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Gram Stain


          Gram Stain for bacteriology

Gram stain pure strain cultures to determine whether Gram negative or Gram positive. Gram staining detects a fundamental difference in the cell wall composition of bacteria.


A.    Prepare a bacterial smear from a pure culture
1.          Put a drop of saline, distilled water, or PBS on a clean glass slide

2.          Using a sterile loop or needle touch an isolated colony and mix in the water drop.

3.          Mix until just slightly turbid (light inoculum is best, excess bacteria will not stain properly).

4.          Let air dry and heat fix. Do not overheat; slide should not be too hot to touch.

5.          Allow to cool.

B.     Flood the slide with crystal violet, and allow to remain on the slide for 60 seconds

C.     Wash off the crystal violet with running tap water.

D.    Flood the slide with Gram’s iodine, and allow to remain on the slide for 60 seconds.

E.     Wash off with running tap water.

F.      Decolorize with 95% alcoholand 5% acetone solution until the solvent flows colorless from the slide (approximate 5-10 seconds). Excessive decolorization should be avoided since it may result in a false gram-negative reading.

G.    Rinse immediately with running tap water.

H.    Counter stain with Safranin for 60 seconds.

I.       Rinse with tap water and allow to air dry.


       RESULTS:Image result for gram stain

1.          Gram negative: cells are decolorized by the alcohol-acetone solution and take on a pink to red color when counterstained with safranin.


2.          Gram positive: cells retain the crystal violet and remain purple to dark blue.

 Image result for gram stain

1.          Positive: Staphylococcus sp.

2.          Negative: Escherichia coli

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