Identification of Unknown Bacterial Samples through Gram Staining

Running head: MICROBIOLOGY
Topic: MICROBIOLOGY
Name of the Student:
Name of the University:
Author’s Note:

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Standard Operating Procedure (SOP):
Title: Identification of unknown bacterial samples through the procedure of gram staining
Standard operating procedure is defined as the set of stepwise instructions which are
compiled for proper carrying out the complex routine operations. The SOP is important as it
fruitfully explains the practices of the practices involved. They are needed for ultimately ensuring
the success of the practical.
The rationale of the experiment is the identification of the organisms from the unknown
samples containing the cultures 1 and 2. Identification is done on the basis of differentiation
between the gram positive and gram negative organism. The difference in the colors of the bacteria
in microscopy is on the basis of the stain retained through the various layers of the bacteria. There
is a distinct difference between the various composition of the cell walls of the gram negative as
well as gram positive bacteria. There is the presence of a thick layer of peptidoglycan which contain
numerous cross linking between the techoic acids. This results in the decolourization of the cell
wall. Among the aqueous solutions, there are various primary stains. In the given procedure of
gram staining, crystal violet (CV) is used. CV+ as well as Cl- ions are there which usually penetrate
through the walls and is present in the membrane of both gram positive as well as gram negative
bacteria. With addition of iodine, there is interaction of the CV+ with the various negatively
charged components present in the bacterial cells as a result of which the cells are stained with
purple dye (Gao et al. 2014).
There are four steps to be done regarding the procedures of gram staining. The first step
involves the application of a primary stain like the crystal violet which is done through heat fixation
of the bacterial culture forming a smear. It is followed by the addition of iodine, which usually
binds to the crystal violet and usually tarps in the cell. This is followed by decolourisation with
acetone as well as ethanol. Then the last procedure is counterstaining with saffranin. Sometime,
carbol fuschin is used in place of safranin as it stains more intensely than the anaerobic bacteria.

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However, it is less commonly utilized a counter stain in the process of gram staining (Hall,
McGillicuddy and Kaplan 2014).
Thus, identification of the type of gram stain retained by the bacteria helps in determining
the gram character of the unknown samples. In the given experiment since the sample has taken up
the purple color it is confirmed that the unknown bacterial sample is gram positive in nature.
Flow chart of the methodologies used in the practical:
Day 1: Plate pouring
technique using BH11 and
CLED plates
Streak plating with the
quadrant method.
Day 2: Biochemical tests
are peroformed; Gram tests,
Oxidase and Catalase tests
Spread plating is done
followed by disk diffusion
assays

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Title: Identification of unknown bacterial samples through gram staining and catalase as well
as oxidase tests and identification of their resistance to antibiotics through antibiotic
susceptibility tests.
Abstract:
The following practical aims in the identification of bacteria on the basis of biochemical
tests. The processes done are biochemical tests like the catalase and oxidase test and lactose
fermentation tests, disk diffusion assays. It has been seen that the colony 1 is Enterobacteriaceae
and colony 2 is Bacillus species.
Introduction:
The aims of the given practical in the first day has been the successful preparation of the
BHI as well as CLED agar plates. The Day 2 practical would focus around the successful
performing of the oxidase, gram and the catalase tests. Thus the hypothesis of the experiment can
be formulated as the difference between the gram and the gram negative bacteria on the basis of
various biochemical tests like the oxidase and the catalase test. Gram staining is defined as the
procedure for the differentiation of the gram negative and positive organism on the basis of the
differences between their cell wall constituents (Burillo et al. 2014). The oxidase test is another
biochemical examination for the identification of organism on the basis of production of
cytochrome C oxidase (Ali et al. 2014). Moreover, the catalase tests would also be used for the
differentiation of the aerotoleratnt strains of Clostridium species mainly. Catalase is the enzyme
present in the bacteria which is used for converting hydrogen peroxide to water whereas Oxidase C
is usually a terminal enzyme which is usually used in the respiratory chain (Tulumoglu, Kaya and
Simsek 2014).