PRESENTED BY : DR. ANINDYA DASGRAM’S STAIN

INTRODUCTIONBefore discussing gram staining procedure I want to discuss

about the history of Microbiology in short.The credit for having first observed and reported bacteria belongs to ANTONY VAN LEEUWENHOEK , a draper in Delft, Holland, whose hobby was grinding lenses and observing diverse materials through them.

But the development of Microbiology as a scientific discipline dates from LOUIS PASTEUR (1822-95).He introduced the techniques of sterilisation and developed the steam steriliser, hot air oven and autoclave.He also established the differing growth needs of different bacteria and contributed to the knowledge of Anthrax, Chicken Cholera and Hydrophobia.

It was Pasteur who coined the term Vaccine. But that time scientists found that bacteria do not show much structural detail under light microscope due to lack of contrast.

This problem was solved by the discovery of staining methods of microbes. Gram staining was devised by the Danish histologist HANS CHRISTIAN GRAM in 1884 as a method of staining bacteria in tissues. This is the most important staining method in Bacteriology. It is used to differentiate bacterial species into two broad groupes , Gram positive & Gram negative based on the physical property of their cell wall.

TYPES OF STAINING TECHNIQUES1.SIMPLE STAINS Methylene blue or basic fuchsin are used. They provide colour contrast but impart the same colour to

all bacteria.

2.NEGATIVE STAINING Indian ink or nigrosin are used. They produce uniformly coloured background against

which the unstained bacteria stand out in contrast. Particularly useful in the demonstration of bacterial capsule

which do not take simple stain and also for spirochetes.

3.DIFFERENTIAL STAINS These stains impart different colours to different bacteria or

bacterial structures. Primary staining is done by one dye Counter staining is done by a different dye of contrasting colour. Example---Gram stain and Ziehl-Neelsen stain.

4.IMPREGNATION METHOD Silver impregnation method Used for structures and cells too thin to be seen under the ordinary

microscope like spirochetes and bacterial flagella.They may be rendered visible if they are thickened by impregnation

of silver on their surface .Example—Fontana’s and Levaditi’s methods of staining

There are several other staining methods like--

ALBERT’S STAIN for volutine granulesGIEMSA’S STAIN for protozoaLEISHMAN’S STAIN for protozoa in blood

filmFLUROCHROME(AuramineO) STAIN for AFBMALACHITE GREEN STAIN for sporesPERIODIC ACID-SCHIFF STAIN for fungi in

tissueSUDAN BLACK B STAIN for intracellular

lipid of bacteria

PROCEDURE OF GRAM STAININGThe Gram procedure requires the application

of four reagents in the following order:1. A basic pararosaline dye such as crystal

violate,methyl violate or gentian violate2.An aqueous solution of iodine3.A decolourizing solvent, like acetone,

alcohol or aniline4.A light red or pink counterstain,like basic

fuchsin, neutral red or safranine

In the original method of Gram(1884) the smear was stained with aniline-gentian violate,treated with Lugol”s iodine, decolourised with absolute alcohol, and counterstained with Bismarck brown.Later modifications give better results

Crystal violate or methyl violate is used in the concentration of 0.5-2%

Solution is facilitated if the dye is first dissolved in alcohol,filtered through filter paper and added to the water

However Gram positive staining can be strengthened by addition of sodium bicarbonate or ammonium oxalate as in the following solutions:

KOPELOFF & BEERMAN’S(1922) MODIFICATION:Sol. A Methyl violate 6B 10 g Distilled water 1 litreSol. B Sodium bicarbonate 50 g Distilled water 1 litreMix 30 vol. of Sol A with 8 vol. of Sol B shortly before

use. Problem of this mixture is that it tends to precipitate within a few days, so cannot be kept.

PRESTON & MORRELL’S(1962) MODIFICATION:Crystal violate 20 gMethylated spirit 200 mlAmmonium oxalate, 1% in water 800 mlIt also strengthen gram positive staining.Another one modification is-JENSEN’S MODIFICATION: Here alcohol is used as a

decolourizer & weak neutral red as counterstain for examination of gonococci & meningococci

IODINE SOLUTION(Gram’s iodine)-Dissolve 20 g of Potassium iodide in 250 ml water & then add

10 g iodine.When iodine is dissolved, make upto 1 lt with water.

In KOPELOFF & BEERMAN’S modification iodine is dissolved in NaOH sol. & then added distilled water.The more alkaline sol. with NaOH is thought to give stronger gram positive reaction.

DECOLOURIZER:Acetone: it is the fastest & most specific

decolourizer.It is applied to smears for only 2-3 sec. However the short period of exposure is difficult to control when many slides are stained simultaneously.

Absolute alcohol(100% ethanol)-It acts more slowly than acetone & should be applied for about 1 min.

Acetone-alcohol-It is a mixture of 1 vol. of acetone with 1 vol. of 95% ethanol.It requires application for about 10 sec.

Iodine-acetone-PRESTON & MORREL(1962) have shown that addition of 0.35% iodine to acetone slows its rate of decolourization & exposure can be lengthened from 2 sec. to 30 sec.

But an irritating aerosol may be produced due to splashing of iodine-acetone sol. into the sink.Acetone quickly evaporates from the droplets & leaves airborne droplet of iodine which irritate the eyes & nose.

To minimise this effect, iodine-acetone sol. should be poured gently on to the slide through a wide nozzle & splashing into the sink should be avoided.

COUNTERSTAIN-1.Safranine-It is commonly used in 0.5% in distilled

water.

2.Dilute carbol fuchsin-It gives the strongest red staining.Colour may be so dark that some gram negative bacteria may be difficult to distinguish from gram positive one.

3.Basic fuchsin-It is weaker counterstain.It is recommended for general use.

4.Neutral red-It is recommended for demonstrating gonococci & other intracellular Gram negative bacteria.

STAINING TECHNIQUE1. Make a smear & dry thoroughly in cool air. Fix

the dried film by passing it briefly through a bunsen flame.

2. Flood the slide with crystal violate sol. for upto 1 min.Wash off briefly with tap water & drain.

3.Flood the slide with gram’s iodine sol. & allow to act as a mordant for about 1 min.Wash off with tap water & drain.

STAINING TECHNIQUE4.Decolourise the smear with acetone for 10-

30 sec. taking care not to overdecolourise & immediately wash off with water.

5.Flood the slide with safranin sol. & counterstain for about 30 sec., wash off with tap water, drain & blot dry with filter paper & examine under oil immersion objective.

STAINING MECHANISM

Gram positive bacteria have a thick cell wall made of as many as 40 sheets of peptidoglycan(50-90% of cell envelope).

Whereas Gram negative bacteria have a thin cell wall made of only one or two sheets of peptidoglycan(10% of cell envelope).

Crystal violate(cv) dissociates in aqueous solution into cv+ & chloride(cl-) ions.These ions penetrate through the cell wall & cell membrane of both gram positive & gram negative bacteria.

The cv+ ion interects with negatively charged components of bacterial cells & stains the cells purple.Iodide(I-) interects with cv+ & form large complexes of crystal violate & iodine(CV-I) within the inner & outer layer of cell.

Iodine is a trapping agent that prevents the removal of CV-I complex & therefore colour the cell.

When a decolourizer like alcohol or acetone is used, it interects with the lipids of cell membrane.Gram negative cell loses its outer lipopolysaccharide membrane & the inner peptidoglycan layer is left exposed.

.The CV-I complexes are washed from the

gram negative cell along with the outer membrane.

In contrast a gram positive cell becomes dehydrated from an ethanol treatment.The large CV-I complexes become trapped within gram positive cell due to multilayered nature of its peptidoglycan.The decolourization step is critical & must be timed correctly.

CV stain is removed from both gram positive & negative cells if decolourising agent is left on too long .

Conversely inadequate decolourization may cause all cells to appear gram positive.

Examples of gram positive bacteria-Staphylococcus,Streptococcus,Actinomycetes,Bacillus,Corynebacteria,Clostridia,Listeria.

Example of gram negative bacteria- Enterobacteriaceae,Pseudomonas,Neisseria,Vibrio etc.

Gram positive bacteria may sometimes appear gram negative in ageing culture & when cell wall is damaged.

Gram indeterminate bacteria or gram variable bacteria do not respond predictably to gram staining.They tend to stain unevenly, appearing partially gram positive & pertially gram negative, or even unstained.

Examples-Micobacterium tuberculosis & leprae & also Acinetobacter

Apart from bacteria, gram’s stain can also be used to identify other microorganisms like Pneumosystis jeroveci cysts, Strongyloides larvae,Toxoplasma gondii trophozoites & Trichomonads etc.

A variety of artifacts can resemble infectious agents in Gram’s stained smear.To overcome this problem a useful maneuver is to stain another smear with acridine orange; with this stain it can be established whether the structure contain DNA & is, therefore, biological.

In direct smear under-decolourization can be monitored by observing the nuclei of inflammatory cells; if they are not completely gram negative, the smear has not been adequately decolourized.

The only recipe for detecting over-decolourization is cross-checking the gram reaction & the morphology of the bacteria.

GRAM POSITIVE COCCI

GRAM POITIVE BACILLI & COCCI IN CHAINS

GRAM NEGATIVE BACILLI

GRAM POSITIVE & NEGATIVE BACILLI

THE END