Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011






Cutting thick and thin within a section due to a minute high pitch vibration in a knife edge (chatter)



On the top is a section of liver stained with Gordon and Sweets method to demonstrate reticulin and on the bottom is a section of endometrium stained Haematoxylin and Eosin, with both sections showing chatter caused by different problems. The chatter in the liver was caused by a loose knife whilst the chatter in the endometrium was caused by hard tissue.


  • Loose knife or block we have already discussed. The solution is to keep it rigid. And the results of a loose knife we saw vividly in the previous slide.

  • Knife clearance angle too great we have also already discussed this at some length. As a matter of interest the clearance angle should be between 3 and 5 degrees. Bancroft and Stevens recommend 2 and 4 degrees but a 2 degree clearance angle, I think is too shallow sometimes leading to the problems we are trying to avoid. For resin sections the angle should be between 5 and 7°. But what is best for your situation with your microtome and your knife is largely a matter of trial and error between those parameters.

  • Excessively hard tissue or wax. If it is the tissue that is excessively hard there are 2 reasons. One is that it really is very hard tissue and the block needs to be soaked in a softening reagent for 20 to 30 minutes before a section can be cut easily. The second cause is over-dehydration. If this is the case then applying moistened cotton wool to the block face for a few seconds is usually sufficient to enable a section to be cut and warm water is often absorbed more quickly than cold water. If it is the wax that is excessively hard, perhaps the block has been on the cold plate too long, then simply allow it to warm slightly. Placing the palm of your hand over the block for a few seconds is usually sufficient to allow a good section to be cut.

  • If areas of calcification are present within the tissue then the block must be surface decalcified for 15 to 30 minutes. Then have another go but remember that after surface decalcification the block needs to be well washed in running water for several minutes otherwise the remains of the decalcifying agent on the block will damage the knife edge. Decalcifying solutions blunt knife edges very quickly.



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© Roy C. Ellis 2002