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(D) Gram-positive bacteria retain the crystal violet stain due to a thick peptidoglycan layer in their cell wall. $1$. $A$ defective staining techniqu

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All the above

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(D) Gram-positive bacteria retain the crystal violet stain due to a thick peptidoglycan layer in their cell wall. $1$. $A$ defective staining technique (e.g.,over-decolorization with alcohol) can wash away the primary stain,making them appear Gram-negative. $2$. $A$ changed $pH$ of the medium can affect the cell wall integrity or the binding affinity of the dye. $3$. In an aged bacterial culture,the cell wall structure often degrades or becomes thinner,losing its ability to retain the crystal violet-iodine complex. Therefore,all these conditions can lead to a Gram-positive bacterium appearing Gram-negative.

Under which of the following conditions does a Gram-positive bacterium appear Gram-negative?

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