Deciphering Gram-positive vs. Gram-negative bacteria

Penicillin is only beneficial when it is administered to patients that are infected with gram-positive bacteria. But how can scientist determine which bacteria are gram positive versus those that are gram negative? The answer...The Gram Staining Procedure! Using two different dyes, this process is able to differentiate which bacteria are composed of 90% cell walls (gram positive) and those that are only made of 10% cell wall (gram negative).

After researching, I found that the Gram Staining Procedure goes as follows:

1. Flood the slide with Crystal Violet (the primary stain).

2. After 1 minute, rinse the slide with water.

3. Flood the slide with Iodine (Iodine is a mordant that binds with Crystal violet and is then unable to exit the Gram+ peptidoglycan cell wall.)

4. After 1 minute, rinse the slide with water.

5. Flood the slide with Acetone Alcohol. (Alcohol is a decolorizer that will remove the stain from the Gram-negative cells.)

6. After 10 or 15 seconds, rinse the slide with water. (Do not leave the decolorizer on too long or it may remove stain from the Gram-positive cells as well.)

7. Flood slide with Safrinin (the counter stain).

8. After 1 minute, rinse the slide with water.

9. Gently blot the slide dry. It is now ready to be viewed under oil immersion (1000x TM) with a bright-field compound microscope.

After this staining procedure, the Gram positive cells will appear purple, having retained the primary stain. The Gram negative cells will appear pink, having retained the counter stain after the primary stain was removed by the decolorizer.