CONCLUSION

From the experiment that we have done, finally, we can conclude that Gram staining
is the method of distinguishing between Gram-positive and Gram-negative bacteria. Grampositive bacteria have a thicker peptidoglycan which makes up their cell walls but have
greater lipid content since these bacteria have an outer membrane. The differences in their
cell wall architecture cause them to look different from one another by performing Gram
staining procedure.
Escherichia coli appeared reddish pink which indicate that these bacteria do not bind
the crystal violet stain. Therefore, it is determined to be a Gram-negative bacteria. The
decolourizer extracted the lipid from outer membrane of Gram-negative bacteria washed off
the crystal violet-iodine complex. Safranin then was used to counterstain Escherichia coli
leaving the colour pink.
Staphylococcus aureus absorbed the purple colour from crystal violet. This indicates
Staphylococcus aureus is Gram-positive bacteria which have thicker peptidoglycan in their
cell wall that trap the crystal violet-iodine complex more effectively, making them resistant to
the decolourizer.
In this experiment, we also have provided some material to help us for reaching the
aim of this experiment such as Huckers crystal violet, Gram iodine, 95 % ethyl alcohol,
safranin, and microscopic slide, staining rack and bibulous paper.
However, before doing the experiment, we absolutely need to pay attention on the
precautions. There are several procedures that we have to do in order to avoid the error in this
experiment, such as prepare smear from cultures of microorganisms, heat fix smears, place
slides on a staining rack and so on.

REFERENCES
1. Leboffe M.Pierce BE. 2010 .Gram Stain, P. 105-109. In Microbiology Lab Theory
and Application, 3rd ed., Morton Publishing CO., Englewood, Co.
2. Cappucino G.James, Sherman Nataline, 7th edition, Pearson Education.
3.

QUESTIONS
1. If the iodine step were omitted what colour would expect a Gram-negative
microorganisms to be? A Gram-positive?
If the iodine in the Grams stain was omitted, the crystal violet enters the cell will
not able to form the crystal violet iodine complex. The crystal violet iodine
complex molecule has a larger size than that of crystal violet molecule and hence
Gram-positive bacteria are able to retain stain. If the iodine were omitted even
Gram-negative cells would get decolourized by alcohol treatment and would be
seen as a Gram-negative cells. Other than that, they would all be the colour of
your counterstain (usually safranin) because without the aid of Grams iodine,
which acts as a mordant (helps to set the dye into the organisms). Crystal violet
would not effectively set into the organisms, and when the decolourizer is added,
the crystal violet will be washed away and the counterstain will be the only stain
effecting the organisms.
2. Could other dyes be substituted for crystal violet Gram stain procedure? For safranin?
Explain your answer.
Crystal violet dye can be replaced with methylene blue. This is because both are
basic dye that have a positive charge that able to bind to negatively charge
particle, such as the teichoic acid at the bacterial cell wall. Therefore, methylene
blue can be used to replace crystal violet dye. Meanwhile, for safranin, it can be
replaced with basic fuchsin. Both dyes are able to stain Gram-negative bacteria

which is a pink colour but based on the information that we have searched, basic
fuchsin give much brighter colour in order for the bacteria to be seen clearly.
3. What role does the mordant play in the Gram stain procedure?
The mordant is the substance that set dyes the iodine in Gram stain fixed the
crystal violet to bacterial cell wall of the Gram-positive bacteria. When Grampositive bacteria are treated with ethanol, the alcohol is thought to shrink the pores
of thick peptidoglycan. Thus, the dye-iodine complex is retained during this short
decolourization step and the bacteria remains purple.
4. What correlation is there between cell wall composition and Gram reaction? Is Gram
reaction correlated with sensitivity too various antibacterial agents?
Crystal violet dissociate into Cl- ions and CV+ which interact with negatively
charged components of the stain in purple. Iodide ion (I-) is than react with CV+,
forming CV-I complex. Alcohol interacts with the lipid of cell membrane, causing
Gram-negative cell lose its outer lipopolysaccharides membrane, exposing the
peptidoglycan layer. Therefore, Cl-I and the outer membrane is washed away.
However, in the Gram-positive cell, the alcohol dehydrated the cell, trapping the
Cl-I because of the thick peptidoglycan. The Gram reaction is somehow correlated
with sensitivity to various antibacterial agents. Gram-positive cells are more
susceptible to mechanical breakage as the amount of peptidoglycan is small.
However, the reaction does not ensure that the same antibacterial agent is able to
destroy the two (2) different species or strain of bacteria even though they showed
the same result in the Gram reaction. This is because types of action such as injury
to the plasma membrane, inhibition of cell wall synthesis and prootein synthesis,
does not only depend on the Gram reaction.