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SUMMARY: A new marine species of Labyrinthula is described for which the name L. minuta is proposed because of its diminutive proportions. This species has been isolated repeatedly from Ulva lactuca and other algae collected from the Atlantic Ocean in the region of Woods Hole, Massachusetts. The vegetative stage, as in other species of Labyrinthula, consists of independent cells interconnected by threads of slime which form a net-like structure called the net-plasmodium. L. minuta differs from all previously described species of this genus in the size, shape, and movement of its cells, in its method of cell division, its colonial morphology and by the formation in aged cultures of multinucleate bodies which resemble miniature plasmodia. No fructification has yet been observed. This species has been grown in pure reproducible laboratory culture on a serum + sea water medium for a period of five years.

Further investigation of the specificity of the steroid growth factor requirement of Labyrinthula vitellina var. pacifica has provided a detailed description of the structural requirements for activity. Active compounds include sterols and products of their autoxidation; the activity of the sterols is not, however, due to autoxidation during the assay period. The data obtained circumscribe the characteristics of the essential metabolite represented by this requirement but do not identify it.

The labyrinthula isolates of Watson will grow over a wide range of pH values in a medium of which the inorganic constituents reflect their marine origin. All are obligately marine, though the less exacting requirements of L. macro-cystis var. atlantica suggest an estuarine habitat. Although only L. minttta var. atlantica requires an amino acid (l-leucine) as a growth factor, no isolate grew well on substrates other than amino acids. All require thiamine. Organic requirements may be obtained from host plants.

SUMMARY: Labyrinthula vitellina var. pacifica requires a steroid as growth factor. Cholesterol, δ4-cholestenone, fucosterol, and β-sitosterol are active. When cholesterol is modified by (1) the C3‡ hydroxyl group becoming trans to the C10 methyl group, (2) esterification of the C3 hydroxyl group, or (3) saturation of the C5, 6 double bond, the resulting compound is inactive. Modification of the side chain of cholesterol or cholestenone by the addition of a C24 ethyl or vinyl group does not destroy activity but unsaturation at C22, 23 does, as do the more marked changes found in diosgenin, deoxycorticosterone acetate, and methyltestosterone. Bile salts are inactive. The specificity of the requirement indicates that active steroids function as essential metabolites rather than solely as protective agents. Active steroids may be obtained by this micro-organism in nature from host plants or from diatoms and bacteria associated with the hosts.