GRAM'S STAINING

Gram staining

AIM:

To stain the bacterial smear by following Gram’s staining procedure and observe their morphology and arrangements

PRINCIPLE

The differences in cell wall composition of bacteria account for the Gram staining. Gram-positive cell wall contains a thick layer of peptidoglycan. In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both Gram-positive and Gram-negative cells. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of bacteria. The outer mem

brane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counter stain, the positively charged dye safranin, is added.

Materials required:

Inoculation loop, slide, Bacterial culture, Bunsen burner, Gram staining kit

PROCEDURE:

Gram Staining Procedure:

1.      Prepare a smear over a clean slide and heat fixed.

2.      Flood the smear with crystal violet and wait for 1 minute.

3.      Wash slide in a gentle and indirect stream of tap water for 2 seconds.

4.      Flood slide with the mordant: Gram’s iodine. Wait 1 minute.

5.      Wash slide in a gentle and indirect stream of tap water for 2 seconds.

6.      Decolorize the smear by Flood with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add decolorizer drop by drop on slide until decolorizing agent running from the slide runs clear.

7.      Flood slide with a counter stain, safranin. Wait 30 seconds to 1 minute.

8.      Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.

9.      Observe the results of the staining procedure under oil immersion (100 x) of a microscope.

Results:

§  Gram-negative bacteria will stain pink/red and

§  Gram-positive bacteria will stain blue/purple.