Question

Describe the color of Gram positive and Gram negative bacteria in each step of the Gram staining procedure. (Be sure to account for the procedure, the reagent, and the colors of Gram negative and Gram positive bacteria in your description.)

Answer 1

Answer

Gram staining is an important process invented by Hans Christian Grams. It is the cornerstone of bacterial identification and taxonomic division. Gram positive bactria are stains purple/violet. Gram negative bacteria stains red/pink.

Protocol -

Initially the Colour of both gram positive and negative are colorless.

1. Add crystal violet which is the primary stain to a heat fixed smear of a bacterial culture. 1 minute is taken with the stain. Both types of bacteria shows dark violet

2. Slide is washed with steam ( gentle and indirect) for two seconds.

3. Grams iodine is added for 1 minute as a mordant. It binds to crystal violet and traps it in the cell. In both cases Colour changes to purple/violet.

4. Slide is washed with steam (gentle and indirect) for two seconds.

5. The slide is flooded with decolorizing agent, acetone - alcohol decolorizer. 10- 15 minutes is waited until the decolorizing agent disappear. Gram negative become colorless and positive remain same.

6. Slide is counterstained with safranine. Waited for 30 - 60 seconds. Gram negative become red/pink and positive remain same that is violet/purple.

7. Slide is washed in gentle and indirect steam until no color appear in the effluent. The slide is blot dry with absorbent paper.

8. Using bright field microscope, the slide is observed under 100x oil immersion.

As mentioned above the gram positive stain violet/purple and gram negative stain pink/ red.