GRAM STAINING
AIM – To stain bacteria by Gram staining (Hookers modification) method
INTRODUCTION – Gram staining is a method of staining used to differentiate bacterial
species into two large groups (gram-positive and gram-negative). The name comes from the
Danish bacteriologist Hans Christian Gram, (1853–1938), who developed the technique while
working with Carl Friedländer in the morgue of the city hospital in Berlin in 1884. Gram's
original method utilised aniline-gentian violet as the first stain, with Lugol's iodine as the
mordant. This was then decolourised with absolute ethanol and counter-stained with Bismarck
brown.
Gram staining differentiates bacteria by the chemical and physical properties of their cell walls
by detecting peptidoglycan, which is present in a thick layer in gram-positive bacteria. Gram-
positive bacteria retain the crystal violet dye, while a counterstain (commonly safranin or
fuchsine) added after the crystal violet gives all gram-negative bacteria a red or pink coloring.
The Gram stain is almost always the first step in the preliminary identification of a bacterial
organism. While Gram staining is a valuable diagnostic tool in both, clinical and research
settings,not all bacteria can be definitively classified by this technique. This gives rise to gram-
variable and gram-indeterminate groups.
FACTORS AFFECTING GRAM STAINING
1. If the smear is too thick, proper decolorizing will not be possible.
2. If the smear is overheated during heat fixing, the cell walls will rupture.
3. Concentration and freshness of reagents may affect the quality of the stain.
4. Washing and drying of the smear between steps should be consistent. Excess water left on
the slide will dilute reagents, particularly Gram's iodine.
5. Gram stain is reliable only on cells from cultures that are in the exponential phase of growth.
Older cultures contain more ruptured and dead cells. Cells from old cultures may stain Gram
negative even if the bacteria are Gram positive.
MECHANISM – Crystal violet (CV) dissociates in aqueous solutions into CV+
and chloride (Cl−) ions. These ions penetrate through the cell wall and cell membrane of both
gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged
components of bacterial cells and stains the cells purple. Iodide (I−or I−3) interacts with CV+ and
forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the
cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of
the CV–I complex and, therefore, color the cell.
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell
membrane. A gram-negative cell loses its outer lipopolysaccharide membrane, and the inner
peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative
cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from
an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell
due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must
be timed correctly; the crystal violet stain is removed from both gram positive and negative cells
if the decolorizing agent is left on too long (a matter of seconds) After decolorization, the gram-
positive cell remains purple and the gram-negative cell loses its purple color. Counterstain,
which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized
gram-negative bacteria a pink or red color
REQUIREMENT - Primary stain: Crystal violet - Crystal violet powder - 5g; Distilled water - 1
liter
Mordant: Iodine solution - Iodine - 10g; Potassium iodide (KI) - 20g; Distilled water - 1 liter
(Dissolve the potassium iodide in 250ml water, then add the iodine. When fully dissolved, add
the remaining water to make 1 liter. Leave to stand overnight before use.)
Decolouriser : 95% ethanol
Counter-stain: Neutral red/Safranin - Neutral red/safranin powder - 1g ; Distilled water - 100ml ;
Acetic acid 1% solution - 2ml
PROCEDURE -
1. Place slide with heat fixed smear on staining tray.
2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
4. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle. The smear will appear as a purple circle on the slide.
5. Decolorize using 95% ethyl alcohol or acetone.
6. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the
alcohol runs almost clear OR for 45 seconds. Be careful not to over-decolorize.
Immediately rinse with water.
7. Gently flood with safranin to counter-stain and let stand1min. Tilt the slide slightly and
gently rinse with tap water or distilled water using a wash bottle.
8. Blot dries the slide with bibulous paper. View the smear using a light-microscope under
oil-immersion.
DIAGRAM-
OBSERVATION –Gram positive (voilet) rods and gram negative (pink) rods are observed in
microscopic field
RESULT – Bacteria are stained by Gram staining method
1. Write 3 names of Gram positive rods
2. Write 3 names of Gram negative rods
3. Write 3 names of Gram positive cocci
4. Write 3 names of Gram negative rods
