[Histonet] Re: Harris hematoxylin weak staining

Pamela Marcum mucram11 <@t> comcast.net
Mon Sep 21 09:09:54 CDT 2009

If you bleach your staining dishes they must be rinsed to excess or some bleach may remain in the container and effect you staining as it leaches into the solution.  Check to be sure everyone takes the time to do this as I have had an issue where one or more people in the lab were more careful tham others about this step.  

Using Harris requires filtering so the type of filter paper can also cause an issue.  Use a more porous paper. 

Pam Marcum 

----- Original Message ----- 
From: "Teri Johnson" < TJJ @ stowers .org> 
To: " histonet " < histonet @lists. utsouthwestern . edu > 
Sent: Monday, September 21, 2009 9:57:13 AM GMT -05:00 US/Canada Eastern 
Subject: [ Histonet ] Re: Harris hematoxylin weak staining 

Aazath , 

There are several strategies you can use to improve your hematoxylin staining. 

1 - use distilled or DI water before putting your slides into the stain. Sometimes the chemicals/minerals/metals in the tap water can weaken your stain. 
2 - replace your stains when they start showing signs of weakening, or routinely in order to keep the quality even. 
3 - try using more than one bucket/dish of hematoxylin and split the total staining time between them. Then you can rotate the last one up to the first position, and put a new change in the second station. This might help keep your staining more consistent. 
4 - does your tap water seem to contain a high level of chlorine? If so, you can use a bluing agent to blue your stained slides, and then use DI or distilled water rinses. 
5 - do not linger in the decolorizing acid alcohol. Usually one quick dip followed by an immediate plunge into water is all you need. 
6 - some have reported that their eosin , if the pH drops too low, can leach out some of the hematoxylin . According to Carson, the ideal pH of eosin is between 4.6 and 5. 
7 - what thickness is your tissue? 3 microns thin tissue needs longer in the stain solution than 5 microns thin tissue. 

There may be others that folks will chime in with, but these are the most obvious ones to me. 

Good luck! 

Teri Johnson, HT( ASCP ) QIHC 
Managing Director Histology Facility 
Stowers Institute for Medical Research 
1000 E. 50th St. 
Kansas City, MO 64110 

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