Gram Staining

Microscopy

Gram Staining Protocol

1. Heat fix sample onto a glass slide. (A dilute sample works best)

2. Flood slide with Hucker’s Crystal Violet for 1 minute.

3. Rinse slide with water (for ~5-10 seconds, until excess stain is removed)

4. Flood slide with Grams Iodine for 1 minute.

5. Rinse with water.

6. Decolourize with 95% ethanol for ~30 seconds. (Acetone may also work as a decolourant but it works much faster than ethanol i.e.: < 7 seconds)

7. Rinse with water.

8. Flood slide with Safranin for 30 seconds.

9. Rinse with water.

10. Blot dry with a paper towel or place in a cool incubator to air dry. (10 minutes at 37°C will suffice)

11. View the slides under 1000x microscopy.

Tips for success

a. Label your slides in pencil so the label won’t get washed away during the decolourising step.

b. Wastewater samples need to be diluted ~ 1 in 20 (depending on the thickness and type of your sample).

c. Acetone use must be optimised such that the primary strain is not completely stripped away.

d. Make up your stain solutions in dark, tightly sealed bottles, and they will last ~6 months.

e. If they cannot be visualized immediately, slides can be stored in a sealed box at room temperature for a week.