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Its function is to impart its color to all

primary stain

establish a color contrast

decolorizing agent

has a contrasting color to that of the primary stain


This is the final reagent, used to stain red those cells that have been previously decolorized


The best Gram stained preparations are made with fresh cultures, that is, not older than

24 hours

some cells will appear purple,
while others will appear red.


Crystal violet, Gram's iodine, 95% ethyl alcohol, and 0.25% safranin.

Gram stain

(substance that combines with the dye to form an insoluble compound


a dilute aqueous solution

Gram's iodine

used in Decolorization in gram step

95% ethanol. (ethyl alcohol)

used Counterstain in gram step

0.25% safranin

Very few bacteria are acid-fast

genera Mycobacterium and Nocardia

the result of having
cell walls with a high content of waxes and other complex lipids


resistant to drying and to many disinfectants


Primary stain of Acid Fast

carbo I fuchsin

Decolorization of Acid Fast

acid-alcohol (I % Hel in 70% ethanol).

Counterstain of Acid Fast

methylene blue

Resulting colors of Acid-Fast

red = acid-fast
blue = non-acid-fast

Primary stain of Spore Stain

malachite green

Counterstain of Spore Stain

0.5% safranin



Spore Stain results

Spores are green
Vegetative cells are red

This violet stain is used first and stains all cells purple

Crystal Violet

killing agent, increases the cells' affinity for a stain.

Gram's Iodine

serves a dual function as a protein dehydrating agent and as a lipid solvent.

Ethyl Alcohol (ethanol), 95%

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