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Basic Principles of Gram Staining

Gram-staining was first described by Christian Gram in 1884. It is a differential staining technique that uses a primary stain (crystal violet) and a secondary counterstain (safranin) to distinguish between gram-positive and gram-negative bacteria based on differences in the thickness and permeability of their cell surface to the dyes (we will discuss these properties in the next lecture). Gram staining is fast and can provide immediate information regarding the presence/absence of bacteria, ultimately guiding the clinician toward the appropriate antibiotic treatment.

UNSTAINABLE ORGANISMS: Some organisms are unable to be identified via the gram staining method. Special dyes or microscopes may be required to visualize the bacteria microorganism.


  • Acid-fast organisms (Mycobacteria & Nocardia)

    • have a high lipid concentration called mycolic acids causing them to stain "acid fast" as they resist decolorization phase.

  • Spirochetes (Treponema, Borrelia, Leptospira & Spirillum)

    • are too thin to be seen under a regular light microscope and often require darkfield microscopy.

  • Intracellular organisms (Legionella, Rickettsia, Coxiella, Ehrlichia & Chlamydiae) 

    • are morphologically similar to gram neg organisms, but won't stain since they are hiding inside other cells.

  • Organisms with no cell-wall ( Mycoplasma & Ureaplasma)

    • If you don't have a cell wall, you can't absorb the gram stain 


Gram stain limitations:

  • UV light, Antibiotics, prolonged heat fixation, crushing of unprotected cells on a slide or autolysis by enzymes can alter the gram stain results (often causing to stain pinker).

  • The age of culture can affect stain results (cells 48 hrs old tend to stain more purple)

Continue to the next topic to learn more details about

the morphology and cell wall characteristics of bacteria!




Bacteria Morphology


Acid Fast Staining

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