Bacteria can be differentiated based on how they react to a a procedure of dying cells called Gram stain. Bacteria are divided into a group that turns purple (gram positive) and a group that turns red (gram negative). Bacteria that are gram (+) include Staphylococcs, Streptococcus, Bacillus and Micrococcus. Gram (-) bacteria include E.coli and Salmonella. The Gram staining procedure is as follows:
Gram Staining Bacteria Procedure
1.Place a drop of distilled water on a slide and, using a swab or inoculating loop, mix the bacteria with the water an smear the mixture on the slide. The mixture will appear cloudy. Using a flame, heat fix the bacteria to the slide (pass the slide through the flame a few times to “dry” the bacteria and affix it to the slide).
2. Using a dropper, add crystal violet to the slide. Let stand for 1 minute.
3. Add iodine to the slide. Let stand for 3 minutes.
4. Decolorize the sample with alcohol. Let stand for 30 seconds.
5. Counter stain the sample with safranin. Let stand 1-2 minutes. Using a dropper, rinse with distilled water.
Gram Staining Results
Gram positive bacteria will appear purple under the microscope. They have a single, thick cell wall. The crystal violet and iodine combine to attach to this wall. The decolorizer (alcohol) dehydrates the cell wall, causing the pores to close, trapping the stain inside. the safranin added in the final step, does not penetrate the wall.
Gram negative bacteria will appear red. The have a cell wall and additional thin layers of fatty sugars. The decolorizer easily penetrates these thin sugar layers, washing away the crystal violet – iodine chemical (purple color). The safranin in the last step attaches to these layers and appears red.