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Gram Stain

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Purpose Statement
To study the gram staining technique.TYPES OF CELL WALLS
The bacterial cell walls are divided in two types. The gram-positive cell wall is composed predominantly of peptidoglycan and is quite thick. The peptidoglycan or murein functions to provide rigidity to the cell and is composed of N-acetyl muramic acid and N-acetyl glucosamine in alternating chains( Silhavy,2015). Their matrix is embedded with polysaccharides, and there is also a presence of teichoic acid and variants which lend it antigenic property.
Figure 1: the structure of gram positive wall.
The gram-negative wall is thin, and peptidoglycan forms a very small part of it, but it has several other components , an outer membrane lies outside the peptidoglycan and is principally composed of proteins and lipopolysaccharides that lend it its negative charge and the lipid A component provides toxicity to the bacteria. The periplasmic space between the outer membrane and the cytoplasmic membrane has many proteins that gather nutrition for the bacteria and help in peptidoglycan synthesis (Silhary et al. 252).
Figure 2: the structure of gram-negative cell wall.
MECHANISM OF GRAM STAINING AND CHANGES IN CELL WALL
In the first step of gram staining, crystal violet is applied to the specimen, and it breaks down into CV+ and Cl-. The CV+ ions and negative charges in the peptidoglycan layer that are present on bacterial cell walls of both kinds interact with each other and the cells are stained purple.

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Iodide is then applied next, the CV+ and I-then form a large complex. In the next step, a rapid decolorizing agent like alcohol or acetone is added. In case of a gram-positive bacteria alcohol causes dehydration trapping the CV+I- and remaining purple colored, whereas the outer lipopolysaccharide component is washed away in gram-negative cells since the alcohol dissolves the lipid present in the outer membrane thus making it porous and allowing the crystal violet to be removed along with CV+I- complexes and so the purple color is also washed away. In the last step, counterstaining with safranin is done, and gram negative cells take it up and are stained pink (Beveridge 848-52).
The main purpose served by gram stain in modern day microbiology is that it acts as a broad classification for different kind of bacteria being quicker than growing a culture. In medical practice, it helps to pinpoint the causative agent and rational prescription of medicines.
This method was produced as a result of work done by Hans Christian Gram and Carl Friedlander in Berlin in the year 1884.
MATERIAL AND METHODS
In the experiment, crystal violet was applied to two different smears, after which iodine was added and then acetone was used as a decolorizing agent and finally the smear were counterstained with safranin.
RESULTS AND CONCLUSIONS
Under the microscope, the bacteria are visible as pink and hence are classified as Gram-negative bacteria. The nature of the stain taken up by the bacteria gives a clue about their classification. These findings can be confirmed by bacterial culture.

Figure 3: View of Gram stain bacteria under the microscope
Works Cited
Beveridge TJ, Davies JA. Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain. Journal of bacteriology. 1983. Print. 156 (2): 846–58
Silhary, Thomas J. Daniel Kahne, and Suzanne Walker. The Bacterial Cell Envelope. Cold spring Harber Perspectives in Biology. 2010. Print.

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