The attachment of the mordant metal (potassium iodide) and crystal
violet to the tissue is by chelation - covalent and coordinate bond
formation. Phosphate hydroxyl groups of the nucleic acids provide a means
for covalent bonding, and other atoms in the vicinity can donate
electrons for the coordinate bond. It is a similar case with proteins,
as there are many hydroxyl and carboxyl groups available.
Nuclear chromatin contains both protein components and DNA, so that two
separate staining events may be happening at the same time. 
That is why some Gram staining methods may still demonstrate nuclear
structures even after all DNA has been decolorized. 

With iodine (trapping agent) the basic principle is that the trapping
agent forms an insoluble compound with the dye (crystal violet) which
precipitates within a structure. Trapping agents are chemicals which inhibit
removal of dyes from tissues. Rather than stop removal of the dye
completely, the usual purpose is to slow it down so that tissues which are
more intensely stained still retain large amounts of dye when the backround
has been decolorized. In this way contrast can be enhanced. Trapping agents
are often confused with mordants, although
their function is completely different.

A solvent is then applied in which the precipitated dye is soluble. The
dye dissolves out easily from most areas, but its dissolution is
resisted for some reason in other structures. The solvent is removed
before these resistant areas are affected. They then appear deep violet
on a relatively colorless background.

The reasons for retention of the dye vary with insufficient
decolorization, air drying during staining and fixation. With structures
such as nuclear chromatin it seems to be just that more dye is present
than in other tissue elements. It therefore, takes longer to remove it
all. 

Eric C. Kellar
Histology/Immunohistochemistry
University of Pittsburgh Medical Center

	----------
	From:  Kathy Wortham [SMTP:kjwortham@yahoo.com]
	Sent:  Thursday, April 22, 1999 9:10 AM
	To:  HistoNet Server
	Subject:  lillie-twort gram stain

	Has anyone run into a problem with blue chromatin staining
(especially
	in lymphoid tissue) with the lillie-twort gram stain?

	Michelle Puette, Pathologist
	m_puette@hotmail.com

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