CARNOY FIXATION - PRACTICAL AND THEORETICAL CONSIDERATIONS

The Carnoy-fixation, paraffin-embedding procedure was modified to minimize shrinkage and to suit the schedule of general histology laboratories. Blocks of tissues were fixed in Carnoy's fluid overnight, transferred to absolute alcohol in the morning and washed in three changes of alcohol for a total of 6-8 hours. Tissues were then left in methyl benzoate overnight, transferred to an Autotechnicon in the morning, cleared in xylene and xylene-paraffin 1 hour each and infiltrated in 2-3 changes of paraffin for a total of 4-5 hours. This procedure has worked well in our hands for nine years. A review of the chemical literature showed that the components of Carnoy's fluid-ethanol, chloroform and acetic acid-can interact by hydrogen bond formation with each other and with various groups in tissues. These association compounds apparently stabilize tissue structures and prevent or minimize shrinkage. Ethanol alone causes collapse of protein structures; exposure must be limited to eight hours or less. Addition of water to an ethanol-acetic acid solution causes considerable swelling of proteins and subsequent shrinkage in absolute alcohol; hence, the ratio fixative: tissue should be not less than 20:1. In our experience, Carnoy's fluid is the fixative of choice for studies of fibrous proteins and associated carbohydrates by histochemical and special staining technics. © 1968 Springer-Verlag.