Gram Stain
Gram Stain
Gram Stain
Staining procedure
1. Flood slide with crystal (or gentian) violet- 60 seconds.
2. Flood with Gram's iodine - 180 seconds.
3. Carefully decolorize with 95% ethanol until thinnest parts of the smear are colorless.
(Wash with water).
This third step is the most critical and also the one most affected by
technical variations in timing and reagents.
4. Flood with safranin (pink color) (10% Fuchsine) - 60 seconds. (Wash with water).
5. Air dry, or blot with absorbent paper.
Results
As shown below, organisms that retain the violet-iodine complexes after washing in
ethanol stain purple and are termed Gram-positive, those that lose this complex
stain red from the safranin counter stain are termed Gram-negative.
Negative
Positive
Mix
Procedure in Gram Stain
Step 1 - Prepare a Smear
• Suspend some of the material to be stained in a drop of water on a microscope
slide, spread the drop to about the size of a nickel.
• Allow to air dry. Heat fix by gently warming above a flame or other heat source.
Step 4 - Rinse
Rinse with water to remove excess Iodine
Step 5 - Decolorize
• Drip 95% Alcohol across the slide about 5 sec
• The effluent should appear pale or clear
Step 6 - Rinse
• Rinse with water to remove excess alcohol
Step 7 - Counterstain
• Flood the slide with Safranin solution
• Let stand 30 sec
The Gram stain differentiates bacteria into two fundamental varieties of cells.
Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-
positive," whereas those that are decolorized and stain red with carbol fuchsin (or
safranin) are said to be "gram-negative." This staining response is based on the
chemical and structural makeup of the cell walls of both varieties of bacteria.
Gram-positives have a thick, relatively impermeable wall that resists
decolorization and is composed of peptidoglycan and secondary polymers.
Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein
bilayer known as the outer membrane, which can be disrupted by decolorization.
Some bacteria have walls of intermediate structure and, although they are
officially classified as gram-positives because of their linage, they stain in a
variable manner. One prokaryote domain, the Archaea, have such variability of
wall structure that the Gram stain is not a useful differentiating tool.
Typical Gram-positive bacteria:
Generally cocci are Gram-positive but there are exceptions. The most significant from a
clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears
as a Gram-negative diplococcus looking very much like a pair of kidney bean.
Importance of a Gram Stain:
The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria
based on staining characteristics, enabling the bacteria to be examined using a light
microscope. The bacteria present in an unstained smear are invisible when viewed
using a light microscope. Once stained, the morphology and arrangement of the
bacteria may be observed as well. Furthermore, it is also an important step in the
screening of infectious agents in clinical specimens such as direct smears from a
patient.
The Gram stain procedure enables bacteria to retain color of the stains, based on the
differences in the chemical and physical properties of the cell wall.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called
Crystal Violet in the cell wall.
Example: Staphylococcus aureus
Bacterial Morphology:
Bacteria are very small unicellular microorganisms ubiquitous in nature. They are
micrometers (1µm = 10-6 m) in size. They have cell walls composed of peptidoglycan
and reproduce by binary fission. Bacteria vary in their morphological features.
Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs
(diplo-bacilli) or chains (streptobacilli).
For example: Bacillus cereus, Clostridium tetani
Spirillum (pleural: Spirilla):
Spiral-shaped bacteria