Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Automated H & E staining problems
We are having a problem with our H & E being inconsistent (sometimes from day to day, sometimes from batch to batch). We have an automated stainer and use bought solutions of hematoxylin and eosin. We do not change program times or reagents, yet sometimes our stain is light and sometimes it is dark (preferred). We have not changed any processes, vendors, or manufacturers, but our stain is continually changing.
The same hematoxylin, eosin, alcohol, and xylene are on our manual stain line. We stain those following the same times as on the auto stainer and they come out perfect every time.
Is your manual stain set-up absolutely identical to your automatic stainer set-up, in time values as well as reagent set-up? If so, the times on the machine may be too short, as explained below.
You commented that when you stain from your manual set-up the staining results are fine. I would recommend that you "manually" stain using your automatic stainer set-up. If you are able to acheive the desired results, then we can identify the mechanical differences between human and machine staining. It would be helpful to compare your stain programs (Manual procedure and automatic times).
Analyzing the stain, is the nuclear stain OK but the counterstain is too light? Is the nuclear stain too light but the counterstain OK? Is the nuclear stain too light and the counterstain too light? Are the stains consistant in their lightness throughout the specimen and throughout all sections on the slide? Do you notice an improvement in the stain after
the new reagents have become somewhat diluted?
One of the biggest differences between hand and machine staining is how the surface tension of the reagent currently on the slide is broken and then replaced by the next reagent. When we stain by hand we exert much more and varied force than a machine does when plunging the slides into the reagent. We also knock off more reagent, so less of the reagent clings to the slide with each move. A stainer (machine, not human) simply lowers the slides slowly, in a single plane, into the reagent. Even the agitation of the machine staining is in that single plane (up and down) movement. When we stain by hand we cause the reagent in the dish to bombard the slide from several angles and with greater force that breaks the surface tension in less time than it takes a machine can accomplish. Therefore longer exposure times (of tissues to stain) may be required on a machine to yield the same results as hand staining.
When programming the machines I find it necessary to watch the hand staining carefully in order to make an accurate translation of a "dip" to a time value that the machine could reproduce. A "dip" in acid alcohol in manual staining may not be able to be reproduced by a machine. I may be able to use 1% acid alcohol in hand-staining but have to use 0.5% acid alcohol on the staining machine with a 2-second timing value to get the same results. Ten "dips" in a manual stain may require 30 seconds on a machine. Ten "dips" in a manual alcohol step may require 1 minute on a machine for the same results.
One of the things we need to remember is that the machine will move the slides exactly the same way for the programmed time. We humans (consciously or unconsciously) adjust our handling of the slides based on how the sections or even the reagents look.
Sakura Finetek USA, Inc.
Torrance, CA 90501