alpha-Difluoromethylornithine (RMI 71,782), a specific irreversible inhibitor of the first step in polyamine biosynthesis, that is, the formation of putrescine from ornithine by ornithine decarboxylase, cures mice infected with a virulent, rodent-passaged strain of Trypanosoma brucei brucei. This parasite is closely related to the trypanosomes that cause human sleeping sickness. The drug, which is remarkably nontoxic, was effective when administered in drinking water or by intubation. The ability of the compound to inhibit ornithine decarboxylase in vitro was demonstrated by the reduced amounts of putrescine synthesized from tritiated ornithine in Trypanosoma brucei suspensions. These observations direct attention to polyamine metabolism as a target for chemotherapy of parasitic diseases.

SUMMARY. An improved assay of vitamin B12 is described. The z strain of Euglena gracilis, which grows more vigorously than the bacillaris strain previously used, is recommended. The pattern of B12 specificity of the two strains appears to be the same. A new medium containing sucrose, aspartic acid, glutamic acid, and glycine at pH 3.6 is well buffered and allows luxuriant growth. Vigorous utilization of sugar appears to depend on readily available nitrogen and a Krebs‐cycle component; these requirements are well met by aspartic acid (or asparagine) and glutamic acid. The proposed procedure is especially suitable for the measurement of B12 in blood serum because rise in pH and precipitation of serum proteins during incubation are minimized. Like bacillaris, the z strain allows the distinction between “combined” and “uncombined” B12 in serum. Serum may be an appropriate test material to tell whether such phagotrophs as Peranema can better utilize bound forms of vitamins than can the related osmotrophs. Patterns of B12 requirements and occurrence are discussed as phylogenetic markers.

Serial passage of six strains of Legionella pneumophila and one strain of Pseudomonas aeruginosa in a liquid chemically defined medium deficient in trace metals resulted in the death of five L. pneumophila strains and very limited growth in the remaining strain and the P. aeruginosa strain. Addition of either iron or magnesium restored growth to almost normal levels in ail of the strains when early-passage inocula were used. A low concentration of magnesium stimulated growth with cobalt, copper, iron, manganese, molybdenum, vanadium, or zinc. When a complete defined medium containing trace metals was used, growth was inhibited by adding the chelators ethylenediaminetetraacetic acid, citrate, or 2,2'-bipyridyl. Chelator inhibition was partly or fully relieved with either calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, nickel, vanadium, or zinc. P. aeruginosa differed from L. pneumophila in that it required higher concentrations of each chelator to inhibit growth and that its growth was stimulated by only four metals: calcium, iron, magnesium, and zinc. A trace-metal supplement for L. pneumophila was designed which included all metals stimulating growth in these experiments and which proved to be sufficient for optimal growth of all the strains.

The chrysomonads are a vast array of brown-pigmented fresh-water, soil, and marine flagellates. Colorless phagotrophic counterparts of several of the pigmented species are common in nature, e.g., Molzus and Oikomonus. The minimal nutritional requirements of 4 fresh-water strains belonging to 2 species are described here. No chrysomonad previously has been grown in chemically defined media. These strains ingest particulate food and are therefore animals (protozoa) and, being to some extent phototrophic, they are plants (algae) as well. They can live indefinitely in darkness or in light on high-molecular dissolved food, as well as in low-molecular chemically defined media. This extraordinary versatility, first thoroughly documented in the pioneering pure culture study of Pring~heirn,~ is accompanied by a remarkable tolerance of variations in media and exceedingly dense growth in appropriate media, including certain simple "synthetic" media [one cannot unambiguously call "synthetic" a medium containing cobalamin (vitamin Biz), whose complete structure is still to be ascertained]. This ease of cultivation-in which respect these chrysomonads contrast sharply with some of the planktonic chrysomonadslO-suggests that the biochemical basis of an intermeshed phagotrophy and phototrophy can profitably be investigated in them.PringsheimQ reviewed the scanty previous work. He demonstrated an active phagotrophy in the same strains on which the present paper is based: They ingested starch grains, oil droplets, casein particles, bacteria, and small algae -including each other. Glucose was utilized in the presence of complex natural materials such as peptone. Liver extracts were strikingly stimulatory. Dark growth was improved by aeration and paralleled that in light. Darkgrown individuals were pale. Cultures accumulated a water-soluble sepia or almost black substance which was also produced by pale growths in the dark.With chemically defined media-the subject of this paper-it should be possible to explore systematically their ability to utilize high-molecular sources of energy and carbon, nitrogen and amino acids, biotin, thiamine, and cobalamin.To identify the growth requirements of aerobic phototrophs, culture methods are needed which combine adequate illumination with freedom from significant chemical contaminations. Such considerations apply with special force to these chrysomonads. Growing densely, they need much illumination. Hence they were cultured in shallow layers of medium, and their sensitive response to certain widely-distributed growth factors such as biotin and cobalamin spurred the development of methods to minimize the confusion introduced by * This investigation was aided by grants from the Lederle Laboratories Division of the American Cyanamid Company, the Atomic Energy Commission [contract AT(30-1)-863] and the Rockefeller Foundition. We are indebted to Doctors E. G . Pringsheim and R. A. Lewin for the geuerdus gift of cultures and unpublished information. Doctor F. C. Bersworth of the Bersworth Chemical Comp...