Ziehl-Neelsen acid-fast staining procedure (2014).Retrieved from. http://www.medical-labs.

net/ziehl-
neelsen-acid-fast-staining-procedure-822/

Young cultures must be used so the crystal violet can stick to the cell walls of Gram positive bacteria.
The cell walls break down in old cultures and the staining process is not accurate. In addition, Old
cultures of some gram-positive bacteria will appear Gram negative. This is especially true for endospore-
forming bacteria, such as species from the genus Bacillus

Virtual Microbiology (n.d). The Gram Stain. Retrieved from.

https://instruction.bact.wisc.edu/instr/book/displayarticle/89

The decolorization step must be performed carefully, otherwise over-decolorization may occur. This
step is critical and must be timed correctly otherwise the crystal violet stain will be removed from the
Gram-positive cells. If the decolorizing agent is applied on the cell for too long time , the Gram-
positive organisms to appear Gram-negative. Under-decolorization occurs when the alcohol is not left
on long enough to wash out the CV-I complex from the Gram-negative cells, resulting in Gram-
negative bacteria to appear Gram-positive.

Gram Stain Technique (n.d). Retrieved from.

https://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1