Question

What is the purpose of heat fixing a slide?

List the 4 major steps of the gram stain and explain what is happening to both Gram positive and Gram negative cells in each step.

Why don’t we heat fix in the capsule stain?

What are the major things that are done in the acid fast stain procedure that allow acid fast cells to be stained?

Hypothetically, let's say you perform an endospore stain and observe that the endospores formed in your specimen exhibit a spherical and subterminal morphology. Why is it useful know this?

Answer 1

1) Heat fixing the slide:-Heat fixing kills the bacteria and makes them fixed to the glass slide so that it they are not washed away while doing the staining. It also makes them take the stain readily.

2) The principle of Gram staining:-

1. Gram positive cells have a more acidic protoplasm, which may account for the retaining the basic primary dye more strongly than Gram negative bacteria.
2. The peptidoglycan of Gram positive bacteria is thick and thus able to retain the dye-iodine Complex.
3. The high lipid content of Gram negative bacteria makes them permeable to secondary dye after decolorization with organic solvents like acetone.

Steps of Gram staining:-

1. Primary staining with para rosaniline dye such as crystal violet, methyl violet, gentian violet. both the Gram negative as well as Gram positive bacteria take up the stain.
2. Application of dilute solution of iodine. Iodine acts as a mordant. It makes complex with the dye so that the dye cannot be removed easily. Both the groups make dye-iodine complex.
3. Decolorization with an organic solvent such as ethanol, acetone or aniline. Gram positive bacteria has thick peptidoglycan therefore they are able to retain the dye-iodine complex. Whereas Gram negative bacteria, couldn't retain dye-iodine complex and get decolourised.
4. Counter staining with a dye of contrasting colour, such as carbol fuschin, safranin, or neutral red. As the Gram negative bacteria are decolorized, they take the counterstain now appearing red. Gram positive bacteria do not take up the stain.
5. Hence by the end of this staining, Gram positive bacteria appear violet and Gram negative bacteria appear pink.

3) Some bacteria have polypeptide cell wall and some have polysaccharide cell wall. The polypeptide cell wall is very moist and slimy, therefore it cannot tolerate heat. Heat fixing such bacteria may cause shrinkage of the capsule and we want fully intact capsule for capsule staining. Therefore, heat fixing is not done in cases of capsule staining.

4) Acid fast staining principle:-

The acid fast bacteria do not stain readily, but once stained resist decolorization with dilute mineral acids, due to the presence of mycolic acid in their cell wall.

Acid fastness has been ascribed to the high content and variety of lipids, fatty acids and higher alcohols found in mycobacterium tuberculosis.

1. A Lipid peculiar to acid fast bacilli, high molecular weight hydroxy acid wax containing carboxyl groups (mycolic acid), is acid fast in the free state.
2. Acid fastness is not a property of lipids alone, but it depends on the integrity of cell wall also.

Procedure:-

1. The smear is a stained by a strong solution of carbol fuschin with the application of heat. Strong carbol fuschin stains the acid fast bacteria.
2. It is then decolorized with 20% sulphuric acid and counter stained with a contrasting dye such as methylene blue. Acid fast bacteria resist the decolorization.
3. The acid fast bacteria retain the fuschin (red) colour, while the others take the counterstain.