Frequently asked questions in staining exercises
How are staining techniques classified?
• Simple stain: where only one stain is used and all bacteria are stained similarly. Eg: Methylene blue, dilute carbol fuchsin
• Differential staining: where different bacteria stain differently to a common staining technique depending on their physiological properties. Eg: Gram’s stain and Acid fast staining
• Special stain: where structures of bacteria like spores, granules, capsule etc are demonstrated. Eg: silver impregnation technique for demonstration of spirochetes, Feulgen stain for demonstration of nucleus, Sudan black stain for demonstration of lipid vacuoles, Ryu’s stain for demonstration of flagella, Albert’s stain for demonstration of metachromatic granules.
• Negative staining: where the background is stained with an acidic dye such as India ink or Nigrosin. Used for demonstration of capsules.

How are stains classified?
• 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).

What is polychrome methylene blue?
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.

Who invented Gram stain?
Hans Christian Gram invented this stain in 1884. The original formulation was Aniline Gentian violet, Lugol’s iodine, absolute alcohol and Bismark brown.

Which are the theories of Gram staining?
• Cell wall theory: Cell wall of Gram positive bacteria are 40 times thicker than those of Gram negative cells, hence they are thought to help retain the dye-iodine complex.
• Lipid Content Theory: Cell envelope of Gram negative bacteria contains an additional membrane (outer membrane), hence containing more lipids than Gram positive bacteria. Acetone or alcohol dissolves the lipid thus forming large pores in Gram negative bacteria through which the dye-iodine complex leaks out. Alcohol/acetone dehydrates Gram positive bacteria shrinking the cell wall and the closing the pores.
• Magnesium Ribonucleate Theory: A compound of magnesium ribonucleate and basic protein concentrated at the cell membrane helps Gram positive bacteria retain the primary dye. Gram negative bacteria do not possess this substance.
• Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are said to be more acidic (2) than those of Gram negative ones (3). Hence the dye is said to bind with more affinity to Gram positive cells.

Which part of the bacteria actually gets stained?
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.

Which are the bacteria or bacterial component that can’t be stained by Gram stain?
• Extremely slender bacteria such as Treponema
• Cells containing waxy substances impermeable to stain such as Mycobacteria
• Minute intracellular bacteria such as Chlamydia and Rickettsia
• Cell organelles such as capsule, spore, flagella etc

Which are the alternatives used in Gram stain?
Primary stain: Crystal violet, Methyl violet and Gentian violet
Mordant: Gram’s iodine, rarely Lugol’s iodine
Decolorizer: Alcohol, acetone, acteone-alcohol mixture (1:1)
Counterstain: Dilute carbol fuchsin, safranin, neutral red, (Sandiford stain for Gonococci)

Which are the positive and negative controls for Gram stain?
Postive control: Staphylococci
Negative control: E.coli, pus cells

What are the conditions when Gram positive bacteria can appear Gram negative?
• When over-decolourized by either prolonged exposure to decolourizer or using acetone alone.
• When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin.
• Old cultures, where cell wall is weakened or action of autolytic enzymes
• Those bacteria that are phagocytosed, where cell wall is acted upon by lysosomal contents

Which is the more important step in Gram stain?
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.

What are the applications of Gram staining?
• Rapid presumptive diagnosis of diseases such as bacterial meningitis
• Selection of empirical antibiotics based on Gram stain finding
• Selection of suitable culture media based on Gram stain finding
• Screening of quality of clinical specimens, such as sputum that should contain many pus cells and few epithelial cells
• Counting of bacteria
• Appreciation of morphology and types of bacteria in a clinical specimen

Name a fungus that is Gram positive?
Candida sps

What are the various modifications of Gram stain?
• Kopeloff and Beerman’s (Primary stain: Methyl violet, decolourizer: acetone or alcohol-acetone mixture 1:1)
• Jensen’s (Primary stain: Methyl violet, decolourizer: absolute alcohol, counterstain: Neutral red)
• Preston and Morrell’s (Primary stain: crystal violet, decolourizer: iodine-acetone)
• Weigert’s (Primary stain: Carbol gentian violet, decolourizer: Aniline-xylol). This is used to stain tissue sections.

What is acid fast staining?
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.

Who introduced Acid fast staining?
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.

Why are Mycobacteria acid fast?
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.

What are the components of Ziehl-Neelsen stain?
Primary stain: Strong Carbol Fuchsin (contain Basic fuchsin and Phenol)
Decolourizer: 20% sulphuric acid
Counterstain: Loeffler’s Methylene blue or 1% Malachite green, Picric acid for color-blind workers

What is acid-alcohol decolourizer?
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.

Which are the various dilutions of sulfuric acid used?
Mycobacterium leprae - 5% H2SO4
Oocysts of Cryptosporidium, Isospora - 1% H2SO4
Tissue sections containing Actinomyctes, Nocardia - 1% H2SO4
Cultures of Nocardia - 0.5% H2SO4
Bacterial spores - 0.25-0.5% H2SO4

What are cold methods of acid fast staining?
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.

Why should the slide be flooded with strong carbol fuchsin?
For uniform distribution of heat, or else the slide may break.

What are the precautions to be taken while preparing or observing smears for AFB?
• A new slide must be used for every specimen, because scratch marks may give false positive.
• A uniform smear from thick portion of the sputum must be made.
• Staining jars should not be used to staining smear as there is risk to cross contamination.
• Fresh blotting paper must be used for each smear for drying the slide to prevent transfer from one slide to another.

How to interpret the smear?
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.

How is the smear graded?
Smears are graded depending on the number of bacilli seen.
3-9 bacilli/entire smear: +
≥10 bacilli/entire smear: ++
≥10 bacilli/in most oil immersion fields: +++

What other methods are available for staining Mycobacteria?
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.

What is beaded appearance of Mycobacteria?
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.

What are metachromatic granules?
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.

Which are the ways to demonstrate these granules?
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.

Why are the bacilli arranged at angles to each other?
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).

What do Albert A(1) and B(2) solution contain?
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.

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