the Gram stain

In your next practical you will be characterising bacterial mixtures. One technique you will be using to do this is the Gram stain.  Here is the Gram stain explained:

The Gram stain differentiates between two major groups of bacteria based on their cell wall structure. So lets take a look at the two types of cell wall structure we are talking about...

 As you can see from the above diagram, Gram-positive bacteria have a thick peptidoglycan layer surrounding the membrane. Gram-negative bacteria on the other hand have a much thinner peptidoglycan layer and in addition they have another membrane surrounding this peptidoglycan layer.

The Gram stain differentiates between these bacteria - hence they are called Gram positive and Gram negative depending on what they look like after being treated by the Gram staining procedure. 

What happens during the Gram Stain procedure?

During this procedure, the bacteria are treated with various chemical solutions in a particular sequence, and the result is either pink or purple bacteria depending on the cell wall structure...

So, going through the diagram above, we can see that there are 5 steps involved in Gram Staining:

1. Fixation - the cells are 'stuck' to a microscope slide using heat
2. Crystal violet - crystal violet dye is washed over the fixed cells and then washed off with water
3. Iodine treatment - iodine solution is washed over the cells and then washed off with water
4. Decolorisation - acetone is washed over the cells and then washed off with water
5. Counter stain with Safranin - Safranin is washed over the cells and then washed off with water

What actually happens is:

For Gram positive bacteria, the crystal violet dye and iodine solutions form a complex in the cell. Treating the cells with acetone then causes the peptidoglycan layer to shrink and this means that the dye complex cannot leave the cells when they are treated with the destain. As a result, the bacteria keep the purple stain.

For Gram negative bacteria, the crystal violet dye and iodine solutions form a complex in the cell but this time, because the peptidoglycan layer is very thin, it cannot prevent the dye leaving the cells again when they are washed with the destain. As a result, the bacteria will show up the Safranin counterstain.

I hope this will help you in lab