Gram Staining:

The Gram stain was first used in 1884 by Hans Christian Gram (Gram,1884). Gram was searching for a
method that would allow visualization of cocci in tissue sections of lungs of those who had died of pneumonia.
Already available was a staining method designed by Robert Koch for visualizing turbercle bacilli. Gram devised his
method that used Crystal Violet (Gentian Violet) as the primary stain, an iodine solution as a mordant followed by
treatment with ethanol as a decolorizer. This staining procedure left the nuclei of eukaryotic cells in tissue samples
unstained while the cocci found in the lungs of those who had succumbed to pneumonia were stained blue/violet. Gram
found that his stain worked for visualizing a series of bacteria associated with disease such as the “cocci of suppurative
arthritis following scarlet fever”. He found however that Typhoid bacilli were easily decolorized after the treatment
with crystal violet and iodine, when ethanol was added. We now know that those organisms that stained blue/violet
with Gram’s stain are gram-positive bacteria and include Streptococcus pneumoniae (found in the lungs of those with
pneumonia) and Streptococcus pyogenes (from patients with Scarlet fever) while those that were decolorized are
gram-negative bacteria such as the Salmonella Typhi that is associated with Typhoid fever.
Purpose
The Gram stain is fundamental to the phenotypic characterization of bacteria. The staining procedure differentiates
organisms of the domain Bacteria according to cell wall structure. Gram-positive cells have a thick peptidoglycan layer
and stain blue to purple. Gram-negative cells have a thin peptidoglycan layer and stain red to pink.
Theory
The Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining
procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated
according to cell wall composition. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan
(90% of cell wall). These stain purple. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of
wall), and high lipid content. These stain pink. This staining procedure is not used for Archeae or Eukaryotes as both
lack peptidoglycan. The performance of the Gram Stain on any sample requires four basic steps that include applying a
primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Gram’s Iodine), rapid
decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, counterstaining with safranin.
Details of the chemical mechanism of the Gram stain were determined in 1983 (Davies et al.,1983 and
Beveridge and Davies, 1983). 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 (I- or I3-) interacts with CV+ to
form large CVI 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 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 (Dmitriev, 2004).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-layered
peptidoglycan 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. At the completion of
the Gram stain the gram-positive cell is purple and the gram-negative cell is pink to red.
Some bacteria, after staining with the Gram Stain yeild a pattern called gram-variable where a mix of pink and purple
cells are seen. The genera Actinomyces, Arthrobacter, Corynebacterium, Mycobacterium, and Propionibacterium have
cell walls particularly sensitive to breakage during cell division, resulting in gram-negative staining of these gram-

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positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness during
growth coincides with in increasing number cells that stain gram-negative (Beveridge, 1990). In addition, in all
bacteria stained using the Gram stain, the age of the culture may influence the results of the stain
Some bacteria do not stain as expected with the Gram stain. For example, members of the genus Acinetobacter
are gram-negative cocci that are resistant to the decolorization step of the Gram stain. Acinetobacter spp. often appear
gram-positive after a well prepared Gram stain. For Mycobacterium spp., the waxy nature of the coat renders the
bacteria not readily stainable with dyes used in the Gram stain, though the bacteria are considered to be gram positive.
Gardnella has an unusual gram-positive cell wall structure that causes bacteria of this genus to stain gram-negative or
gram-variable .
The reagents listed below can be made or purchased commercially from biological supply houses
1. Primary Stain: Crystal Violet
Staining Reagent: Solution A for crystal violet staining reagent
Crystal violet (certified 90% dye content), 2g
Ethanol, 95% (vol/vol), 20 ml
Solution B for crystal violet staining reagent: Ammonium oxalate, 0.8 g and Distilled water, 80 ml Mix A and B to
obtain crystal violet staining reagent. Store for 24 h and filter through paper prior to use.
2. Mordant: Gram's Iodine : Iodine, 1.0 g, Potassium iodide, 2.0 g , Distilled water, 300 mlGrind the
iodine and potassium iodide in a mortar and add water slowly with continuous grinding until the iodine is dissolved.
Store in amber bottles.
3. Decolorizing Agent: Ethanol, 95% (vol/vol)
*Alternate Decolorizing Agent: Some professionals prefer an acetone decolorizer while others use a 1:1
acetone and ethanol mixture. Commercially, a variety of mixtures are available, most using 25 – 50% acetone with the
ethanol. A few include a small quantity of isopropyl alcohol and/or methanol in the formulation.(Acetone, 50 ml ,
Ethanol (95%), 50 ml)
4. Counterstain: Safranin (Stock solution: 2.5g Safranin O 100 ml 95% Ethanol )
Working Solution: 10 ml Stock Solution and 90 ml Distilled water

PROTOCOL
1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note
that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram's iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent. Wait 15 seconds or add drop by drop to slide until decolorizing
agent running from the slide runs clear.
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then
blot dry with absorbent paper.
8. Observe the results of the staining procedure under oil immersion using a Brightfield microscope. At the
completion of the Gram Stain, gram-negative bacteria will stain pink/red and gram-positive bacteria will stain
blue/purple.