Stains are classified based on the pH of their chromophore (color bearing ion) into acidic, basic and neutral. Acidic dyes have anionic chromophore eg., sodium+ eosinate-. Basic dyes have cationic chromophore eg., methylene blue+ chloride-. Acidic dyes combine more strongly with cytoplasmic components of bacteria, especially the nucleus that is basic in nature. Neutral dyes have both acidic and basic component that nullify each other. They are Romanowsky’s stain and are used in staining parasitic forms. Stains can be either natural (eg: carmine and hematoxylin) or coal-tar derivatives /aniline stains (eg: methylene blue, crystal violet). Supravital (cells removed from the body) and intravital (cells still a part of the body).
Loeffler’s methylene blue solution treated with Potassium hydroxide turns into Polychrome methylene blue after prolonged storage with shaking. Used in McFadyean’s reaction for Bacillus anthracis in blood films and demonstration of metachromatic granules of Corynebacterium diphtheriae.
Hans Christian Gram invented this stain in 1884. The original formulation was Aniline Gentian violet, Lugol’s iodine, absolute alcohol and Bismark brown.
It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Presence of an intact cell wall is important for retaining Gram positivity. Cell wall deficient forms such as Mycoplasma and L forms are Gram negative.
Decolourization is the most important step as this step differentiates between Gram positive and Gram negative bacteria. Over-decolourization can result in Gram positive bacteria appearing Gram negative and under-decolourization can result in Gram negative bacteria appearing Gram positive.
Certain bacteria or their structures have the ability to retain the primary dye (strong carbol fuchsin) and resist decolourization by weak mineral acids such as H2SO4, HCl. Such bacteria or their structure are termed acid fast and this property is termed acid fastness. There are two types of acid fast staining, the hot method and the cold method. The hot method (Ziehl-Neelsen) involves heating the slide while the cold methods such as Kinyoun’s and Gabbett’s do not involve heating the slide.
Ehrlich in 1882 discovered acid fastness. The original method involved staining with aniline-gentian violet and decolourization with strong nitric acid. It was later improved by Ziehl and Neelsen.
The cell walls of Mycobacteria are made up of waxy substance, Mycolic acid that is relatively impermeable to ordinary staining techniques. But, by application of heat and a mordant (phenol), the cell can be stained. The purpose of heating is to soften the waxy material of the cell wall and allow the stain to enter the cell. Basic fuchsin is more soluble in phenol and phenol is a better solvent for lipids and waxes.
3% HCl in 95% alcohol (methylated spirit). This is useful in differentiating saprophytic Mycobacteria from pathogenic Mycobacteria. Pathogenic Mycobacteria are both acid and alcohol fast but saprophytic Mycobacteria are only acid-fast. Saprophytic Mycobacteria can get declourized by alcohol. 95% alcohol can be used as a secondary decolorizer after decolourizing with acid. Especially used in staining smears prepared from urine that may contain Mycobacterium smegmatis.
The two methods namely Kinyoun’s and Gabbett’s don’t involve heating of slides, hence called cold methods. Heating is substituted by increased concentration of phenol and prolonging the duration of staining. Kinyoun's method is favoured for detection of Cryptosporidium oocysts in fecal samples. Gabbett’s method has decolourizer and counterstain in one solution.
For uniform distribution of heat, or else the slide may break.
At least 100 oil immersion fields must be viewed before declaring the smear as negative. The sensitivity of smear is low because it requires the presence of 104 bacilli/ml to be smear positive. If the number of bacilli is less than this, the chances of detecting them are less. In such a case, the sample should be subjected to concentration techniques such as Petroff’s method. If the smear is positive for AFB, it should be counted/graded. Failure to detect any AFB does not rule tuberculosis. Grading of smears has prognostic value.
Smears are graded depending on the number of bacilli seen.
Sputum smears for Mycobacteria can be stained by fluorescent dyes such as Auramine and Rhodamine as they have affinity for mycolic acid in their cell walls. The fluorescent microscopy is useful in screening large number of specimens. Large area of smear can be quickly observed that too under high power dry objective.
Beaded appearance is used to describe the appearance of Mycobacteria when the cell doesn’t stain uniformly, showing stained and unstained regions. These forms are common in Mycobacterium tuberculosis while Mycobacterium bovis stains uniformly. Most saprophytic Mycobacteria stain uniformly.
Metachromatic granules are polymetaphosphate reserves produced by Corynebacterium diphtheriae in nutritious medium. These granules are also known as Babes Ernst granules, Volutin granules, Polar bodies etc. They are called metachromatic granules because of they exhibit metachromasia, a property where the granules appear in a colour different from that of the dye used. When stained with polychrome methylene blue, they appear purple. They are produced in abundance in serum containing medium such as Loeffler’s serum slope.
Albert’s stain, Neisser’s stain, Ponder’s stain and Pugh’s stain. They can be demonstrated as refractile bodies in wet mount or slightly more gram positive structures in Gram stain.
The bacilli are arranged at angles to each other resembling English letter V or L or Chinese letter (cuneiform) pattern because the daughter cells don’t separate completely after cell division (binary fission).
Solution A(1) contains Toluidine blue, Malachite green, Glacial acetic acid and Alcohol while solution B(2) contains iodine and potassium iodide in distilled water.