Protocol for Gram staining

Solutions:

Hucker's crystal violet reagent is made by mixing two solutions:

This mixture is stable and can be kept at room temperature for months. An iodine solution and counterstain are also required:

Procedure:

Prepare a light suspension of cells from very young cultures grown on appropriate agar medium. If the suspension prepared is too turbid, dilute with distilled water.

1.       Add one drop to a clean glass slide and spread the drop with a loop over the surface of the slide. Allow to air-dry.

2.       Carefully fixate the cells by moving the slide into a flame (bacteria upwards).

3.       Flood the slide for 1 minute with Hucker's reagent.

4.       Wash the slide by dipping the slide into slow running tap water.

5.       Flood the slide with iodine solution for 1 minute.

6.       Place slide diagonally in glass box and rinse of iodine solution with safranin. 

7.       Add excess amount of fresh safranin and wait for 35 seconds.

8.       Rinse slide with water as described under (5).

9.       Allow the slide to air-dry.

10.   Examine the preparations with the oil immersion objective of the bright field microscope (do not use the phase-contrast objective!). A drop of oil can be placed on the slide directly.

11.   Gram-positive cells appear purple and Gram-negative cells pink. (The iris of the microscope condenser should be opened as wide as possible. With a closed condenser colours can hardly be discriminated.).