For microscopical examination the sample is fixed and stained. The Gram staining is often the first step in the identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacterisa can be definitely classified by this technique.

Gram staining (or Gram’s method) is an empirical method of differentiating bacterial species into two large groups (Gram positive and Gram negative) based on the chemical and physical properties of their cell walls. Gram-positive bacteria appears purple whereas the Gram-negative bacterial species appear pink in spinal fluid sample.

There are four basic steps of the Gram stain, which include applying primary stain (crystal violet) to a heat fixed smear of a bacterial culture, followed by the addition of a trapping agent (Gram’s iodine or Lugol iodine solution), rapid decolorisation with acetone+spirit solution, and counterstanding with safranin. All of the necessary reagents are included in the Reagena Gram staining.

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