# 2.1: Gram Stain Procedure

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The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.

## Preparation of microscope slide:

1. Clean slide with a Kimwipe and alcohol to remove any fingerprints.
2. Draw two circles with your Sharpie on the bottom of the slide.
3. Using your inoculation loop, put two small drops of water in each circle.
4. Using aseptic technique, remove a very small amount of bacteria from the culture tube. Make sure you flame the tube before and after you enter.
5. Smear the bacteria in the drop of water on your slide. You may go out of the perimeter of your circles!
6. Let the slide air dry completely.
7. Heat-fix the slide by running it through the flame 3-4 times with the ‘smear’ side up. Do not flame the side with the bacteria!
8. Let the slide cool completely and you are ready to stain it.

## Staining procedure:

1. At the back sinks, place crystal violet on each smear for 1 minute.
2. Rinse the crystal violet off of the slide by swishing the slide gently in the large beakers labeled ‘Crystal Violet’.
3. Tap slide on paper towel to remove most of the water.
4. Place Gram’s iodine on each smear for 1 minute.
5. Rinse by running water from the tap very slowly over the surface of the slide while holding it at an angle.
6. Tap slide on paper towel to remove most of the water.
7. Place ethanol on each smear and for 15-30 seconds. This is the most variable step.
8. Rinse with water and tap dry.
9. Place safranin on each smear for 1 minute.
10. Rinse with water and tap dry.
11. Blot gently with bibulous paper.
12. Dry the bottom of the slide before placing it on the stage of the microscope and view with the oil immersion lens.

This page titled 2.1: Gram Stain Procedure is shared under a CC BY license and was authored, remixed, and/or curated by Molly Smith and Sara Selby (GALILEO Open Learning Materials) .