Question

1. How does background stain works?

2. State the use of the primary and secondary stain in Gram staining procedure?

3.Describe the detailed procedures used in the application of gram stain

Answer 1

Ans 1- Some bacteri ado not take get colored by dye, but the background takes up the color. Negative staining produces an outline or silhouette of the organisms against a colorful background.

Ans 2- Gram stain permits the separation of all bacteria into two large groups, those which retain the primary dye (gram-positive) and those that take the color of the counterstain , secondary dye (gram-negative).  The primary dye is crystal violet and the secondary dye is safranin O.

Ans 3-

The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. The steps of the Gram stain procedure are listed below.

1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet–iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye.

Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish between gram-positive and gram-negative bacteria.