Gram positive vs gram negative color manual#

Which are the bacteria or bacterial component that can’t be stained by Gram stain? Cell wall deficient forms such as Mycoplasma and L forms are Gram negative. Presence of an intact cell wall is important for retaining Gram positivity. It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Which part of the bacteria actually gets stained? Hence the dye is said to bind with more affinity to Gram positive cells. Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are said to be more acidic (2) than those of Gram negative ones (3).Gram negative bacteria do not possess this substance. Magnesium Ribonucleate Theory: A compound of magnesium ribonucleate and basic protein concentrated at the cell membrane helps Gram positive bacteria retain the primary dye.Alcohol/acetone dehydrates Gram positive bacteria shrinking the cell wall and the closing the pores.

Acetone or alcohol dissolves the lipid thus forming large pores in Gram negative bacteria through which the dye-iodine complex leaks out.

Lipid Content Theory: Cell envelope of Gram negative bacteria contains an additional membrane (outer membrane), hence containing more lipids than Gram positive bacteria.

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.

The original formulation was Aniline Gentian violet, Lugol’s iodine, absolute alcohol and Bismark brown. Hans Christian Gram invented this stain in 1884. Used in McFadyean’s reaction for Bacillus anthracis in blood films and demonstration of metachromatic granules of Corynebacterium diphtheriae. Loeffler’s methylene blue solution treated with Potassium hydroxide turns into Polychrome methylene blue after prolonged storage with shaking. Supravital (cells removed from the body) and intravital (cells still a part of the body). Stains can be either natural (eg: carmine and hematoxylin) or coal-tar derivatives /aniline stains (eg: methylene blue, crystal violet). They are Romanowsky’s stain and are used in staining parasitic forms. Neutral dyes have both acidic and basic component that nullify each other. Acidic dyes combine more strongly with cytoplasmic components of bacteria, especially the nucleus that is basic in nature. Basic dyes have cationic chromophore eg., methylene blue+ chloride. Acidic dyes have anionic chromophore eg., sodium+ eosinate. Stains are classified based on the pH of their chromophore (color bearing ion) into acidic, basic and neutral.

Negative staining: where the background is stained with an acidic dye such as India ink or Nigrosin.

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.

Special stain: where structures of bacteria like spores, granules, capsule etc are demonstrated.

Differential staining: where different bacteria stain differently to a common staining technique depending on their physiological properties.

Eg: Methylene blue, dilute carbol fuchsin

Simple stain: where only one stain is used and all bacteria are stained similarly.

and Hopps, H.C., Geographic Pathology Division Armed Forces Institute Of Pathology, Washington, D.C.

Gram positive vs gram negative color manual#

This procedure does not work well on Bouin's fixed tissues.ĪFIP Manual of Histologic Staining Methods ed.Steps 4, 5, 9 and 10 are critical with regard to timing.Preferably, all stains and solutions are applied to the slide while it is in a horizontal position, except for steps 4, 10 and 11, where the slide should be dipped into the solutions contained in coplin jars.Slides may remain in Xylene until ready for mounting. Xylene, three changes, for 10 dips each.Cellosolve three changes, for six quick dips in each.Immediately blot away excess, but not to dryness). Stain for three seconds in Tartrazine Solution.Rinse thoroughly in distilled water and blot lightly to remove excess water (not to dryness).(Differentiates and 'fixes' the Basic Fuchsin). Gallego's Solution, for five minutes.Stain in Basic Fuchsin Solution, 0.5%, Aqueous for five minutes.Differentiate in Cellosolve, until blue color no longer streams away from the section (approximately 5-10 seconds).Mordant in Gram's Iodine for five minutes.Place slides on staining rack and pour on Crystal Violet Solution, 1%, Aqueous for two minutes.Deparaffinize and hydrate to distilled water.Brown-Hopps Method For Gram Positive And Gram Negative Bacteria EMS Catalog #26106 Fixation