Most of these investigations concerned organisms belonging to the genus Streptomyces. Such problems as the properties of the cell wall, septation of hyphae, the nature of the cytoplasm and its contents, and the manner of cell division and spore formation have been considered. The extreme narrowness of filaments (about 1 micron) renders internal structures difficult to study; thus decisions regarding their nature have in some cases been controversial and inconclusive. The present studies were undertaken to add to the information of the cytology of actinomycetes by means of both light and electron microscopy and by the use of microchemical tests. The organisms investigated belong to the genus Nocardia (Proactinomyces), and for the most part one strain, Proactinomyces ruber (Casab6) Baldacci,8 was used. This strain was selected for detailed study because it grows well and is intermediate in morphology between forms that are bacteriumlike and those that are Streptomyces-like. METHODS AND RESULTS Morphology of old cultures. Stained preparations of 6-month-old cultures of ' This organism would now be placed in the genus Nocardia; however, the old name Is retained here, as matters of redesignation are beyond the scope of the present work. 589

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This study waz supported in part by a grant from the General Research Fund of the University of Kansas. 168 12 hrs. Short rods elongating and bending. 24 hrs. Young colonies composed of long fragmented hyphae. Few branches produced.Fragmentation of types 1 and 2 begins in center of small colony. 36 hrs. Large colonies consisting of long hyphae with few branches. 72 hrs. Large dense colonies which fragments in center, peripheral hyphae continue to grow, branch and fragment STOCK No. 15. Nocardia sp. 12 hrs. Refractive rods germinating by elongation. 24 hrs. Young colonies formcd by fragmentation into long hyphae. Few branches and fragmentation is of types 1 and 2. 36 hrs. Colonies formed by long unbranched hyphae continue to elongate a t the edges.Fragmentation produces shorter cells. 70 hrs. Circular colonies, dense in center, radiating, nonbranched hyphae around periphery. STOCKNo. 16. Nocardia sp. FIGURES 17, 18, 19 and 20. 12 hrs. Bent rods, some forming short branches. 24 hrs. Small colonies consisting of fragmented hyphae. 36 hrs. Circular colonies with short fragments in the center. around periphery. 70 hrs. Large circular dense colonies, short bent peripheral hyphae. STOCK No. 57. 12 hrs. Rods elongating and bending, few short branches produced. 24 hrs. Small colonies consisting of sharply bent hyphae in center, with longer curved hyphae around edges. Branches few. Fragmentation of type 1 Nocardia sp. FIGURES 13, 14, 15 and 16. Fragmentation frequently of type 1. FIGURES 21, 22, 23, and 24. Few short branches produced. Hyphae mostly unbranched Jensenia canicruria Risset and Moore. FIGURES 25, 26,27 and 28. 72 hrs. Small compact colonies with fragmented center and short peripheral hyphae. STOCK No. 76. 12 hrs Rods elongating, bending, and producing short branches. 24 hrs. Small colonies with secondary branching, type 2 fragmentation beginning in center. 36 hrs. Small colonies with hyphae at periphery with few branches, fragmentation in center. 72 hrs. Small compact colonies with short fragments in the center, short peripheral hyphae produced. STOCK No. 78. N . corallirta (Bergey et al.) Bergey et al. FIGURES 61, 62, 63, and 64. 12 hrs. Short elongating rods, some bending. 24 hrs. Branched hyphae forming small colonies. Fragmentation of type 2 beginning in center. 36 hrs. Colonies consist of fragmented central hyphae, peripheral hyphae sparsely branched. 72 hrs. Central hyphae fragmented to short rods, arranged a t angles. Peripheral hyphae characteristically branched. STOCK No. 79. N . convoluta (Gray and Thornton). FIGURES 141, 142, 143, and 144 12 hrs. Curved elongating rods, occasional short branches produced. 24 hrs. Rods elongating and branching. Branches are produced so as to suggest dichotomous branching. 36 hrs. Circular colonies with curved branching hyphae a t periphery. 70 hrs. Large colonies with dense center, long branched hyphae at periphery. STOCK NO. 87. 12 hrs. Short rods not germinating. 24 hrs. Rods elongating with short branches. 36 hrs. Rods forming small branched colonies which are fra...

DNA hybridizations, base composition determinations, and tests for physiological reactions were performed on 10 isolates of Nocardia asteroides differing in microscopic colonial morphology, carbon compound utilization, and virulence for mice. One strain was eliminated from the species by all three methods and identified as N. otitidis-cavarium (N. caviae). Relatedness among the remaining 9 isolates, expressed as relative percentage binding at two temperatures, fell into two groups. The range of base composition of the DNA from these isolates was 67.7 to 69.2% GC. Formal subdivision of the taxon based on variations in physiological reactions and DNA homologies was judged to be premature.Recent developments in microbiology have made the identification of Nocardia asteroides more certain. Physiological reactions (1-3) and chemical analyses of cell components (4-6) can be used to separate this important veterinary and human pathogen from other actinomycete genera and from other nocardiae.N. asteroides was found to be heterogeneous based on morphological and biochemical criteria (1, 7-9), physiological characters (10; Franklin and McClung, 1972, unpublished data), serological properties (11,12; Schneider, Franklin, and McClung, 1971, unpublished data), and virulence for mice (13). The numerical taxonomic studies of TsUKAMURA (14) and GOODFELLOW (15) also indicate species heterogeneity.This study was undertaken to determine if physiological reactions, DNA base composition determinations, and DNA hybridization studies would aid in deciding whether the taxon N. asteroides can accommodate a number of isolates, or whether members of this species are truly heterogeneous and might better be split into different species.

Cellular fatty acids of the six Nocardia asteroides strains grown on glucose, glucose and amino acids, glycerol, and Dubos oleic albumin complex were determined by gas–liquid chromatography. Cells grown on each medium contained saturated, unsaturated, and branched-chain fatty acids. The fatty acids consisted of normal saturated C13, C14, C15, C16, C17, and C18; monoenoic C16 and C18; branched-chain C14, C15, and 10-methyl C18. Composition of the media affected cellular fatty acid content of N. asteroides strains qualitatively and quantitatively. Five of the six strains closely resembled each other, but one strain appeared to be different. The fatty acid pattern of Nocardia may be a useful criterion in differentiation of this genus from the closely related Mycobacterium and Streptomyces, which have a different fatty acid composition.