SUMMARY: Six strains of Actinomyces israelii were trained to grow in continued subculture on a medium composed of 99 volumes of 1 yo casein hydrolysate + 1 volume of heart broth, the mixture containing 0.5 yo (w/v) glucose. Poor growth of limited viability was obtained in a medium consisting of 1 yo casein hydrolysate + 0.5 Yo glucose (w/v) only. The addition of a wide variety of different amino-acids, fatty acids, growth factors and mineral salts to certain of the less complex media did not compensate for the partial or complete omission of the heart broth. Of the glucose supplied in relatively complex media about 40% was utilized, and lactic acid equivalent to 30-60 yo of the glucose utilized appeared in the medium.Actinomyces israelii, the anaerobic organism which is the causal agent of actinomycosis in man and in some animals, has a well-established reputation for being difficult to maintain in laboratory culture. Alternation of media such as cooked meat + serum, nutrient agar +sterile horse blood, concentrated sheep heart broth + glucose, has been recommended (Erikson, 1940). Rosebury (1944) noted the advisability of a 'mixed diet', and found brain+heart infusion of especial value. Later workers (e.g. Thompson, 1950;Holm, 1950) used brain broth, ' hormone ' agar, blood agar or broth supplemented with ascitic fluid. Brewer's (1940) thioglycollate (mercaptoacetate) medium, in common use for this and other anaerobes, contains pork infusion solids, Such media are amongst the richest used for the cultivation of micro-organisms, yet short life, frequent dying out and a t best lessened activity attend the prolonged use of any one of these complex media for the cultivation of A . israelii. The following report describes an attempt to produce simpler media, and the growth characteristics obtained thereon. METHODS Organisms.Five human strains, typical Actinomyces israelii, and one porcine strain closely resembling these, were selected for detailed study. All six strains had been freshly isolated from cases of actinomycosis.Apparatus. Except where otherwise mentioned all liquid media were dispensed into rimless test-tubes 12 x 75 mm., plugged with cotton-wool.Automatic siphon-burettes of 2 or 10 ml. capacity which could be sterilized and also dismantled for cleaning were devised to fit 500 ml. (20 oz.) screwcapped ' medicine flats ' and 25 ml. screw-capped bottles. These burettes were used for dispensing all media.

No abstract

SUMMARY: When first isolated on soil extract agar, soil actinomycetes consistently produce aerial spores in surface colonies. They retain this property when maintained in sterile soil, or when grown on washed suspensions of common soil bacteria, living or dead, in a water agar medium. In soil, when the composition, moisture content and temperature are kept constant, the initial stimulus towards the production of aerial mycelium is free access of air: the quantity and nature, vegetative or sporogenous, of the inoculum, and, within a broad range, the pH of the soil are of minor importance. Once growth is established, the next most important factor stimulating sporulation in the soil is also physical, namely dehydration. In natural and sterilized soils of different origins, and in a 'synthetic' soil containing 250 p.p.m. of nitrogen as nitrate, the modes of growth of different actinomycetes strains are similar, and generally uncharacteristic of their species.The stress laid upon the variability of the actinomycetes in general, as reflected in their remarkable responsiveness to changes in media, in the extensive studies of Waksman (1919) and of Lieske (1921), has been echoed by most succeeding workers in the field. The two outstanding gross variations are in pigment production and the appearance of the aerial, spore-bearing apparatus. Soluble pigment production, whether of the yellow-brown (Plotho, 1940) or of the red-blue (Oxford, 1946) type has been shown in mass cultures to depend on the pH reached in the varying stages of metabolism of different nutrient substances. The striking difference between the smooth, cartilaginous, dense-textured colonies composed of vegetative hyphae only, and the ' powdered ' growth characteristic of an abundant production of aeria.1 mycelium arising from ramified substratum filaments (see P1. 1, figs. la,b), is not so readily attributable to single factors. Nevertheless, the ability of the organism to reproduce itself by means of spores is biologically one of its most important properties. This paper reports an investigation of some of the factors which affect this property. Composition of the mediumThe effect of readily available nitrogen. Afanasiev (1937), working with parasitic and saprophytic strains of Actinomyces scabies, attempted to find the C : N ratio which favoured the development of the vegetative growth only. I n a synthetic solid medium containing glucose as the main source of carbon, a high proportion of nitrogen, supplied either as KNO, or asparagine, partly suppressed the development of aerial mycelium. The organic nitrogen compound was particularly effective, a phenomenon noted also by Erikson (1935) in a group of actinomycetes isolated mainly from human pathological material. In

Representatives of the genus Micromonospora have so far been reported mainly from soil or, in the case of thermophiles, from composts. The type species, M. chalceae, described by ,2rskov (1923) who erected the genus, was first isolated by Foulerton (1905) under the name Streptothrix chalceae. This remained the only known strain among the Actinomycetes bearing the generic name Micromonospora until Jensen (1930, 1932) described a large number of soil micro-organisms which corresponded with Qrskov's description: "A branched unicellular mycelium is formed, consisting of very delicate hyphae with short lateral branches, each of which bears a single terminal spore." Recently, Waksman, Umbreit, and Cordon (1939) observed thermophilic strains of Micromonospora in composts and concluded from a study of the literature that some of the thermophiles isolated from soil or manure by Tsiklinsky (1899), from composts by Miehe (1907), and from decomposing clover hay by Schiitze (1908) should also come into this genus.The N.C.T.C. strain no. 4582 (Lister Institute) isolated by Gibson from blood culture in a case of Banti's disease and described by the writer (1935), which was tentatively classed as an Actinomyces (Qrskov's Group I) because of a sparse de-velopment of aerial mycelium, should probably be regarded as a Micromonospora of slow growth with scant lateral spore formation, resembling M. parva Jensen. Another strain corresponding with M. fusca Jensen was observed by the writer (1940) in a

SUMMARY:The only thermophilic actinomycetes found in composts made from lawn cuttings were members of the genus Micromonospora. The isolates obtained agreed in the main with the description of M. vuZguris (Tsiklinsky) Waksman, Umbreit & Cordon. Three strains have been studied in detail. They are true thermophiles, with an optimum range of growth 45-60', and appear in abundance at the high-temperature phase of the compost, with a profuse production of aerial mycelium and spores. Spore gemination is induced by heat activation, 5 min. a t 85' securing growth under suboptimal conditions. Thin suspensions of spores in 1 yo sucrose or in very dilute nutrient broth withstand 100' for periods of up to 45 min. Only very dense aqueous or saline suspensions are tolerant of these exposures. Surface growth on cellophan bearing a high density of aerial mycelium and spores withstands dry heating a t 100' for 150 min. or a t 106' for 5 min.In an extensive study of 480 cultures of thermophilic and thermoduric microorganisms isolated from the dust, grain, hay, etc., as well as from the milk obtained on various dairy farms, Prickett (1928) found that the only true thermophiles were either spore-forming bacilli or actinomycetes. Gordon & Smith (1949), making a taxonomic survey of 216 presumed thermophiles collected from many sources, were able to reduce the sporing bacilli to two main species, Bacillus coagulans and B. stearothermophilus. The thermophilic actinomycetes have not yet been studied extensively, but from the accounts so far published it would seem that the forms occurring in grass composts belong predominantly to the genus Micromonospora. Tsiklinsky (1899) isolated an actinomyces-like thermophile from decaying straw, manure, etc. which she termed Thermoactinomyces vulgaris, and which was renamed Micromonospora vulgaris by Waksman, Umbreit & Cordon (1939) as the result of finding similar forms on contact slides in dunged composts. Schutze (1908) was the first to describe in detail the development of a thermophilic actinomycete occurring in spontaneously heated clover hay, which he named Actinomyces monosporus, and which from his pictures is clearly a species of Micromonospora. In their study of the decomposition of the protein and hemicellulose fractions in grass composts, Forsyth & Webley (1948) noted that the extensive chalk-white encrustation of the upper layers of the compost during the high-temperature phase was attributable to the growth of a species of Micromonospora.The present study deals with the thermophilic actinomycete flora obtained from a series of grass composts put up during two seasons in Webley's (1947) composting container, and in collaboration with Dr Webley. All isolates are micromonosporae, have marked features in common, and, apart from a broader temperature range and lack of growth on potato, agree in the main with the description of M . vulgaris.