During the study of the growth of Mycobacteriurn rhadochnous NCIB 9905 in a minimal medium with n-decane as the sole carbon source, a growth factor and a hydrocarbon emulsifying factor were discovered. The factors weie heat stable, were released from cells by autolysis or by physical disruption and functioned independently of one another. Inoculations of < 10' cells/ml of culture medium routinely failed to grow unless an autoclaved cell suspension or purified extract was added. This growth factor requirement was not satisfied by exogenous trace metals, vitamins, amino acids, intermediates of decane metabolism or Mycobactin, but was satisfied by an extract from cells of an unrelated species growing on n-decane. The extract of strain 9905 did not satisfy the sideramine requirement of Arthrobactev JG9 and was not associated with ferric ions. Some evidence was obtained suggesting that the growth factor may be of a new type involving the chelation of calcium ions. DURING THE ISOLATION of hydrocarbon-oxidizing organisms from various soil and water samples it was clear that the predominant bacteria belonged to morphologically related groups including Mycobacterium, Arthrobacter and Nocardia. Most of the strains exhibited considerable variation in daily subcultures and often lost the rapidity of growth observed when originally isolated. These variations included passage of a large proportion of the culture from the smooth tb the rough phase, as shown by the dry wrinkled appearance of isolated colonies on agar media and by the appearance of pellets of cells in liquid culture. The pellets reached 5-10 mm in size and posed a serious problem in the industrial exploitation of the bacteria concerned. With one strain, Mycobacterium rhodochrous NCIB 9905, the inability to initiate growth in a mineral salts medium unless large inocula were used was particularly striking, and the nature of the phenomenon was investigated. Materials and MethodsCultures The hydrocarbon-utilizing organisms used were isolated from spillage area soils in the Oil Works of I.C.I. Heavy Organic Chemicals Division, Billingham, Co. Durham, using enrichment cultivation methods and the medium and carbon source described below. Two specimen cultures were deposited in the National Collection of Industrial Bacteria and designated respectively as Pseudomoms aeruginosa (NCIB 9904) and Myco. rhodochrous (NCIB 9905).

S L~K R Y . The ultrastructure of free spore coats of Bacillus megaterium 9885 was studied by exploitation of the spontaneous lysis of newly germinated spores in the presence of phosphate and absence of Ca*+, and the subsequent release of coat subunits. The subunits appeared as the middle layer of a sandwich. The primary size of the subunits was c. 20nm. Negative staining suggested the presence of peripheral charged groups (carboxyl?) surrounding a hexagonal-shaped subunit containing an acentric electron opaque spot ; where subunits had associated into monolayers, the peripheral rims disappeared but the number and position of subunits mas located from the acentric spots which remained visible. The newly germinated cell remained attached to the spore coat via a short connecting structure of amorphous material. Caa+ protected against lysis and also largely prevented coat disruption. Subsequent EDTA treatment did not produce the normal effects of Ca deficiency; instead, thin bridges of elastic material of apparently high tensile strength and remarkably uniform diameter were seen to connect neighbouring spore masses. The significance of these observations is discussed in relation to the luiown chemical composition of coats of related strains.

Summary. Micromanipulation of single spores on an agar surface allowed the observation of normal and heat damaged spores in microculture. In tests for viability on different media, spores recorded as ‘dead’ in some media were fully viable when nutrients were supplied by diffusion from agar cylinders. In microculture of heat‐damaged spores, only those phase‐bright spores which exhibited considerable delay in becoming phase‐dark were eventually capable of forming visible colonies. Rapidly germinating spores were incapable of any outgrowth, except for a small minority which developed to a maximum of 4–8 cells and then lysed.

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