Why is Gram-Staining Important?
Gram-staining separates bacteria into gram-positive and gram-negative groups, based on the microorganisms' abilities to retain the dye crystal violet during decolorization with alcohol. The color difference between the two reflects differences in the outer surface structure of the microorganisms.
Using a slide that is already smeared with the bacteria, cover the smear with the primary stain, Crystal violet. Leave on for 20 seconds.
Rinse off with distilled water and drain off excess water.
Pour the mordant, Gram's iodine solution, on smear and leave on for 1 minute.
Drain off excess Gram's iodine and pour the decolorizing agent with 95% ethyl alcohol over the smear for 10 to 20 seconds or until the solvent flows colorlessly from the slide.
Rinse the slide with distilled water for a few seconds.
Put the counterstain, Safranin, on the smear for 20 seconds.
Rinse with distilled water for a few seconds.
Blot the slide between pages of bibulous paper and allow to dry.
Slide may be observed under oil immersion immediately.
Interpreting the results:
So how do you tell which bacteria you observe under the microscope are gram-negative and which are gram positive? Gram-negative bacteria have a pink color because they lost the purple color of the crystal violet during the decoloriztion process and were left with only the pink of the safranin. Gram-positive bacteria have a purple color since they retained the dye from the crystal violet, even after decolorization.
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