ABSTRACT
Coelomic fluid of Echinus esculentus was prevented from clotting by drawing it from the animal into a chilled syringe containing EGTA. The mixture was layered onto a chilled 6-step gradient of Ficoll + Triosil (ranging from 2.3 to 45% Triosil + Ficoll) dissolved in filtered seawater in a plastic centrifuge tube and centrifuged for 6 minutes at 2000 r.p.m. The coelomocytes separated into 4 distinct bands: a lowest layer of red spherule cells, then a band of colourless spherules cells, followed by a band containing a mixture of phagocytic leukocytes plus some red and colourless spherule cells, with a band of vibratile cells at the top of the gradient.
By puncturing the tube, the gradient was drawn off dropwise and the cells counted in a Coulter Counter to determine the concentration of each cell type (“coelomocyte profile”). Different spécimens of Echinus esculentus showed considerable variation in the coelomocyte profile. In fecting the animals with 109 marine Pseudomonad strain 111 two hours before removal of coelomic fluid caused a général decline in all 4 coelomocyte bands. An infecting dose of 105 bacteria caused no change in 3 of the bands but affected the phagocytic leukocytes. Studies with the separated bands of cells from normal urchins indicated that clotting was primarily a function of the phagocytic leukocytes. The powerful haemagglutinin for rabbit erythrocytes is localized principally in the colourless spherule cells, while bactericidal activity against marine Pseudomonad strain 111 appears to be due to the phagocytic leukocytes and red spherule cells.
