Ilda McVeigh

Nutritional studies of Histoplasma capsulatum.

A chemically defined medium, composed of inorganic salts, glucose, asparagine, cystine, and a vitamin supplement, has been devised for growth of the yeast phase ofHistoplasma capsulatum. Growth in this medium was abundant and compared favorably with that in media containing complex natural material. Conversion of each of the 20 strains examined was accomplished by one or more passages on agar slants of the medium and incubation of the cultures at 37° C. Yeast phase cultures on this medium have been stored for 6 months or more at approximately 4° C without conversion or loss of viability. Of the 20 strains examined for vitamin requirements of the yeast phase, all were partially deficient for thiamine; nine for inositol; five, either partially or completely deficient for niacin; and one, completely deficient for biotin. No specific amino acid was required for growth of the yeast phase, but an organic source of sulfur and one of nitrogen were essential. Cystine and cysteine were equally effective for growth of the yeast phase when supplied on an equivalent sulfur basis and very little difference in growth occurred in media which contained equal amounts of nitrogen in any one of the following compounds: asparagine, glutamic acid, aspartic acid, and proline, Of the 20 strains, all but one, which requires biotin, were capable of continued growth in the mycelial phase when subcultured on an agar medium containing only inorganic salts and dextrose, but growth was improved significantly by asparagine or casein hydrolysate.

Growth requirements of Haemophilus vaginalis.

A medium, composed of peptone, maltose, dextrose and phosphate buffer, which supports good growth ofHaemophilus vaginalis has been devised. Growth was about equally good with soluble starch substituted for maltose. Optimal growth occurred in the media adjusted to pH 6.8 before sterilization and the media sterilized at 112 C for 12 minutes supported better growth than did corresponding media sterilized at 121 C for 15 minutes. The organisms grew both aerobically and anaerobically. For growth in a semi-defined medium, containing enzymatically hydrolyzed vitamin-free casein, inorganic salts, carbohydrates and six purine and pyrimidine bases, the organisms were found to require the following B-vitamins: thiamine · HCl, riboflavin, niacin (or niacinamide), pteroylglutamic acid, and biotin. They failed to grow in the above medium lacking purine and pyrimidine bases, but the single omission of any one of the six bases (adenine sulfate, guanine·HCl, cytosine, uracil, thymine, and xanthine) except adenine sulfate did not affect growth of either strain tested. One strain failed to grow in the medium lacking adenine sulfate. Since the strains investigated, including the type, do not require X and/or V factors or otherwise definable coenzymelike substances, they do not qualify as members of the genusHaemophilus on the basis of its current circumscription. Thus, reconsideration must be given to the classification of the organisms now known asHaemophilus vaginalis.

Factors affecting mycelial to yeast phase conversion and growth of the yeast phase of Histoplasma capsulatum.

A procedure for rapid induction of mycelial to yeast phase (M→Y) conversion ofHistoplasma capsulatum has been devised. Exposure of mycelial fragments to low oxidation-reduction (O/R) potentials (+5 to+65 mv) either aerobically (ascorbic acid treatment) or anaerobically (nitrogen atmosphere) for 18 to 24 hours at 37° C resulted in induction of the M→Y conversion process whether or not an organic sulfur source was available. However, aerobic conditions and a suitable organic sulfur source, such as cysteine, cystine or lanthionine were found essential for outgrowth and maintenance in the yeastlike phase.

The growth of Trichophyton mentagrophytes and five of its variants as affected by several nitrogen sources.

The phenomenon of variation in the imperfect fungi has long been recognized. It is especially important because the identifica? tion of the various species depends largely on their appearance in culture, and failure to take cognizance of the variation displayed by most species in laboratory culture has led to a great deal of confusion in their taxonomy. Skinner, Emmons, and Tsuchiya (4) estimate that the number of true species can perhaps be reduced to one tenth of the reported number by a critical comparison of strains and a proper evaluation of variability. Like many other imperfect fungi, Trichophyton mentagrophytes gives rise to pleomorphic variants as the culture increases in age. These can be distinguished by rate of growth, morphology of colony, pigment production, microscopic appearance, and physiological characteristics. Nine such variants were isolated by Robbins and McVeigh (3) and have been maintained in culture apparently unchanged since their isolation. Little work has been done on the nutrition of variants of imper? fect fungi from a comparative standpoint. Robbins and Ma (1)

Development of a chemically defined medium for growth of Neisseria gonorrhoeae.

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO2 during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.

Inhibitory effects of organic sulfur compounds on Histoplasma capsulatum.

Eight classes of organic sulfur compounds, comprising 42 substances, were tested in vitro for activity against the yeast phase ofHistoplasma capsulatum Darling. Activity was found in the classes of thiols, thio acids, disulfides, and thiolsulfonates. The classes of sulfinates, sulfenamides, penicillamine analogs, and reaction products of thiols with aldehydes or ketones were unpromising. Tentative conclusions are drawn of structure-activity relations within classes as a guide to coupling of classes to form disulfides.

Effects of various compounds on growth of yeast cells of Histoplasma capsulatum.

A number of compounds were screened for their effects on growth of the yeast cells ofHistoplasma capsulatum. Included were penicillin and related compounds, sulfhydryl inhibitors, various organic sulfur compounds recently synthesized for the first time, and compounds structurally related to the required metabolites, thiamine and cystine or cysteine. Cephalothin was the only one of the penicillin related compounds which inhibited growth. This occurred only when a high concentration (8.3 × 10−4 M) was used. Of the analogues of cystine tested, allylglycine had the greatest inhibitory effect on growth of the yeast cells in the synthetic medium, but it failed to inhibit growth in a complex medium containing peptones and plasma. Among the sulfhydryl inhibitors, the maleimides were the most effective, producing complete inhibition of growth in the peptone medium at 10µg/ml or less. At subinhibitory concentrations the cultures tended to become mycelial. The action of the maleimides was reversed by cystine over a range of concentrations. At low concentrations, some of the disulfide derivatives of thiamine stimulated growth equally as well as thiamine, but at concentrations of 100 to 150µg/ml, they completely inhibited growth. On the basis of results obtained to date, three classes of the new organic sulfur compounds being tested offer promise as sources of potentially useful chemotherapeutic agents. These classes, which differ widely in structure, are as follows: the benzyl decylaminoethyl disulfides, the acyl disulfides, and the trithiopercarbamates.