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Differential and Cytological Staining Techniques




  • To study and gain expertise on differential and cytological staining techniques.




Differential staining is a technique that helps to characterize the microorganisms depending on the difference in the physical and chemical nature of the microorganism. The differential and cytological staining techniques discussed in this chapter help to differentiate between acid fast and non acid fast cells and to visualize the intracellular constituents of the microbial cells including endospores, capsules, metachromatic granules, and flagella. In this technique the differential stains applied on the bacterial smear reveals the different types of cells at one point, reveals one part of the cell with one color and other parts a different color.


Differential Staining

Gram stain technique is a differential staining technique, which separates bacteria into two groups (discussed in earlier chapters), Gram-positive bacteria and Gram-negative bacteria. Another differential staining technique is acid-fast technique which differentiates species of Mycobacterium from other bacteria. Mycobacterium species due to its special cell wall resist the effect of the decolorizer acid-alcohol and retain the color of the primary stain carbolfuchsin stain and stains the acid fast cells in bright red color. Other bacteria lose the stain and take on the subsequent color of the counter stain methylene blue stain and stain the cell as blue. Endospore staining is a special staining technique, to observe bacterial spores, where the spores take the color of the primary stain Malachite green, while the counterstain, safranin, give color to the non-spore forming bacteria. Specific stains such as nigrosine, Indian ink etc help to visualize the bacterial stains which cannot be stained by usual staining methods. The metachromatic granules, the characteristic feature of Cornybacterium diphtheriae, can be differentiated from the bacterial cells with the help of Albert staining techniques. Flagella are the thin delicate structure for bacterial motility. The thin structures of the bacterial flagella make it difficult to observe under bright field microscope. Special stains and mordents such as Leifson’s stain are required for staining the bacterial flagella.


Endospore Stain:


Schaeffer-Fulton Method


Endospore staining is used to visualize specialized cell structures. The endospore stain is used to determine the highly resistant spores of certain microorganisms within their vegetative cells. The multiple thick coats of the spore made the endospore resistant to stain with most dyes. In Schaeffer-Fulton method, the primary stain, Malachite Green, is added over the heat fixed bacterial smear and heated over a steam bath for few minutes. This will soften the hard outer coverings of the spore and the primary stain gets stick to the spore. When taken from the steam bath followed by further cooling hardens the outer layer of the spore. During this stage both the spore and vegetative cells appear as green in color. But later the thick outer layer makes the spore resistant to the action of decolorizing agent (water), but however, water can easily decolorize the vegetative cells.  When counterstained with Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink color.


Dorner Method


Dorner method is an alternative method for staining the endospores. In this staining process, 2- 3ml broth culture of microorganism and equal volume of  Basic fuchsin is heated in a water bath at 100o C for 10 minutes. The extensive boiling softens the structure of the bacterial spores and the basic fuschin get into the spores. After the boiling process, the microbial culture-basic fuschin stain is allowed to cool for sometime which hardens the outer covering of the spore. In order to give a color to the background and to differentiate between the vegetative cells and endospores, a thin film of loopful of the microbial culture-basic fuschin stain and 2 nigrosin on a clean glass slide. Since nigrosin is negative charged, the bacterial cells cannot easily taken up by the cells. After staining the vegetative cells appear become colorless, the endospores stains as red which can be present as terminal or sub terminal. The background is stained as dark by the Negrosin stain.


Acid Fast Stain:


Ziehl Neelsen Method


The Ziehl Neelsen Method is used for staining the Mycobacterium in clinical specimens. The thick outer waxy covering (mycolic acid) of the Mycobacterium cell walls act as a barrier  and does not allow all the stains to enter into the cell. In order to visualize these cells higher concentrations of the staining solution is needed and once this stain enters the cell, it is too difficult to remove the stain using a decolorizer. When the clinical specimen is stained with basic dyes such as carbolfuchsin (primary stain) with the continuous application of heat, softens the waxy lipid outer covering of the cell wall and the stain readily enters the cell and stains the cell cytoplasm. When decolorizing agents such as acid-alcohol is added over the primary stain, some bacterial cells cannot be easily decolorized and such bacterial cells are called as acid fast bacteria. The bacteria with high concentration of lipid are easily decolorized by the decolorizing agent and are said to be non-acid fast bacteria. Finally, the addition of the counter stain, Methylene blue, dyes the colorless non acid fast cells as blue thus differentiating them from the pink acid fast bacteria which are unaffected by the Methylene blue.


Capsule Stain:


Capsules are the gelatinous outer layer of the bacterial cells and these structures cannot retain the color of the staining agents. The capsules can be visualized by means of two methods.


 Positive Capsule Staining


Since capsule is water soluble in nature, it is too difficult to stain the capsule with normal staining methods. The positive capsule staining method (Anthony Method) uses two reagents to stain the capsular material. The primary stain Crystal violet is applied over a non heat fixed bacterial smear so that both the bacterial cells and capsular material take up the color of the primary stain. The ionic nature of the bacterial cell binds the crystal violet stain more strongly while the non-ionic nature of the capsule get adhere with the crystal violet stain. When the decolorizing agent copper sulfate is added over the bacterial smear, the loosely adhered crystal violet stain is washed off from the capsular material without removing the tightly bound crystal violet from the cell wall. The capsular material absorbs the light blue color of the copper sulfate in contrast to the purple bacterial cell.


Negative Capsule Staining


Another simple method to visualize the bacterial capsules is by using negative staining Technique. During staining the non heat fixed bacterial smear with the acidic stains such as Nigrosin will not penetrate the bacterial cells (since both acidic stain and bacterial surface has negative charge). Instead the acidic stain deposits around the bacterial cells and create a dark back ground and the bacteria appear as unstained with a clear area around them, capsule.

Note: If you heat fix the bacterial smear for capsule staining, the cells will shrink creating a hallow zone around the bacterial cell and will be mistaken for the capsule.


Metachromatic Granule Staining: Albert’s Staining


Albert’s staining is specially demonstrates the presence or absence of the metachromatic granules, a characteristic feature of Cornybacterium diphtheriae. During gram staining if a smear appears as purple rods with straight or slightly curved with clubs at the end, with a characteristic V shape then it is suspected as Cornybacterium diphtheriae. The further confirmation can be done by Albert’s staining technique. This techniques employ two stains Albert Stain A( combination of Toluidine blue, Malachite green, Glacial acetic acid, alcohol and distilled water) and Albert Stain B(Iodine, potassium iodide and distilled water). After the staining process the metachromatic granules appear as bluish black where as the cell appears as green color.


Flagella Staining


Bacteria are motile by means of flagella. The flagella are too thin to be seen in ordinary stains, special stains and techniques needed to visualize the flagellum enough stain to obtain a visible thickness. Specialized stains are usually found in microbiology laboratories to detect the presence or absence of flagella to indicate the nature of that bacterium (motile/non-motile).


Liefson Stain


When a bacterial culture is stained with Liefson stain, the tannic acid component of the stain produce a colloidal precipitate which can be taken up by the bacterial flagella will become colorized which can be easily visualized using microscopy. The concentration of the tannic acid and dye is important in staining the bacterial flagella while the alcohol concentration in the Liefsons stains helps in maintaining the solubility of the components. On microscopic observation, the bacterial cells and flagella will stain red and the flagellar arrangement can be visualized easily. The age of the bacterial culture, condition of staining solutions, concentrations of the staining solution etc can also affect the staining reaction.




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