Gram Stain

 The Gram staining method, named after the Danish bacteriologist who originally devised it in 1882 (published 1884), Hans Christian Gram, is one of the most important staining techniques in microbiology

1. Perform a bacterial smear. 2 1. Perform a bacterial smear . 2. Saturate the smear with crystal violet for 1 minute. 3. Rinse the slide gently with water. 4. Saturate the smear with iodine for 1 minute. 5. Rinse the slide gently with water. 6. Decolorize with Gram decolorizer (acetone/alcohol) for 3-5 seconds 7. Rinse the slide gently with water. 8. Counterstain with safranin for 1 minute. 9. Rinse the slide gently with water. 10. Carefully blot the slide dry with bibulous paper. 11. Observe the slide under the microscope

Gram positive bacteria will stain purple Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink

Gram positive Gram positive bacteria have a thick mesh-like cell wall made of peptidoglycan

Gram positive bacteria will appear purple under the microscope Gram positive bacteria will appear purple under the microscope. They have a single thick wall. The crystal violet and iodine combine to attach this wall. The declorizer (alcohol) dehydrates the cell wall, causing the pores to close, trapping the stain inside, the safranin added in the final step, does not penetrate the wall.

gram negative bacteria does not have peptidoglycan

Gram negative bacteria will appeared Gram negative bacteria will appeared. The have a cell wall and additional thin layers of fatty sugars. The decolorizer easily penetrates these thin sugar layers, washing away the crystal violet-iodine chemical. The safrain in the last step attaches to these layers and appears red.

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