Smears of Orconectes virilis hemolymph were prepared by fixation in 10% formalin immediately upon withdrawal, and after an interval of 1, 5, or 15 minutes. These smears were then stained by a variety of histochemical means designed to identify a material which is released from both hyaline and granular cells during the coagulation process. Based upon reactions to various carbohydrate, lipid and protein tests, this material appears to consist of a glycoor mucwprotein, and its localization and activity coincide with the "fibrin ferment" described by Hardy (1892). Its release is probably initiated from the hyaline cells, followed shortly thereafter by a similar activity of the granulocytes. While it cannot be ascertained by the methods used in this study whether or not this material participates actively in clotting, its release is nevertheless coincidental with coagulation.
As long ago as 1892, Hardy suggested that the "explosive corpuscles'' of Astacus contained the "fibrin ferment" (Halliburton, 1885) which initiated, or at least participated in the clotting process of this crustacean, He observed the release of this ferment in cytoplasmic blebs from h e pseudopodia into the plasma. Most students of arthropod hemolymph coagulation have attributed at least some function in the process to the hemocytes (Howell, 1885; Loeb, '04, '10; Tait and Gunn, '18; Gregoire, '51; Gregoire and Florkin, '50; Levin and Bang, '64).
Several reports have described histological and histochemical staining charac teristics of arthropod hernolymph cells (Tait and Gunn, '18; George and Nichols, '48; Dumont, Anderson and Winner, '66; Wood and Visentin, '67). None, however, has attempted a specific identification of Hardy's ferment and recorded its behavior during clot formation. The present study is an attempt to relate intra-cellular substances with hemolymph coagulation in the crayfish Orconectes virilis.
Methods
Adult Orconectes virilis obtained from E. G. Steinhilber & Co. of Oshkosh, Wisconsin were maintained in twenty-gallon aquaria containing aerated tap water to a