Procedures for the isolation and characterization of Metha7zonzo?zas n~ethatto-oxidans Brown and Strawinslii are described. Isolates from varied sources are alike in cellular morphology, inasmuch a s they form only microcolonies, and in their dependence on methane or methanol as carbon and energy sources for growth. Both organic and inorganic nitrogen sources are used. The organism is a Gram negative non-sporeformillg rod, 1.5 to 3.0 p by 1.0 p in size, and motile by means of a single polar flagellum. In growing cultures the osygen/methane ratio was approsinlately 1.1 and in resting cells 1.7. The R.Q. for methane with resting cells was 0.43. Resting cells were unable t o oxidize organic compounds other than methane, methanol, formaldehyde, and formate. Formic acid was detected in test solutions after cell suspensions had metabolized methane, methanol, and formaldehyde. Using sodium sulphite as trapping agent for formaldehyde, it was found that 60 t o 70y0 of the methane or methanol consumed was converted to formaldehyde. I n the presence of iodoacetate, 70% of the methane consumed was present terminally as methanol. Thus i t was shown that methanol, formaldehyde, and formic acid are sequential intermediates in the osidation of methane by these organisms.In 1905 Kaserer concluded that inethane was oxidized by bacteria but he did not attempt t o isolate the responsible organisms. Shortly thereafter Sohngen (16) described a methane-oxidizing bacterium which he named Bacillus methanicz~s. Since that time, bacteria capable of utilizing inethane have been reported by inany ~vorlters including Munz (13), Giglioli and Masoni (9), Aiyer (I), A4ogilevsltii (12), Boltova, Kusnetsova, and I

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Pure cultures of methane-oxidizing bacteria isolated from soil, from the rumen of a fistulated cow, and from coalmine water were found to be identical in morphological, cultural, and physiological characteristics with Methanoojonas otiethanooxidoans of Brown and Strawinski. Two of the isolates were serologically related to the organism of Brown and Strawinski. All the strains required methane for good growth, but a delayed moderate growth occurred on methanol. No other substanices were utilized as carbon and energy source. Nitrogen re(luirements were satisfied by nitrates, anuimonium salts, peptone, or certain amnino acids. The taxonomic position of the species is discussed. Since the first description of methane-oxidizing bacteria by S6hngen (1906), numerous investigators have relported studies dealing with microbial oxidation of methane and other gaseous hYdrocarbons. Among the more recent rel)orts are those of Hutton and ZoBell (1949), Dworkin and Foster (1956), Strawinski and Brown (1957), B3rown and Strawinski (1957, 1958), and Leadbetter and Foster (1958). Recent reviews of hydrocarbon oxidation by microorganisms have been provided 1y Fuhs (1961) and Foster (1962). In most reports dealing with methane-oxidizing bacteria, the descriptions of the organisms involved have been inadequate to afford detailed coml)arison with those described by other investi'rators, and the cultures are not available for coml)arative studies. This rep)ort describes the methods employed in the isolation of three additional strains of methane-dependent organisms, an(d compares them with the organism named Alethanomonas nmethanooxidans bv Brown and Strawinski (1958).

Baton lRouge), AND C. S. MCCLESKEY. Identity of the pink-pigmented methanol-oxidizing bacteria as Vib:-io extorquens. J. Bacteriol. 88:1065-1070. 1964.-Pink-pigmented bacteria isolated from enrichnment cultures of methane oxidizers were found to possess similar morphological, cultural, and physiological characteristics. All the strains utilized methanol, formate, oxalate, succinate, glycerol, and benzene as sole carbon sources; methanol, formate, and glycerol afforded best growth. Most strains utilized fructose and ribose; other carbohydrates tested were not available as carbon and energy sources. There was strain variation in the use of hexane, heptane, n-propanol, n-butanol, acetate, and propionate. Methane, ethane, n-propane, and n-butane were not utilized. Our isolates, and Pseudonmonas tnethanica of Harrington and Kallio (not the methane-de

Corynebacterium nephridii was found to reduce nitrate (contrary to the original description) at a rapid rate. In the conventional 0.1% nitrate broth, neither nitrite nor nitrate was detected after 24 hr. There was no assimilation of nitrate nitrogen, and the final product of nitrate reduction was nitrous oxide. Manometric studies and growth experiments indicated that the organism is incapable of reducing nitrous oxide. C. nephridii is gram-negative, grows on bile salts (5%) agar, EMB Agar, and MacConkey Agar. It was proposed that this species be transferrrd to the genus Achromobacter and designated Achromobacter nephridii (Bulsing, D611, and Freytag) comb. nov.