Nematodes

Gram Staining

Principle

It is the most common technique used in microbiology for differential staining. It is named after its inventor Dr. Christian Gram. It’s a differential staining which requires the use of at least three chemicals that are applied sequentially to a heat-fixed smear. The first reagent is called primary stain. Its function is to provide a basic stain to all types of cells present in the smear. Then a second reagent is used, called a decolorizing agent. Based on the chemical composition of the cell wall it can decolorize the primary stain. Then a final reagent is added which is called counterstain which can provide contrasting color to the non-colored cells. Counterstain can not be absorbed into the already stained cells (Primary stain). It will only colorize the cells if the primary stain is removed by the decolorizing agent.  In this way, cell types can be distinguished from each other on the basis of the stain they will retain. By this technique, we can divide bacterial cells into two groups: gram-positive and gram-negative which is one of the essential tools for bacterial classification and identification.  This staining method is based on the chemical composition of the bacterial cell wall. Gram-positive cells have thick peptidoglycan while gram-negative has a very thin peptidoglycan layer. Moreover, the gram-negative cell wall is further surrounded by a lipid-containing layer. Peptidoglycan is the basic structural block of the bacterial cell wall. It is composed of two chemical subunits N-acetyl glucosamine and N-acetyl muramic acid. These are cross-link to each other which provide rigidity to the gram-positive cell wall. While gram-negative cell wall is not so much rigid due to high lipid contents. This is why primary stain is retained in gram-positive cells while gram-negative cell loses their stain upon decolorizing.
Primary stain: Crystal Violet (give purple to bluish color) 
Mordant: gram’s iodine. This reagent serves as a mordant which has the property of intensifying the primary stain. It binds to crystal violet and forms an insoluble complex crystal violet-iodine (CV-I). this serves to intensify the color of the primary stain. At this point, cells will appear purple.
Decolorizing Agent: Ethyl Alcohol (95%). This reagent serves a dual function as a hydrating agent and lipid solvent. Due to lipid solvent properties, it can weaken the lipid coating in the gram-negative cells by making it porous. Which will ultimately remove the CV-I complex from the cell wall. Upon washing of primary stain gram-negative bacterial cells are rendered colorless at this point. While gram-positive cells retain primary stain due to no washing effect of ethyl alcohol due to their rigid peptidoglycan structure.
Counterstain: Safranin. This is the final reagent use to stain the remaining unstained bacterial cells to red/pink colored. As in the decolorizing step only gram-negative cells are decolorized, they will be stained red while gram-positive cells are already stained in purple color.

Materials 

24h nutrient agar culture of S. aureus and E. coli, crystal violet, Gram’s iodine, ethyl alcohol (95%), safranin, glass slides, staining rack, inoculation loop, Bunsen burner, blotting paper, microscope.

Procedure

1. Clean the slide with alcohol to render it oil-free.
2. Perform a smear by placing a drop of water, then mix culture to drop. Then, with other slides spread the mixture to the other end.
3. Allow the smear to air dry and heat fix the smear.
4. Gently flood the smeared area with crystal violet and wait for one minute.
5. Wash the smear with tap water.
6. Flood the smeared area with Gran’s iodine and wait for one minute.
7. Wash with tap water.
8. Decolorize with 95% ethyl alcohol. Add drop by drop until the alcohol becomes clear.
9. Wash with tap water.
10. Counterstain with safranin by flooding the smeared area for 45 seconds.
11. Wash with tap water.
12. Blot dry the slide and examine under a microscope.