From:Pam Marcum <>

Although it is not specifically stated in most protocols I was trained to
always add the materials in the order given with a good mix after each.  The
person who trained me Histology did not explain why, just said they don't
work if you mix wrong.

While working at Sigma in stain development I went back to very old German
protocols and early procedures where I found we did indeed need to add in a
specific order.  I no longer have the references (it's been 20 years and I
had to leave them with Sigma)

The original German stain manufacturers did give very good instruction and
list problems that occurred if the order was changed.  I know at least two
stains we did, did not work if mixed out of order.
Pamela A Marcum
Product Development Manager
Polysciences, Inc
-----Original Message-----
From: Lee & Peggy Wenk []
Sent: Sunday, October 15, 2000 7:41 PM

Does it matter what order the reagents are added together to
make an aluminum salt-sodium iodate hematoxylin, such as

I always add them in the same order, with lots of stirring
between reagents. Got wondering if it would make a difference
if they were added in a different order. Since we're making
hematoxylin into hematein, and making hematein into
aluminum-hematein, it seems like it should make a difference.

(This is for my School. I still have the students make Mayer
hematoxylin. It helps them to learn what's in their
hematoxylin, and hopefully helps them learn to

Peggy A. Wenk, HTL(ASCP)
William Beaumont Hospital
Royal Oak, MI 48073

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