Introduction
Gram staining is the most important differential staining method used in microbiology. Gram stain or Gram staining, also called Gram’s method, is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique.
1) Gram-positive Bacteria:
After staining a bacteria with crystal violet, if its cell wall resists decolourisation by washing with a decolourising agent (ethanol or acetone), it is a gram-positive bacteria. Examples: Bacillus, Staphylococcus.
(2) Gram-negative Bacteria:
After staining a bacteria with crystal violet, if its cell wall allows to undergo decolourisation by washing with a decolourising agent (ethanol or acetone), it is a gram-negative bacteria. Examples: Escherichia, Salmonella, Vibrio.
Differentiation of bacteria into gram-positive and gram-negative groups provides the most important clue for proceeding further in proper direction for identification of unknown bacteria. It is also useful for simple differentiation of bacteria into gram-positive and gram-negative groups. It also gives an idea about the shape and arrangement of the bacteria cells.
Steps Of Gram Staining
- Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material).
- Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple.
- Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine (CV-I) complex; all cells continue to appear blue.
- Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells.
- The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled, gram-positive bacteria. The gram-negative bacteria appear colorless and gram-positive bacteria remain blue.
- Application of counterstain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.
Principle
In gram staining, the bacteria cells are first stained with a primary stain, crystal violet that enters into the cells and stain them purple-blue. Then, the cells are treated with a mordant, gram’s iodine, which enters into the cells and binds to the primary stain forming an insoluble dye-mordant complex (crystal violet-iodine complex), thus making the escape of the stain difficult.
Then, the cells are treated with a decolourising agent, such as ethanol or acetone. It acts as a lipid solvent and as a protein- dehydrating agent. In gram-positive cells, the decolourising agent acts initially as a lipid solvent, dissolving out the little quantity of lipid present in the cell wall, thereby forming minute pores in it. Subsequently, it dehydrates the cell wall proteins (peptides), which close the pores.
Now, the stain- mordant complex is difficult to be removed by the decolourising agent and the cells retain the purple- blue colour of the primary stain. When counter-stained by a pink-red coloured stain, safranin, it cannot enter into the cells, as the pores have been closed.
The cells finally retain the purple-blue colour of the primary stain. On the other hand, the gram-negative cell wall contains large quantity of lipid (LPS), which is dissolved out by the decolourising agent, resulting in the formation of large pores. These pores do not close appreciably on dehydration of cell wall proteins.
That is why; the stain-mordant complex escapes through the pores, when washed by the decolourising agent and the cells appear colourless. When counter-stained by safranin, it enters into the cells through the pores and finally the cells take the pink-red colour of the counter-stain.
Experiment
Reagents And Materials Required
- Slide
- Loop
- Primary stain (crystal violet)
- Mordant (gram’s iodine)
- Decolourising agent (95% ethanol)
- Counter-stain (safranin)
- Broth/slant/plate culture of bacteria
- Microscope
- Immersion oil.
Procedure
- A slide is cleaned properly under tap water, such that water does not remain as drops on its surface.
- The adhering water is wiped out with bibulous paper and the slide is air-dried.
- A smear of bacteria is prepared at the center of the slide in two methods as follows.(a)If a bacterium grown on agar plate or agar slant is to be observed, a drop of water is put at the center of the slide and a loop of bacteria from the plate or slant is transferred to it by a loop sterilized over flame. Then, by slow rotation of the loop in the drop, a bacteria suspension is made and it is spread till a smear is obtained.(b)If a bacterium grown in liquid broth is to be observed, a drop of the bacteria suspension is directly placed at the center of the slide by a flame-sterilized loop and a smear is made by spreading.
- The smear is air-dried.
- The smear is fixed by heating. Heating results in coagulation of the cellular proteins, due to which the cells stick to the slide surface and do not get washed away during staining, Heat- fixation is done by quickly passing the slide high above a flame 2-3 times, with the smear surface facing upward, so that the smear does not get heated up.
- The slide is kept on a staining tray and flooded with the primary stain, crystal violet, for 1 minute.
- Excess stain is washed away from the smear under gently-flowing tap water, in such a way that, water does not fall directly on the smear.
- The smear is flooded with the mordant, gram’s iodine, for 1 minute.
- Excess mordant is washed away from the smear under gently-flowing tap water, in such a way that, water does not fall directly on the smear.
- The smear is flooded with the decolourising agent, 95% ethanol, for 5 seconds, taking care, so that the smear is not over-decolourised.
- The ethanol is quickly washed away from the smear under gently-flowing tap water, so as to prevent over-decolourisation. Care is taken, so that, water does not fall directly on the smear.
- The smear is flooded with the counter-stain, safranin, for 1 minute.
- Excess counter-stain is washed away from the smear under gently-flowing tap water, in such a way that, water does not fall directly on the smear.
- The slide is blotted dry with bibulous paper.
- The slide is clipped to the stage of the microscope and the smear observed under low power and high dry objectives.
- A drop of immersion oil is put on the smear.
- The smear is observed under oil-immersion objective.
Observations (Under Oil-immersion Objective)
1. Colour of the cells:
Purple-blue: Gram-positive bacteria
Pink-red: Gram-negative bacteria
2. Shape of bacteria:
Spherical (coccus)
Rod-shaped (bacilli)
Comma-like (vibrio)
Spiral (spirochetes)
3. Arrangement of bacteria
Pairs (diplobacillus/diplococcus)
In fours (tetrads)
In chains (streptococcus/streptobacillus)
Grape-like clusters (staphylococcus)
Cuboidal (sarcinae or octet)
Gram-variable Reaction
In some cases, the gram-positive cells appear as gram-negative cells. This is called gram-variable reaction.
The reasons are as follows:
(a) Over-decolourisation may occur, due to which the gram-positive cells also lose their violet colour.
(b) Over-fixation may occur, due to which the gram-positive cells lose their ability to resist decolourisation
(c) If old culture of bacteria is used, the components of the cell wall may change with the age of the cells allowing decolourisation.