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It is possible to decolourise gram positive bacteria and thus get false results of all gram negative organisms. Similarly, it is possible to under-decolourise and get all gram positive organisms. Ideally controls of known organisms should be run with each stain.

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Q: Why is decolorizing the most crucial step?
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What is the most crucial step in the gram stain procedure?

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 long.


Which step is most crucial or most likely to cause poor results in the gram stain?

Gram staining is a type of differential staining in which two types of bacteria are differentiated on the basis of their cell wall either gram positive or gram negative although all the steps in gram staining are crucial, the most important step the most crucial step in the performance of the Gram staining procedure is the decolorization step which is the Acid-Alcohol (3% HCl and 95% Ethanol) and must be timed correctly; the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).


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