Gram Stain

Gram stain is a empirical method named after its

inventor, Hans Christian Gram, that is used to

divide bacteria into two large groups, gram-negative

and gram-postive bacteria.

The way that this is done is relatively simple:

1. Make a slide or dish of bacteria that you are going to stain. Heat the slide/dish until the bacteria are fixed to it
2. Add primary stain; crystal violet.
3. Add Gram's iodine. This does not stain the bacteria, but fixes the violet stain in the bacterial cell wall.
4. Wash the slide/dish with ethanol alcohol and acetone, which decolorizes it. If the bacteria is gram-positive it will retain the stain in the cell wall, staying purple, if it is negative, it will go back to being colorless.
5. Add the second stain,safranin and incubate it for about a minute. Wash it with water for 5 seconds, maximum. If the bacteria is gram-positive then the cell will retain the primary stain, will not take the secondary stain, and will appear black-violet. If it's negative, then the cell will lose the primary, take the secondary, and turn a pink-red color.

    

    As we stated before, the purpose is to divide the bacteria into two groups; gram-positive and gram-negative.

A few characteristics of a gram-positive bacteria are a cytoplasmic membrane and a thick cell wall made of peptidoglycan. It also has capsule polysaccharides and a two-ring  supported flagella. A gram negative bacteria also has a cytoplasmic membrane, but in contrast, it has a thin cell wall made of peptidoglycan. There is also a space between the layers of peptidoglycan and the secondary cell membrane called the periplasmic space. Also, the flagella, when present, has a four-ring support instead of two.

    If you learn well with visual aid, there is a video that you can watch on gram staining technique below. This man was a little bit sloppy with his technique, but it will help you to get a basic visual idea on how to do a gram stain.

Gram Stain Procedure