Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011






Did you know that during fixation with compound fixatives, like Helly or Zenker for instance, the most rapid penetrator will determine the fixation image because penetration is determined by the law of diffusion?



The 'Law of Diffusion', is D equals K times the square root of T


where d is the distance penetrated in time t and K is a constant depending upon the fixative used.


The K value does also vary depending upon the density of the tissue to be fixed. The denser the tissue the slower the rate of penetration. So the value of the constant K is affected by the type of fixation agent and the density of the tissue undergoing fixation.


When choosing a fixative another factor to consider is how rapidly the rate of penetration falls off with time. For example – with a K value of 0.84 mercuric chloride penetrates into liver a distance of 20 microns in just over 2 seconds. That is to say a penetration rate of 36 mm in 1 hour: but actually it will only penetrate 0.84mm in 1 hour because of the density of the tissue. But into albumin-gelatin gel mercuric chloride will penetrate 2.27mm in 1 hour. The gel being far less dense than liver.


It is similar story with other fixatives. This emphasises the importance of using small pieces of tissue for processing. Small is good - Big is bad!


But the bottom line is that potassium dichromate penetrates more quickly than other fixation agents, followed by formaldehyde (K=3.6), acetic acid (K=2.75), ethanol and mercuric chloride (K=2.2) which penetrate at a similar rate. Then Picric acid and glutaraldehyde which are relatively slow (half the speed of alcohol); whilst Chromium trioxide (K=1) and osmium tetroxide (K=1) are the slowest.


So a solution containing formalin and potassium dichromate would see the potassium dichromate penetrate almost twice as quickly as formalin. Meaning that the primary fixation is done by Potassium Dichromate with formalin acting as a secondary fixative.


So to fully understand what is happening within the tissue, especially if you are involved in research, it is often best to use a simple fixative solution like 10% formalin rather than a complex fixative made up of several chemicals which penetrate tissue at different rates with each chemical having a different effect upon tissue proteins.


It is interesting to note that glutaraldehyde at different concentrations acts differently. The weaker solution will penetrate far quicker than the stronger solution. I haven’t been able to find an explanation why that should be nor have I been able to find similar data for other fixatives but - I suspect it is to do with molarity.




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© Roy C. Ellis 2002