. Then, the slide is removed from the flame until the steaming
stopped and gently reheated which is being done for 5 minutes. After 5
minutes, the excess of stain is being washed with water and blot with blotting
paper. Then the slide is being flooded with safranin red for about 1 minutes.
Then the excess is being washed and allowed to air dry. The slide is being
viewed under immersion oil and the observation is being recorded.