CONDITIONS FOR ATTACHMENT AND DEVELOPMENT OF SINGLE CELLS RELEASED FROM MECHANICALLY-DISRUPTED THALLI OF PRASIOLA STIPITATA SUHR

¹ Experimental Marine Botany Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
² Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02154

Methods are described for the nearly quantitative release of the single cells composing the thallus of Prasiola stipitata Suhr. by mechanical grinding in a hand homogenizer. The released cells have walls and attach to substrates quantitatively by means of a mucoid material containing acidic, possibly sulfated, polysaccharides. A simple assay for measuring attachment of these cells is described. A comparison of the size distributions of the cells immediately after grinding and after attachment indicates that both the large numbers of vegetative cells and the smaller numbers of aplanospores are attaching and developing. Normal development of the attached cells under laboratory conditions has been observed through the stage of two-dimensional division. Cell attachment is optimal up to about 5 x 10⁵ cells per ml; higher densities are inhibitory. Low temperatures interfere with attachment in darkness but not in light; inhibition of attachment with 3, (3,4 dichlorophenyl) 1,1 dimethyl urea (DCMU) indicates that photosynthesis is necessary for attachment at low temperatures. At higher temperatures energy can probably be obtained from respiration as well. Inhibitors of protein synthesis in the cytoplasm (cycloheximide) and plastid (chloramphenicol) inhibit attachment, the inhibition by cycloheximide approaching 100%. Molybdate is a good inhibitor of attachment and is reversed by sulfate as might be expected from its action as an inhibitor of sulfate activation in the formation of sulfated polysaccharides. Cells already attached can be released from attachment by treatment with molybdate, indicating that the materials involved in attachment are subject to metabolic turnover. Algal material obtained from various places at different times show some variability in the rate of attachment but material from all collections ultimately showed complete attachment of cells.