Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011





Smudgy or unusual haematoxylin staining when using hand staining or an automatic staining machine.
This is generally not a staining technique failure be it manual or automatic. The problem is most commonly associated with fixation, processing or drying a slide. And there are many factors which can contribute to poor nuclear staining.

Some things to check are:
  • Drying out of the specimen before fixation which is a form of mummification. This usually occurs in the theatre where the specimen can be left for some minutes before being placed into fixative. Drying out of the specimen and the beginnings of autolysis and putrefaction can occur all of which can have a bearing on the H&E.

  • The fixative. Is your fixative a fixative? Who made it up? Is it made up correctly? Even commercial organisations have been known to produce bad batches of fixatives and dye solutions. If the fixative is not as it should be it too can affect the H&E.

  • Poor staining is most often seen in small biopsies and it is almost always due to over-processing – using long processing schedules when very short schedules should be used. Over-processing causes other problems as well such as sections can be difficult to obtain because of hard, brittle tissue.

  • Re-cyclers do not produce absolutely pure 100% ethanol. There is always a little water left behind. So if this re-cycled product is used as absolute ethanol there will be too much water retained in the tissue which will affect subsequent processing and staining. Re-cycled ethanol should only be used as a 95% ethanol.

  • The slides should only be dried at just above the melting point of the wax. At over 70 degrees C the tissue starts to cook, which alters the tissue characteristics and usually results in dark smudgy staining.



Click the button to return to the Home Page

© Roy C. Ellis 2002