Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011






Inadequate staining and/or leeching of eosin from tissue sections after washing in water and before dehydrating.


This was a problem I had some years back and it nearly drove me nuts. My laboratory used, and still uses a linear stainer for staining sections with H&E. At times there was virtually no eosin in some of the sections whilst at other times staining was perfect and then again there were times when the eosin was just too heavy. There seemed to be no consistency in the way the eosin solution would stain. We changed the source of the dye several times, we changed the concentration several times we tried adding things like phloxine and even using alcoholic eosin but the problem kept recurring.


Our solution!

Buffer your eosin
Eventually we tracked our problem down to Adelaide tap water which is notoriously acid. The pH of water leaving the tap is often around 4.0 to 4.5 and depending upon the time of year can contain all sorts of heavy salt deposits which raise the pH. And our slides were receiving a brief wash in Adelaide tap water after staining in eosin. So we started to buffer our eosin and the problem disappeared.




We buffered with sodium acetate and acetic acid, starting off with two stock solutions.


Stock solution 1

  • 0.575% aqueous acetic acid

Stock solution 2

  • 0.82% sodium acetate

In truth we are never quite that accurate with our weighing. Close enough was good enough.

Then we mixed 295 ml of acetic acid with 705 ml of sodium acetate solution to which we added 5 g eosin Y

The final pH should be 5. In fact good differential staining within the cytoplasm is best between a pH of 5 and 5.3. The solution is stable and we stained for 20 seconds in our linear stainer.

This first section is a Haematoxylin and Eosin washed in acid water after eosin staining which is too red.


The next section is a Haematoxylin and Eosin washed in alkaline water after eosin staining which has no red since alkalinity hinders eosin staining.


The third section is a Haematoxylin and Eosin washed in Adelaide tap water after staining in buffered eosin and it is just right.

Another related problem that has been reported, although it isn't one I have experienced is eosin bleeding out of the section into mounting media once the section has been dehydrated, cleared and coverslipped.


This is more common where there is high humidity and is due to atmospheric moisture being absorbed by alcohols and particularly xylene substitutes, which do absorb atmospheric moisture. If there is moisture still present in the section after coverslipping and moisture in your alcohols and clearing agents, albeit in small amounts, eosin will bleed from the section.


And yet another related problem in relation to stains bleeding out after coverslipping is that Van Gieson stain also has this tendency to bleed out of the mounted section if water is present in your final xylene and small amounts of residual water are present in the coverslipped section. Van Gieson is also removed from a section by alcohol - so if you use Van Gieson as a counterstain blot the section dry then let it air dry after staining and before mounting under a coverslip.




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© Roy C. Ellis 2002