Now let us study gram staining which is the most commonly used staining technique in microbiology. Here's an outline for gram staining. First we will look at the technique. Then we will see each of the seven steps in detail including the washes. Next we'll review the differences between gram positive and Gram negative cell walls. This review will help us understand the reasoning behind gram staining.

And finally, we will know that apart from identifying gram positive and Gram negative cells by the color, the stain helps in identifying other characteristics of the cell. As with other stains let us focus on the gram staining technique first It starts by fixing the cells onto the slide by applying heat. At this point in heat fixation since both cells that is grand positive and Gram negative cells are unstained they cannot be identified yet under the microscope. Next, crystal violet is used to stain the cells at this step, both gram positive and Gram negative cells are stained in blue. Then, the cells are treated with iodine which is used to form a precipitate with the stain. Following iodine treatment, a D colorizer is used.

The D colorizer removes the crystal violet stain from negative cells Gram negative cells that is, but not from the gram positive cells. Finally, in order to visualize the gram negative cells, a counter stain has to be used such as saffron. And at this point, Gram negative cells are stained as pink to red, whereas gram positive cells are remained stained in blue, due to the crystal violet. Thus, gram positive and Gram negative cells can be distinguished easily. Let us look at each step in detail. After he fixed in the cells.

The crystal violet dyes applied to the slide specimen beginning the gram staining procedure Next, the slide is washed for about 10 seconds. Then Graham's iodine is applied. After one minute the solution is washed off with tap water. Next slide is washed with tap water. Then a D colorizer. Usually a mixture of ethanol and acetone is used.

Then the counter stain suffering is applied to the slide, causing the gram negative cells to be stained in pink. And at last slide is allowed to dry before being visualized under the microscope. This is how a gram staining containing both gram positive and Gram negative bacteria cells would look like. In figure a. You see the blue stained gram gram positive cells that are circular known as coxide. And in pink are the gram negative cells that are rod shaped known as last lie.

In figure B, you have two types of bassy lie. One is the gram positive, so stained in blue and the other gram negatives self stained in pink. Just as a review of the differences in the cell wall of gram positive and Gram negative cells You can notice that both cell types have an inner membrane, the gram positive and Gram negative. That is the inner cell membrane. Both cells have a peptidoglycan layer, which is shown here in this light, orange orangish color. However, the gram positive cells have a much thicker peptidoglycan layer compared to the gram negative 50 dislike and layer which is much thinner for the more the gram positive cells like an outer cell membrane, whereas the gram negative cells have an outer cell membrane.

Finally, the grandpa's two cells have less lipid content compared to the gram negative cells. So, if you are wondering, what is the reasoning behind gram staining it has to do with the chemical and physical properties of the cell wall. Mainly the lipid content and the thickness of the peptidoglycan, also known as PG layer. gram positive cells have a low lipid content and a thick peptidoglycan layer, whereas Gram negative cells high have a high lipid content and a thin PG layer. The D colorizer, also known as a solvent is a solvent that easily removes a crystal violet dye from the thin PG layer and dissolves the liquid layer of the gram negative cells, which further enhances the leaching of the primary stain. So, the crystal violet, along with the iodine form a precipitate that is retained by the gram positive cells but not by the gram negative cells.

Gram staining apart from distinguishing bacterial cells as gram positive and Gram negative cells, based on the color allows us to identify the shape of the bacterial cells as we have seen before and their sizes. As to most dyes that's giving details of the cell morphology. Just as a review of gram staining techniques, cells are stained with crystal violet then iodine, which stains the cell as purple and blue respectively. Then the dye is washed off with a de colorizer which is usually an alcohol wash or a mix of acetone. And saffron is finally used to counter stain the gram negative cells. gram positive cells then retain the crystal violet whereas the gram negative cells do not.

Therefore, gram staining is based on the ability of a cell in retaining crystal violet during solvent treatment. The difference in the microbial cell wall is clearly noticeable by using grams staining. This concludes our study of the Gram stain