NaturalGreen

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<b><span style="font-size: 14.0pt; line-height: 115%;">Gram staining</span></b><span style="font-size: 14.0pt; line-height: 115%;"> is a differentiate staining
technique which is used to differentiate bacteria into two groups such as gram
positive and gram negative based on their ability to retain color during
staining. <o:p></o:p></span></div>
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<span style="font-size: 14.0pt; line-height: 115%;">                              <b>Gram negative bacteria </b>have outer lipid
membrane which surrounds the cell membrane. Only 10% of their cell wall is
comprised of peptidoglycan.  Ex:
Salmonella, Shigella, Escherichia coli (E. coli). Gram negative bacteria are
stained in pink color. <o:p></o:p></span></div>
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<span style="font-size: 14.0pt; line-height: 115%;">                         
<b>Gram positive bacteria</b> have
no outer lipid membrane. 80-90% of their cell wall is comprised of
peptidoglycan.</span> <span style="font-size: 14.0pt; line-height: 115%;">Ex:  Bacillus, Clostridium, Lactobacillus,
Listeria, Taphylococcus, Streptococcus, and Streptomyces. Gram positive
bacteria are stained in purple color.</span></div>
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<b><span style="font-size: 14.0pt; line-height: 115%;">Procedure-</span></b><span style="font-size: 14.0pt; line-height: 115%;"> A loopful from a broth or agar plate
is taken into a glass slide. Then the smear is prepared by heat fixing at
bunson flame. As the proteins of the cell wall are denatured by heat, it
supports the bacterium cells to adhere to the slide. The smear is stained by
flooding with crystal violet. It is allowed crystal violet to react with smear
for 2 minutes. Gram’s iodine is added to from large Crystal violet iodine
complex. It is allowed iodine to react for 1minutes. Excess</span> <span style="font-size: 14.0pt; line-height: 115%;">Crystal violet iodine complex is
drained from the slide.  The smear is
decolorized by adding 95% ethyl alcohol without washing for 3o seconds. The
stain from the cell wall is rinsed by water. Safarin is added as the
counterstain. It is allowed safarin to react with smear for 30 seconds. It is
rinsed by water and allows air dry. The stained smear is observed under dry
high, mid or lower power objectives or oil immersion objectives. <o:p></o:p></span></div>
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<span style="font-size: 14.0pt; line-height: 115%;"><b><u><span style="font-size: 20.0pt; line-height: 115%;">Observations:</span></u></b></span></div>
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<b><span style="font-size: 14.0pt; line-height: 115%;">Crystal violet</span></b><span style="font-size: 14.0pt; line-height: 115%;">- Crystal violet is added as the
primary stain. It is a basic stain. Its positively charged ions penetrate the
cell walls of both gram positive and gram negative and bond to the negatively
charged cell components. So both gram positive and gram negative cells are
stained in purple color. <o:p></o:p></span></div>
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<b><span style="font-size: 14.0pt; line-height: 115%;">Gram’s Iodine</span></b><span style="font-size: 14.0pt; line-height: 115%;">- It is added as the mordant. It
increases the affinity of ions of the primary stains to cell wall via reaction
with crystal violet ions. It forms large Crystal violet iodine complex. Both
gram positive and gram negative cells are still stained in purple color.<o:p></o:p></span></div>
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<b><span style="font-size: 14.0pt; line-height: 115%;">95% Ethyl alcohol</span></b><span style="font-size: 14.0pt; line-height: 115%;">- In the case of gram negative
bacteria, ethyl alcohol is able to dissolve the outer lipid membrane. It left
the cell membrane exposure. It increases the porosity of the cell wall. So
after addition of 95% Ethyl alcohol, water is able to wash away large Crystal
violet iodine complex from the thin peptidoglycan layer.  So after water resining, the smear appears in
colorless.<o:p></o:p></span></div>
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<span style="font-size: 14.0pt; line-height: 115%;">                                    Ethyl
alcohol acts as dehydrate agent in gram positive bacteria. It causes the pores
in the cell wall to shrink. So water is unable to wash away large Crystal
violet iodine complex from the thick peptidoglycan layer. So after water
resining, the smear still remain in purple color. <o:p></o:p></span></div>
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<span style="font-size: 14.0pt; line-height: 115%;"><b><span style="font-size: 14.0pt; line-height: 115%;">Safranin</span></b><span style="font-size: 14pt; line-height: 115%;">- Safranin is added as counter stain.
Gram negative bacteria appear in pink color. Gram negative bacteria are unable
to stain in safranin and still appear in purple color.</span> <o:p></o:p></span></div>

Gram staining

Gram staining is a differentiate staining technique which is used to differentiate bacteria into two groups such as gram positive and gram...

Gram staining

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