Emended descriptions of these species and subspecies are given.In 1972, Terasaki (14) published a morphological, physiological, and biochemicd characterization of 49 strains of spirilla, including a number of new strains isolated from putrid infusions of shellfish (13) and a strain isolated from mosses collected in the Antarctic (see Table 1). In 1973, eight of the new strains were placed by T e r d (15) in five new species of Spirillum (S. psychrophilum, S. hiroshimense, S. pelagicum, S. pusillum, and s. multiglobuliferun), and three of the strains were placed in two new subspecies (S. itersonii subsp. nipponicum and S. peregrinum subsp. integrum) of previously established species. In the same year, Hylemon et al. (6) proposed that the genus Spirillum Ehrenberg 1832 be divided into three genera: SpirilZum, Aquaspirillum, and Oceanospirillum. Subsequently, additional characters were determined for the 11 new isolates mentioned above, and the results of both the original and the supplementary tests indicated that the freshwater strains possessed the salient characteristics of the genus Aquaspirillum (6, 9, 10) and that the marine strains possessed those of the genus Oceanospirillum (6,9,10). In the present paper, the new nomenclatural combinations necessitated by the transfer of these organisms and emended descriptions of the species and subspecies are provided. MATERIALS AND METHODSBacterial strains. The strain designations of the 11 isolates used in this investigation, their identification when they were originally described (15), their sources, and their dates of isolation are listed in Table 1. Maintenance of cultures.The Spirillum strains were maintained as agar stab cultures at room temperature (15 to 33OC) with monthly transfers, except for strain CA1, which was maintained at 4°C with trimonthly transfers. Freshwater spirilla were maintained on a nutrient agar of the following composition (in grams per liter of distilled water): Arei-peptone (Kyoei Seiyaku & Co., Ltd., Tokyo), 5.0; Arei-meat extract, 3.0; and agar, 15.0. For marine strains, a seawater agar was used, with seawater (drawn from Kairo-en, Itsukaichi-cho near Hiroshima) being substituted for the distilled water in the above nutrient agar. In those cases where the ceUs of certain strains were apt to "straighten out" during maintenance, smooth colonies were occasionally selected so as to maintain stocks of typical morphology. The method used for freeze-drying the spirilla has been reported recently by Terasaki (16).Inocula. With the exception of the tests for determination of substrates that can serve as sole carbon 130

Pages 132-133, Table 3: "f" on the left of the figures 1.0-1.2 of Spiral depth for S. pusillum IF6 Pages 132-133, Table 3: Nitrate reduced to nitrite (7 days) for S. pusillurn IF6 should read "+." Pages 132-133, Table 3: Nitrate reduced beyond nitrite stage (7 days) for S. pusillum IF6 should read "-." Pages 132-133, Table 3: Acid from carbohydrates (14 days) for S. multiglobuliferum OF1 should read "-." Page 133: "Temperature of incubation. With the exception of CA1, which was incubated at 2O"C, all cultures were incubated at 30°C unless otherwise noted." should be inserted between lines 9 and 10. pelagicum UF1, S. pusillum IF6, and S. multiglobuliferum OF1 were measured only at 30°C.

On Two New Species of Spirillum by Yasuke TERASAKI*

Pure culture isolation of aerobic chemoheterotrophic spirilla from mud and sand samples collected from various freshwater and seawater areas in Japan was accomplished by applying the following three methods in the sequence shown: (i) boiled shellfish infusion method for enrichment of spirilla; (ii) glass capillary method for selection of spirilla; and (iii) ordinary streak plate method for pure culture isolation of spirilla. The aerobic spirilla grew well in all enrichment cultures in the 20 experiments performed. Crude cultures containing abundant spirilla were obtained from all enrichment cultures by use of glass capillaries. Pure culture isolation of spirilla from the crude cultures was reliably achieved by the ordinary streak plate method.The putrid infusion of crushed raw shellfish was appropriate for the enrichment of freshwater as well as marine spirilla, and many strains were isolated by direct inoculation onto plates of nutrient agar or nutrient agar using seawater (1,2). Effective enrichment cultures of water spirilla were also obtained by adding the infusion of shellfish prepared by boiling to mud samples collected from freshwater or seawater areas (2), and glass capillaries were also used for the selection of spirilla in that study at the suggestion of Watanabe. Subsequently, the present author found that aerobic chemoheterotrophic spirilla could easily be isolated from mud or sand samples by combining the following three methods in the sequence presented: (i) boiled shellfish infusion method for enrichment of spirilla; (ii) glass capillary method for selection of spirilla; and (iii) ordinary streak plate method for pure culture isolation of spirilla. Then, the present study was carried out to ascertain whether effective enrichment cultures are acquired by chance or the methods are reliable for the enrichment and isolation of aerobic chemoheterotrophic spirilla. This paper deals with the details of the methods and presents the results obtained by applying the methods to mud and sand samples collected from various locations in Japan. 395

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