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Gram Staining
What is Gram Staining?

Gram staining is a common technique used to differentiate two large groups of bacteria
based on their different cell wall constituents. It is the most important and widely used
differential staining technique in microbiology. In this process the fixed bacterial smear is
subjected to the following staining reagents in the order listed, crystal violet, iodine solution,
alcohol (decolorizing agent), and safranin or some other counterstain. The Gram stain
procedure distinguishes between Gram positive and Gram negative groups by coloring these
cells either red or violet. Gram positive bacteria stain violet due to the presence of a thick
layer of peptidoglycan in their cell walls, which retains the crystal violet. Alternatively, Gram
negative bacteria stain red, which have a thinner peptidoglycan cell wall, which does not
retain the crystal violet during the decoloring process.

History

The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938)
in 1884.

The Gram-positive includes many well-known genera such as Bacillus, Listeria,

Staphyllococcus , Streptococcus, Enterococcus, and Clostridium.

Most Gram-negative includes the Cynobacteria, Neisseria, Spirochaetes , and

Enterobacteriales etc.

How Does Gram Staining Work?

Gram staining involves three processes:

 staining with a water-soluble dye called crystal violet,

 decolorization, and
 counterstaining, usually with safranin.
Due to differences in the thickness of a peptidoglycan layer in the cell membrane
between Gram positive and Gram negative bacteria, Gram positive bacteria (with a
thicker peptidoglycan layer) retain crystal violet stain during the decolorization
process, while Gram negative bacteria lose the crystal violet stain and are instead
stained by the safranin in the final staining process. The process involves three steps:

1. Cells are stained with crystal violet dye.

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2. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a
complex between the crystal violet and iodine (CV-I complex). This complex is a larger
molecule than the original crystal violet stain and iodine and is insoluble in water. In
Gram Positive cells only, this CV-I complex binds to the magnesium-ribonucleic acid
component of the cell wall . The resultant magnesium-ribonucleic acid crystal violet-
iodine (Mg-RNA-CV-I) complex is more difficult to remove than the smaller CV-I
complex.
3. A decolorizer such as ethyl alcohol or acetone is added to the sample, which
dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal
violet-iodine complex (CV-I complex) is not able to penetrate this tightened
peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.
Conversely, the outer membrane of Gram negative bacteria is degraded and the
thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal
violet-iodine complex and the color is lost.
4. A counterstain, 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.

A Gram stain of mixed Staphyllococcus aureus (Gram positive cocci, in purple) and Escherichia coli ( Gram
negative bacilli, in red), the most common Gram stain reference bacteria

How To- Staining :

Reagents:

1. Crystal violet (primary stain)

2. Iodine solution/Gram's Iodine (mordant that fixes crystal violet to cell wall)
3. Decolorizer (e.g. ethanol)
4. Safranin (secondary stain)
5. Water (preferably in a squirt bottle)

Staining Procedure and concerns:

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Steps:
1 Make a slide of cell sample to be stained. Heat fix the sample to the slide by carefully
passing the slide through a Bunsen burner three times.
Please note that the quality of the smear (too heavy or too light cell
concentration) will affect the Gram Stain results.
2 Add the primary stain (crystal violet) to the slide and incubate for 30 sec to 1 minute.
Rinse slide with a gentle indirect stream of tap water for a maximum of 5 seconds to
remove unbound crystal violet.

3 Add Gram's iodine for 1 to 2 minute- this is a mordant, (a substance that forms an
insoluble complex by binding to primary stain) or an agent that fixes the crystal violet
to the bacterial cell wall.

4 Wash slide in a gentle and indirect stream of tap water for 5 seconds.

5 Flood or rinse slide with acetone or alcohol. Wait 10-20 seconds or add drop by drop
to slide until decolorizing agent running from the slide runs clear (The alcohol will
decolorize the sample if it is Gram negative, removing the crystal violet. However, if
the alcohol remains on the sample for too long, it may also decolorize Gram positive
cells).

6 Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute and wash with a
gentle and indirect stream of tap water until no color appears in the effluent and then
blot dry with absorbent paper. (If the bacteria is Gram positive, it will retain the
primary stain (crystal violet) and not take the secondary stain (safranin), causing it to
look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the
primary stain and take the secondary stain, causing it to appear red when viewed
under a microscope).

6 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.

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FIG. 1. shows gram-positive (blue/purple) rods and FIG. 2. shows gram-negative (pink/red) rods.

Details of the chemical mechanism of the Gram stain:

crystal violet penetrate through the wall and membrane of both gram-positive and
gram-negative cells staining the cells purple. When added, iodine 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 .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

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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, 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.

Cell wall:
Cell wall in a bacteria cell is meant for its;
 shape, rigidity, ductility.
 porousity and permeablility.
 taking part in cell division
 Possessing target site for antibiotics, lysozymes, bacteriophases.
 antigenicity that are important in virulence and immunity.
Cell wall is 10-25 nm thick and weighs 20-30 % of the dry weigh of the cell.

Chemically the cell wall of Gram positive bacteria is simpler than that of Gram negative
bacteria.
Peptidoglycan ( murein) is the rigid part and principal component of the cell wall . It
is more abundant in Gram positive bacteria than in Gram negative. It is made of
alternating strands of N-acetylmuramic acid and N-acetylglucosamine linked eachother by
an peptide chain (of four amino acids).

Difference between cell wall of Gram + and Gram – bacteria;

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 Gram negative bacteria have double cell walls, with a thin inner wall of peptidoglycan
and an outer wall of carbohydrates, proteins, and lipids. Such bacteria do not stain
purple with crystal violet. Most bacteria have the Gram-negative cell wall. The outer
membrane in Gram negative bacteria is connected by lipoprotein layer.
 The peptidoglycan layer of Gram positive bacteria is much thicker than that in Gram
negative bacteria (multiple layers). Most Gram positive cells contain significant amounts
of teichoic acids which is antigenic in property.
Cell wall is essential to the survival of many bacteria, and the antibiotic penicillin is
able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan.

There are 4 conditions to be followed for a valid Gram staining

procedure: 

Young cultures - Must be young within 18-24hrs old

(Older cultures lose their Gram staining properties
due to changes in the CWs as the cells get older)
Thin smear
Thicker or uneven smears will result in uneven staining
and decolourization (When staining the thin smear a short decolorizing
time 10 to 20 seconds should be used).
Fresh reagents - Of proper strength. (With older staining reagents, filter stains
before use). 
Control cultures - For a known Gram Positive bacterium and Gram Negative
culture (S.aureus & E.coli)
View slides using brightfield microscopy- Gram-stained bacteria should be
viewed with a bright-field microscope at 1000X magnification with oil immersion.
If the smear of cells is crowded it will be difficult to note cell shape and
arrangement. Adjust the brightness sufficiently to reveal the colour of the
specimen.