#Aim - To Perform Gram Staining
# Materials Required –
Fresh bacterial cultures, Glass slides , Inoculating loop, Burner or spirit lamp , Tissue paper,
Compound microscope.
# Chemicals Required -
• Crystal violet.
• Ethyl alcohol (95%)
• Distilled water
• Iodine Solution (KI3)
• Safranin
#Theory -
Gram staining is a differential staining method that was devised by a Danish physician Hans
Christian Gram in 1884 while working in Berlin. He was examining lung tissue sections from patients
who died of pneumonia and wanted to differentiate Pneumococci (now known as Streptococcus
pneumoniae). During these investigations he observed that some bacterial species readily lose the
primary stain on treatment with ethanol while others resist decolourisation which led to
development of this two step staining procedure with an intervening ethanol step. It is simple
staining method that allows investigators to identify bacteria under the microscope and easily
subdivide them into two broad groups- Gram positive and Gram negative. The method is still in use
as a first step in identification of generally pathogenic bacteria.
#Difference Between Gram-Positive and Gram-Negative Bacteria -
Feature Gram-Positive Bacteria Gram-Negative Bacteria
Blue or purple after Gram
Staining Color Pink or red after Gram staining
staining
Thick peptidoglycan layer Thin peptidoglycan layer (8-10
Cell Wall Structure
(20-80 nm) nm)
Outer Membrane Absent Present
More resistant due to outer
Resistance to Generally less resistant
membrane
Antibiotics
Cocci (spherical) or bacilli
Morphology Spherical, rod, or spiral-shaped
(rod-shaped)
Staphylococcus,
Examples Escherichia coli, Salmonella
Streptococcus
Permeability to Less permeable due to thick More permeable due to porins in
Substances wall the outer membrane
#Principle –
Gram reaction is one of the commonly used methods to characterise bacteria, based on differences
in cell wall composition. Gram positive bacteria have a cross linked multilayered peptidoglycan. On
the other hand Gram negative bacteria have a thin single layer peptidoglycan and an outer
membrane of lipopolysaccharides (LPS), with or without a capsule.
The staining technique uses two basic stains that are called primary and counter stain. The former
is crystal violet or methylene blue while the latter is safranin. Between the two staining steps is
addition of mordant (Gram’s iodine) for better interaction between the cell wall and the primary
stain and decolorization with ethanol. The decolorizing agent is both a protein dehydrating agent
and lipid dissolver. It increases the porosity of the cell wall by dissolving the outer LPS layer of Gram
negative bacteria and effectively dehydrates the thick Gram positive cell wall that results in cell wall
shrinkage and reduced permeability.
All bacterial cells are stained with crystal violet and appear purple coloured. The Gram positive
bacteria retain the primary stain after decolorization whereas it is easily removed from Gram
negative bacterial cells. In the final step only Gram negative bacteria are stained with the counter
stain and appear reddish / pinkish. The difference in color between two types of cells allows
characterisation.
#Procedure –
• Clean 3-4 slides with 70% alcohol and allow them to air dry. Do not touch the cleaned surface;
hold them from the edges.
• Prepare a smear of each of the given cultures using aseptic technique.
• A smear may be prepared either from broth cultures or colonies on semisolid medium.
• A broth culture is first resuspended by mild tapping and then a loopful of the culture is
aseptically picked with an inoculating loop. Next it is transferred to the centre of a clean slide
and spread evenly.
• Leave the smear to air dry.
• Fixed the dried smear by passing the slide rapidly few times over the flame.
• Immerse / flood the smear in crystal violet for 1 min.
• Gently rinse off excess stain with water.
• Carefully add Gram’s iodine and leave the slide for 1 min.
• Gently rinse with water.
• Decolourise with 95% Ethanol .
• Rinse gently with water.
• Counter stain with safranin for 1 min.
• Finally rinse with water, let it dry and observe the slide(s) under the microscope.
#Result –
The Gram-Positive Bacteria retain the primary stain (Crystal Violet) after decolourization meanwhile
the Gram-Negative Bacteria cannot retain the primary stain. Hence, it was observed that Gram-
Positive bacteria had purple appearance meanwhile Gram-Negative Bacteria had reddish/pinkish
appearance.
#Precautions –
• Avoid using older cultures. They tend to give Gram variable reaction. It is best to subculture
before the experiment.
• The smear should be thin, less dense and uniformly spread. Try to use only small amount of
culture.
• Take care not to overheat the smear during fixation as this may kill the cells and affect your
results.
• Sterilise the inoculation loop after each transfer. Use cooled sterilised loop.
