Gram stain

Author: Nat Pernick, M.D. (see Authors page)

Revised: 18 December 2015, last major update September 2013

Copyright: (c) 2003-2015,, Inc.

PubMed Search: Gram stain[title]
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Cite this page: Gram stain. website. Accessed December 19th, 2018.
Definition / general
  • Stain to detect and differentiate bacteria

  • Apply crystal violet, then iodine, then decolorize by alcohol/acetone, then counterstain by safranin/fuchsin
  • Gram positive bacteria retain the crystal violet-iodine complex after decolorization, are not counterstained, and appear purple
  • Gram negative bacteria have a different cell wall structure, don’t retain the crystal violet-iodine complex after decolorization, and so are counterstained by safranin/fuchsin and appear pink/red

Paraffin sections:
  • Use neutral red instead of safranin; gram negative organisms usually stain poorly because their bacterial wall lipid is removed in tissue processing
  • Note: with hematoxylin and eosin staining on paraffin sections, bacteria appear as blue rods or cocci regardless of gram reaction; colonies appear as fuzzy blue clusters
  • Rapid diagnostic strategy for bronchioalveolar lavage samples consists of Gram stain and bacterial ATP assay (Arch Pathol Lab Med 2005;129:78)
  • Not suitable for burn wound surfaces (Arch Pathol Lab Med 2003;127:1485)
Microscopic (histologic) images

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Fusarium in peritoneal fluid

Gram positive tissue sections-

Bacillus anthracis in CSF

Lactobacillus osteomyelitis

Mycobacterium abscessus (soft tissue)