Answer / visweswara rao

Gram's staining is the one used for staining the bacteria
to determine whether the bacteria is Gram positive or Gram
negative.

the process of Gram staining is
1) Take a loopful of bacterial culture and place it on
the clean and sterilised slide.
1) Fix smear by heat.
2) apply crystal violet.
3) Wash with water
4) Cover with Gram’s iodine
5) Wash with water. do not blot.
6) Decolourize for 10-30 sec with gentle agitation in
acetone(30ml)and alcohol(70ml).
7) Wash with water. do not blot.
8) Cover for 10-30 sec with safranin(2.5% solution in
95% alcohol.)
9) Wash with water and let dry.

The bacteria depend on the colour absorbtion by the cell
wall is classified into two types. They are 1.Gram psoitive
bacteria (purple) 2. Gram negative bacteria(red or pink
colour)

Answer / r.rajani

Gram staining is based on the ability of bacteria cell wall
to retaining the crystal violet dye during solvent
treatment. The cell walls for Gram-positive microorganisms
have a higher peptidoglycan and lower lipid content than
gram-negative bacteria. Bacteria cell walls are stained by
the crystal violet. Iodine is subsequently added as a
mordant to form the crystal violet-iodine complex so that
the dye cannot be removed easily. This step is commonly
referred to as fixing the dye. However, subsequent treatment
with a decolorizer, which is a mixed solvent of ethanol and
acetone, dissolves the lipid layer from the gram-negative
cells. The removal of the lipid layer enhances the leaching
of the primary stain from the cells into the surrounding
solvent. In contrast, the solvent dehydrates the thicker
Gram-positive cell walls, closing the pores as the cell wall
shrinks during dehydration.

Answer / biomaster

1) Fix smear by heat.
2) Cover with crystal violet.
3) Wash with water
4) Cover with gram’s iodine
5) Wash with water. do not blot.
6) Decolorize for 10-30 sec with gentle agitation in
acetone(30ml)and alcohol(70ml).
7) Wash with water. do not blot.
8) Cover for 10-30 sec with safranin(2.5% solution in
95% alcohol.)
9) Wash with water and let dry.

Answer / megha

Gram staining is one such technique which is used to
differentiate between gram-positive and gram-negative
bacteria.
itis also called as differential staining.
it involves the followong steps:
1. take a clean greese free slide
2.place a drop of sterile water
3.please a loopful of culture on it and make a thin smear
4.heat the smear,till it gets dried
5. add a drop of crystal violet stain and allow it to stand
for 1min.
6.wash the slide, with a distilled water
7.now add, few drops of gram's iodine and allow itto stand
for 1 min.
8.wash the slide
9.add a counterstain saffranin and allow it to stand for a
min.
10.Wash the slide andallow to aid dry/
11. add a drop of cedarwood oil & observe under oil
immersion microscope.

Answer / santhosh kumar guptha

Classic Gram staining techniques involves following steps:  

Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material).

Application of the primary stain (crystal violet).Crystal violet stains all cells blue/purple

Application of Mordant: The iodine solution (mordant) is added to form a crystal violet iodine (CV-I) complex; all cells continue to appear blue.

Decolorization Step: The decolorization step distinguishes gram-positive from gram-negative cells.The organic solvent such as acetone or ethanol, extracts the blue dye complex from the lipid-rich, thin walled gram negative bacteria to a greater degree than from the lipid poor, thick walled, gram-positive bacteria.  The gram negative bacteria appear colorless and gram positive bacteria remain blue

Application of counter stain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.

Principle of Gram Stain

Cell wall of Gram Positive and Gram Negative Bacteria

The differences in cell wall composition of Gram positive and Gram negative bacteria accounts for the Gram staining differences. Gram positive cell wall contain thick layer ofpeptidoglycan with numerous teichoic acid cross linking which resists the decolorization.In aqueous solutions crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both gram-positive and gram-negative cells. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple.When added, iodine interacts with CV+ to form large crystal violet iodine (CV-I) complexes within the cytoplasm and outer layers of the cell. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram negative bacteria.The outer membrane of the gram-negative cell (lipopolysaccharide layer)  is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.The highly cross-linked and multi-layeredpeptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell.After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added. 

Answer / kaynat chainwala

FIRSTLY TAKE A LOOPFUL OF CULTURE ON A GLASS SLIDE,THEN ADD
FEW DROPS OF CRYSTAL VIOLET FOR 5 MINS......THEN DISCARD
EXCESS STAIN AND THEN FEW DROPS OF ACETOCARMINE
STAIN.........N THEN KEEP IT FOR 2 MINS.......WASH THE SLIDE
N PUT A DROP OF CEDARWOOD OIL AND OBSERVE IN OIL IMMERSION
MICROSCOPE{100 X}

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