1. Gram stain

2. Purpose: to differentiate between G+ve bacteria and G-ve bacteria
- This stain is a differential stain ,it consist of at least 3 reagents :

3. 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

6. *Mordant: iodine is mordant which fixes the primary stain to bacteria

8. *Which is the most important step of Gram stain ?(why?)

9. 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

10. 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

11. 10-Dry with filter paper
11- Examine under microscope