[Histonet] Wright-Giemsa stain

[John Kiernan]

Using a combination of two traditional blood stains such as Giemsa's and Wright's seems an antiquated approach. According to G. Clark's "Staining Procedures" (3rd ed, 1973, p.131) it dates from Artur Pappenheim (1912). It is certainly irrational to use in succession two stains that ought to be closely similar.

The staining mechanism for this group of methods has been pretty well understood for more than 30 years. All the desired colours can be obtained using a solution containing just two dyes (azure B and eosin Y) that are available as pure compounds. There are simple staining methods (eg the "standardized Romanowsky-Giemsa method" in Boon & Drijver's "Routine Cytological Staining Techniques", 1986) that use only the two pure dyes. 

Good eosin Y with high purity, has been around for years. Pure azure B (made by direct synthesis and sold as azure B isothiocyanate) is expensive. It is not the same as certified azure B, which is made by partial demethylation of methylene blue and also contains some azure A and methylene blue (see Penney et al. 2002 Biotech. Histochem. 77: 237-275.). 

Certified dyes are powders that have been tested by an independent non-profit organization, the Biological Stain Commission (BSC) and are used by vendors of solutions. In addition to methylene blue, azures A, B & C, methylene violet (Bernthsen) and eosins Y and B, the BSC also certifies mixed dye powders for some of the traditional blood stains: Giemsa's, Wright's, Jenner's and Macneal's tetrachrome. 

Ideally, a standardized Romanowsky-Giemsa method with pure azure B and eosin Y should be used, as described by Wittekind & Kretschmer 1987 Histochem. J. 19: 399-401. It is cheaper to use a traditional blood stain, which always contains various unnecessary thiazine dyes in addition to azure B, provided that the stock solution has been made from certified ingredients. The BSC does not certify staining solutions, but the vendors of solutions should state that their products were made using certified dyes.
For rational troubleshooting, see Horobin and Walter 1987 "Understanding Romanowsky-Giemsa staining" Histochemistry 86: 331-336 or the book "Troubleshooting Histology Stains" by RW Horobin & JD Bancroft (1998).

John Kiernan
Anatomy, UWO
London, Canada
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----- Original Message -----
From: "Marshall, Kimberly" 
Date: Friday, January 22, 2010 16:27
Subject: [Histonet] Wright-Giemsa stain
To: histonet <@t> lists.utsouthwestern.edu

> I am from a small Hospital where due to the size, Histology does the
> Bone Marrow's. We go to the procedure, make the smears, as 
> well as do
> the Wright-Giemsa stain. I am having a hard time 
> getting the Wright's
> stain dark enough for my Pathologist. I have added 
> stain time, checked
> the pH of my stain, tried different rinse's and buffers, but 
> still not
> getting it dark enough. I do go back to the stain for 45 
> mins or more
> and that will get it at least close to what they want. Is 
> there anyone
> out there that can give me some advise on this 
> stain? Thanks in
> advance for your time
> 
> Kimberly