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Steps to perform gram-staining

  1. Apply crystal violet on the slide and keep it for 60 seconds. Wash the slide with water.

  2. Apply iodine on the slide and keep it for 60 seconds. Wash the slide with water.

  3. Apply 95% ethyl alcohol on the slide and keep it for 5 to 10 seconds. Wash the slide with water.

  4. Finally, apply safranin on the slide and keep it for 45 seconds. Wash the slide with water.

Four stages of gram staining experiment. In the first stage, the content of the bottle with a purple substance, called crystal violet, is poured on the glass slide and left there for 60 seconds. As a result, the spherical and rod-shaped structures inside the circle, representing the microscopic view of the bacteria on the glass slide, are colored in purple. In the second stage, the content of the bottle with an orange substance, called iodine, is poured on the glass slide and left there for 60 seconds. As a result, the bacteria under the microscopic view remain purple. In the third stage, the content of the bottle with a light blue substance, called 95% ethyl alcohol, is poured on the glass slide and left there for 5 to 10 seconds. As a result, the rod-shaped bacteria under microscopic view are decolorized, and the spherical ones remain purple. In the last stage, the content of the bottle with a pink substance, called safranin, is poured on the glass slide and left there for 45 seconds. As a result, the rod-shaped bacteria under microscopic view are colored in pink, and the spherical ones remain purple. The rod-shaped bacteria are named gram-negative, and the purple ones are named gram-positive.

Figure 1: Workflow of gram staining method