Gram Stain\ Acid Fast Stain

• Since bacteria can’t be seen by naked eye,

microscope is used. And to facilitate the
observation of bacteria, stains are used.
•
• Stains or dyes are divided into to groups:
1. Acidic dyes: negatively charged
2. Basic dyes: positively charged

• Basic dyes such as crystal violet, sufranin, basic

fuchsin and methylene blue are used for staining
bacteria since bacteria are negatively charged.
Staining process is divided into tow groups:

Simple staining Complicated staining

• One type of dye is used • Tow or more of dyes are
• To study the arrangement used
and morphology of • To study the arrangement
bacterial cells. and morphology of
bacterial cells and
differentiate between
different types of bacteria.
• Gram stain is an example
of this type of staining.
Gram stain steps:
• First of all, smear must be prepared and
then go to gram stain.

1. Crystal violet (1 min)

washing
2. Iodin (1 min)
Washing
3. Decolorization by alcohol
(8-10 sec.)
washing
4. Safranin (1 min)
washing
• False gram negative result could be resulted
from the over decolonization of gram positive
bacteria, where weakly and strongly bounded
crystal violet-iodin complexes are removed by
alcohol. So the cell wall of gram positive
bacteria is empty from crystal violet and ready
to receive safranin and therefore appear pink
under microscope.
 Acid fast stain
• Acid fast microorganisms are the microorganisms that
resistant to acid and, therefore, acid do not affect them at
all.

• Such as mycobacterium that cause tuberculoses.

• Acid fast microorganisms can’t be stained by gram stain

although they are similar to gram positive bacteria
regarding the several layers of piptedoglycan, due to the
high amount of lipids in their cell walls which by turn make
the penetration of crystal violet to the cell wall very hard.
Acid fast stain steps:
1. Carbol fuchsin contains red dye
washing
2. Decolorization: using acid-alcohol
washing
3. Methylene blue
washing

• Acid fast microorganism appear red under

microscope, while any other type of bacteria appear
blue.