2.3.2: Gram Stain Activity

In this activity you will prepare and stain bacterial samples using Grams stain procedure.

1. Obtain five microscope slides, clean and dry the microscope slides using lens cleaner and Kim wipes
2. Label the slides using a China marker as seen below

   ***NOTE*** the circle must be placed on the bottom of the slide to prevent the marker from being mixed with the bacterial sample

   

3. Obtain 1-2 loopfuls of bacteria from the appropriate broth culture and introduce the inoculum to the appropriate microscope slide, spreading it into a thin film
4. Allow the slides to air dry completely
5. Fix the smears by passing the air-dried samples 2-3 times through the flame of the Bunsen burner smear side up
6. Cover the smears with crystal violet for 1 minute
7. Rinse the slides slowly with DI water for 5 seconds or until the water runs clear
8. Rinse the smears with Gram’s Iodine and then cover the smear with iodine and allow the slides to set for 1 minute
9. Rinse the slides slowly with DI water for 5 seconds or until the water runs clear
10. Apply Gram decolorizer slowly. Continue to add the Gram decolorizer until the dyes does not run off from the smears

    ***NOTE*** Gram decolorizer is a mixture of ethanol and acetone, which is highly flammable!!!

11. Rinse the slides slowly with DI water for 5 seconds or until the water runs clear
12. Cover the smear with safranin for 30 seconds
13. Rinse the slides slowly with DI water for 5 seconds or until the water runs clear
14. Use Kim wipes to blot the slides, smear side down. Blot, do not rub!!!
15. Use your compound microscope to visualize your stained slides.

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