Gram Staining Procedure

There are different steps in this procedure which is needed to be followed step by side to successfully complete this technique. Those steps are given below:

1. Firstly, get a free slide which should be clean and free from grease.
2. With the loop pour the sample and make a smear of it.
3. Dry the slide with air and then provide heat to the slide. This heat is given to the sample to fix the sample on the slide so that it doesn’t get washed off too easily. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly.
4. Now after the heat finally the dye (crystal violet) is poured onto the slide and minimum for 30 seconds and maximum for 1 minute it is kept on the slide and then it is washed with water. This time is given so that the bond is formed between the dye and the cell membrane of the bacteria. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly. 
5. The mordant or it can be said the iodine solution is applied and it is also kept for a maximum 1 minute and then the slide is washed with normal water. It gets trapped in the cell after making a bond with the crystal dye.
6. Now it’s the turn for acetone or alcohol to be used on the slide and then after 10-20 seconds, the slide is washed again with water. These two agents work as decolorizers.
7. The last step is the application of safranin for 1 minute and then again it is rinsed with water.
8. The slide undergoes blot and air dry and now it is ready for observation under the microscope.

Gram stain is a technique to impart color to the bacterial cell to differentiate between gram-positive bacteria and gram-negative bacteria based on cell wall composition. Gram Staining is a laboratory procedure that consists of four reagents crystal violet (primary stain), iodine (mordant), decolorizer (ethyl alcohol), and safranin (counter stain) to stain the bacterial cell. Hans Christian Gram is a Danish bacteriologist who named this stain and developed this method in 1884.

The basic function of this technique is to differentiate between bacteria based on the chemical and physical properties of the cell walls. The difference in the cells can be identified by the cell wall as the gram-negative bacteria has a thin cell wall due to which the violet stain gets washed out with ethanol whereas the cell wall of gram-positive bacteria is thicker because of which violet stain stays out and give pink color to the bacteria.