EXPERIMENT No.

09
GRAM STAINING OF BACTERIA

APPARATUS/ MATERIALS REQUIRED:

 Slide
 Bunsen burner
 Loop wire
 Dropper
 Microscope
 Cover slip
 Laminar flow cabinet
 Bacterial cultures
 Different stains

THEORATICAL BACKGROUND:
Gram Staining
The technique used to differentiate between gram positive and gram negative bacteria. In this
method, we use the primary stains, a decolorizer and a counter stain. Gram positive bacteria
will be stained purple while gram negative bacteria will be stained pink or red.
Most commonly used stains are:
 Crystal violet
 Methylene blue
 Carbol fuchsin
 Feulgen stain
 Safranin
 Hematoxylin etc.

PRINCIPLE:
Primary stain (Crystal Violet)

The primary stain of the Gram's method is crystal violet. Gram staining is based on the ability of
bacteria cell wall to retain dye i.e., the crystal violet during solvent treatment. The cell walls for
Gram-positive bacteria have a higher peptidoglycan and lower lipid content than gram-negative
bacteria.
By applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat
fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don’t
rinse out during the staining procedure. The addition of iodine binds to crystal violet and traps
it in the cell.

Iodine treatment
Iodine is used in gram staining procedure to differentiate gram positive and gram negative
organisms. Iodine acts as a mordent to form the crystal violet complex that enhances the dye
(crystal violet) to penetrate through the pores present in the cell wall/ cell membrane. So the
dye cannot be removed easily.
Decolorizer
Ethyl alcohol or acetone can be used as the decolorizing agent. The alcohol dissolves with lipids
found in the outer cell membrane of gram negative bacteria, allowing the crystal violet – iodine
complex to leak out of the thinner peptidoglycan layer.
Counter stain – Safranin
A counter stain such as the weakly water soluble safranin is added to the sample, staining it
red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in
gram positive cells. However, the decolorized gram negative cells are stained red.

PROCEDURE:
1. I Prepared a bacterial smear from specimen of bacterial culture (bacterial suspension).
I. Add one lope full of specimen of bacterial suspension onto a glass slide.
II. Allow it to air-dry.
III. Heat fix the specimen onto the glass slide, unless the specimen is heat-fixed, the
bacterial smear will wash away during the staining procedure.
2. I saturated the smear with crystal violet for 1 minute and rinsed the slide with tap water
to remove the excess crystal violet.
3. After that I saturated the slide with iodide for 1 minute. Again rinsed with tap water to
remove the excessive iodide.
4. Then I saturated the slide with decolorizer (Alcohol/ acetone) for 30 seconds.
5. After that I counter stained the slide with safranin for 1 minute and rinsed slide with
water.
6. After drying, observed the slide under microscope and noticed either the sample was
gram-positive or gram-negative bacteria.
Result/ Inference:
I performed gram staining of bacterial culture and observed the nature of bacterium (either
gram + ive or gram – ive) after preparing the slide and observed it under microscope.

Precautions:
1. Smear should be accurately fixed.
2. Do not put excessive stain, use dropper for this purpose.
3. Wash with iodine and decolorizer using dropper.
4. Let the stain dry then wash it.
5. Focus the microscope appropriately and observe the color carefully.