Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011





To obtain a section from tissues which are hard to cut after conventional fixation and processing


This is more of a problem for those who work in a vet lab cutting animal tissues or help researchers who cut animal tissues - nevertheless you never know when the knowledge might come in handy.

Tissues such as vertebrate eye and yolky eggs. They tend to harden excessively and can be very difficult to cut if normal fixation and processing schedules are used. Sectioning in particular can be very difficult. Now we are not talking here about brittle tissue just tissue that can be excessively hard.

One solution which works very well is to fix tissues in Perenyi’s fluid for 10 to 12 hours. If you can spare the time. But tissues like vertebrate eye and yolky sac are much more likely to be research on animal tissues than routine human material so time is not quite so critical.

Then place in 70% ethyl alcohol for 2 to 3 hours after fixation. Then process using normal processing schedules.

Perenyi’s fluid
150 ml of 1% Chromic acid
400 ml of 10% aqueous Nitric acid
300 ml of 95% ethyl alcohol
150 ml of Distilled water
Mix in the order given immediately before use.

When disposing of Perenyi's fluid: Never dispose of Perenyi’s fluid into a waste system containing mercuric chloride. Because ethanol, mercuric chloride and chromic acid can cause spontaneous combustion.

Perenyi’s fluid is actually a slow decalcifying agent, good for small calcium deposits. Both nitric and chromic acids have a softening effect on tissues which are naturally hard or tend to harden excessively during fixation and processing and ethanol is a coagulant fixative. When all of the ingredients are combined and the fluid used for fixation it does allow for a reasonable section to be obtained from these difficult tissues.

The fixative is particularly good for eyes as it fixes the retina really well with very little separation of the layers and does not harden the lens.

Another good fixative which can be used is Kleinberg’s solution, which is especially good in marine histology for creatures such as crabs, lobsters, crustaceans and the like which have a crusty shell. This is another of those solutions which can be used as a mild decalcifying or softening agent.

Kleinberg’s solution
1000 ml of Distilled water
20 ml of Conc sulphuric acid
approximately 15 g of Picric acid
Add the acid to water slowly then slowly add the picric acid.
Tissues go into 70% ethanol for at least 2 hours after fixation and before processing.
Then process normally.

Staining is often quite good because of the picric acid component.




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© Roy C. Ellis 2002