Download Gram stain procedure

Purpose: to differentiate between •
G+ve bacteria and G-ve bacteria
- This stain is a differential stain ,it
consist of at least 3 reagents :
1- primary stain: (crystal violet) : all bacterial cell
will be stained
2- decolorizing(alcohol) : some of the bacteria will
be effected by the alcohol and will lose their
color ,other remain stained
3- secondary or counter stain(safranin):
the bacteria that lost their color with alcohol will
be counter stain with safranin
*Mordant: iodine is mordant which
fixes the primary stain to bacteria
*Which is the most important step of
Gram stain ?(why?)
Gram stain procedure:
1- Put one drop of water in the
middle of plain slide
2- Inculcate the bacteria
3- Do the smearing
4- Allow to air dry
5- Do heat fixation for 3x
6- Stain crystal violet (1min.),then wash
with water
7-Iodin on the slide(1min.),then wash with
water
8- Alcohol (wash it directly)
9-Safranin(1min.) ,then wash it with water
10-Dry with filter paper
11- Examine under microscope