Why Is Decolorization The Most Important Step In Gram Staining

The decolorizing stage is the most important because it is at this stage that the principle of the staining method manifest. The decolorizing step is the most critical in gram staining.

Ex 8 Gram Stain Scientist Cindy

It is critical to leave the slides exposed to the decolorizer alcohol long enough to remove the crystal violet stain from the thin peptidoglycan layer of the gram negative bacteria cells but not so long that it removes all of the crystal violet from the thick peptidoglycan layer of the gram positive bacteria cells.

Why is decolorization the most important step in gram staining. Decolorization is important because it will rinse the color out of the gram negative cells so they can be dyed with a secondary dye so you can see the difference between the gram positive and gram negative cells. Organisms that are easily decolorized are gram negative while those that resist are positive. Why is it recommended that gram staining be performed on young bacterial cultures not older than 24 hours.

It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The outcome in this step is based on how easily the cell releases the crystal violet iodine complex. Dividing bacterial cells in two major groups gram positive and gram negative it is a critical tool when classifying and differentiating bacteria.

Over time gram positive cells lose their ability to retain the crystal violet stain after decolorization. This step is 20 seconds long and it is the most important step in the gram stain procedure. After the decolorizing step a counterstain is applied usually safranin but sometimes fuchsine to color the bacteria pink.

The most critical step of gram staining is the decolorization step as crystal violet stain will be removed from both g ve g ve cells if the decolorizing agent e g alchohol is left on too. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between gram positive and gram negative bacteria gram positive bacteria with a thicker peptidoglycan layer retain crystal violet stain during the decolorization process while gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. Heat fixing the cells when done to excess alters the cell morphology and makes the cells more easily decolorized.

The thick cell wall of gram positive cells is dehydrated by the decolorizing step causing them to shrink and trapping the stain iodine complex inside. Bohumil drasar emeritus professor of bacteriology. Hans christian gram in 1884 the gram stain is a differential stain that is used in identifying bacteria.

The particular concerns in this step are listed below reviewed in mcclelland 2001 excessive heat during fixation.

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