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Gram Stain. Differential stain (Hans Christian Gram, a Danish doctor ). He developed a new method to stain bacteria so they can be visible in specimen samples. Differentiate bacteria into two large groups (the Gram Positive and the Gram negative).

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gram stain
Gram Stain
  • Differential stain (Hans Christian Gram, a Danish doctor ). He developed a new method to stain bacteria so they can be visible in specimen samples.
  • Differentiate bacteria into two large groups (the Gram Positive and the Gram negative).
  • Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics.

Almost all bacteria are described by their Gram stain characteristics.

  • Based on differences of Cell wall structures
reagents for gram stain
Reagents for Gram Stain
  • Crystal Violet (purple).
    • Primary stain; positive stain
    • Stains cell wall purple
  • Iodine
    • Mordant
    • Combines with primary stain to form an insoluble complex that gets trapped in bacterial cell wall


    • Decolorizer
    • CV-I complex washed out of Gram negative organisms because it cannot be trapped by lippopolysaccharide layer; flows right through outer membrane
  • Safranin (pink)
    • Counterstain
    • Simple positive stain that provides contrasting dye for decolorized cells (Gram negative)
    • Stains all cells, but only the negative ones actually appear pink.
gram staining procedure
Gram Staining Procedure
  • After air drying and heat fixation.
errors during staining
Errors During Staining
  • Never ever used old culture.
  • Never ever used sample for patient take antibiotic.
  • Time of Decolorizer:
      • Over: G + see as G -.
      • Low: G- see as G +.
  • Time of fixation:
      • Over: G + see as G -.
      • Low: no sample on slide.
acid fast stain ziehl neelsen stain
Acid-fast stain (Ziehl-Neelsen stain)
  • The acid-fast stain is another differential staining method.
  • In this case, the target cells are usually members of the genus Mycobacterium.
  • The cell walls of these bacteria contain an unusually high concentration of waxy lipids, thus making conventional simple stains and Gram stains useless.
  • The genus Mycobacterium contains two important human pathogens, M. tuberculosis and M. leprae,which cause tuberculosis and leprosy, respectively.

The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol, and methylene blue.

  • Acid-fast bacilli will be bright red after staining.

Acid-fast stains are useful in identification of acid-fast bacilli (AFB)and rapid, preliminary diagnosis of tuberculosis (with greater than 90% predictive value from sputum samples).

  • It also can be performed on patient samples to track the progress of antibiotic therapy and determine their degree of contagiousness.
acid fast reagents
Acid Fast Reagents
  • Carbolfuchsin (red), a phenolic stain: is the primary stain in the acid-fast test. It is soluble in the lipids of the mycobacterial cell wall.
  • Heating the specimen, or adding a wetting agent such as Tergitol, increases the penetration of the carbolfuchsin.
  • Following application of the carbolfuchsin, the specimen is cooled and decolorized with a solution of 3% hydrochloric acid and 95% ethanol (acid-alcohol).

Since carbolfuchsin is more soluble in waxy cell

  • lipids than in acid-alcohol, the acid-alcohol removes
  • the carbolfuchsin from non-acid-fast organisms, but
  • not from acid-fast organisms.

Following decolorization, the sample is counterstained with methylene blue which Cannot penetrate mycolic acid; provides contrast to non acid fast cells.

  • Prepare a smear organism and a on glass slides.
  • Allow the slides to air dry, and then heat fix the organisms.
  • Apply enough of carbolfuchsin with Tergitol to cover the bacteria. Allow it to set for five minutes.
  • (Alternate) If Tergitol is not available, apply enough carbolfuchsin to cover the bacteria. Place the slide on a pre-warmed hot plate set on low for 8 minutes. Do not allow the stain to evaporate or Boil. Add additional stain, if necessary. Remove the slide and allow it to cool.
Apply enough methylene blue to cover the bacteria.

Allow it to set for 30 sec.

Gently rinse the slide with water.

Blot (don't wipe) the slide dry with bibulous paper.

Allow the slide to air dry.

Examine the slide under oil immersion.

Positive organisms will appear pink or red;

Negative organisms will appear blue.

Rinse the slide with acid-alcohol (15-20 sec), drop by drop, just until the alcohol runs clear.

Gently rinse the slide with water.



The reddish-pink color and “cording” (sticking together in long ropy masses) of the Mycobacterium cells due to the excess lipids of the cell wall