The document describes the Gram staining procedure, which is used to classify bacteria as either gram-positive or gram-negative. The procedure was developed by Hans Christian Gram in 1884 and involves staining bacteria samples with crystal violet dye followed by iodine to form a complex within the cell. Gram-positive bacteria retain the stain due to their thicker peptidoglycan layer, while gram-negative bacteria wash out the stain more easily due to their thinner peptidoglycan layer. The document also discusses several modifications of the Gram staining technique.

1. BY-: Abhishek Singh
Medical Microbiology

2.  Most bacteria are classified as gram –positive or
gram-negative according to their response to the gram
staining procedure . This procedure was named for the
histologist Hans Christen Gram (1884)who
developed this differential staining procedure in an
attempt to stain bacteria in infected tissues of lung of
a patient who died from pneumonia.
 This is a differential staining technique.

3.  The Gram positive cells have a more acidic protoplasm,
which accounts for retaining the basic dye more strongly
than Gram negative bacteria . Iodine makes the protoplasm
more acidic and serves as mordant, i.e. iodine combines
with dye to form a dye-iodine complex and fixes the dye in
bacterial cell. The Gram positive cell wall or cytoplasmic
membrane being less permeable the dye-iodine complex
gets trapped within the cell. Gram negative cell wall has
increased permeability to acetone or alcohol, permitting the
outflow of complex during decolorization.

4.  Fix the smear by heat.
 Cover with crystal violet.
 Wash with water. Do not blot.
 Cover with Gram’s iodine.
 Wash with water. Do not blot.
 Decolorize for 10-30sec with gentle agitation in acetone and alcohol.
 Wash with water.
 Cover for 10-30sec with safranine.
 Wash with water.

6. The smear given to me shows GPC in
pairs.

7. There have been several modifications of Gram's stain. These are:
 Kopeloff and Beerman's modification: Primary stain solution consists of freshly
constituted methyl violet with sodium bicarbonate in distilled water. Mordant consists of
iodine dissolved in 4% NaOH solution. Basic fuchsin is used to counterstain the smear. This
method may be modified to stain tissue sections.
 Jensen's modification: This method involves use to methyl violet as primary stain,
iodine and potassium iodide in water as mordant, and neutral red as counterstain. For Neisseria
spp, Sandiford's counterstain is useful.

8.  Weigert's modification: This modification is particularly useful for staining tissue
sections. The primary stain carbol gentian violet is prepared using saturate alcoholic solution
of gentian violet and 5% phenol solution. Gram's iodine is used as a mordant and aniline-xylol
is used as a decolorizer. The counterstain carmalum (carminic acid and potassium alum in
water), however is used ahead of primary stain. This method may be used to stain
Pneumocystis cysts.
 Preston and Morrell's modification: The primary stain used in this modification is
ammonium oxalate-crystal violet. The smear is washed in Lugol's iodine and further treated
with iodine solution. The smear is decolorized using iodine-acetone decolorizer and
counterstained using dilute carbol fuchsin solution. This method has been further modified to
overcome the irritating iodine in aerosols by reducing the iodine concentration to one-tenth
and shortening the duration of decolorization to ten seconds.

9.  Use actively growing cells (18-24 hrs old)-->old cells lose their ability to hold the stain;
appear Gram negative.
 Prepare thin smears and adjust decolorization time-->when cells are crowded, they resist
decolorization. Bacteria in thin smears decolorize faster than thick smears.
 Avoid overheating cells-->excessive heat disrupts cell walls, G+ appear G-.
 Don't rinse too long-->water is a decolorizer.

10.  Violet dye
 Crystal violet - 10g
 Absolute alcohol - 100 ml
 Distilled water - 1L
Iodine solution
Decolourizer
Counter stain.

11. Staining is restricted to only bacteria and
few yeast.

12.  Identification of bacteria in clinical specimens.
 Demonstrating the morphology of bacteria on the basis of
colour & shape.
 It distinguish two categories
 Gram positive
 Gram negative
 Choice of culture media for inoculation.