The Cultivation of Actinomyces israelii in a Progressively Less Complex Medium

Abstract

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.

Comparison of Physiological and Biochemical Characters of Actinomyces spp. with those of Lactobacillus bifidus

“…Erikson & Porteous (1953) found that on a heart broth casein digest medium, Actinomyces strains produced lactic acid equivalent to only 30-60 yo of the glucose utilized; other products were not reported.…”

The Growth Factor Requirements of the Wills Strain of Actinomyces israelii Growing in a Chemically Defined Medium

“…israelii (Erikson & Porteous, 1953). Thus the fact that neither coenzyme A (Christie & Porteous, 1962) nor pantothenic acid was required for growth was surprising for a reputedly fastidious anaerobic heterotroph.…”

“…Every precaution was taken in the present work to avoid contamination (Christie & Porteous, 1962) and no evidence of contamination of experimental cultures was found; Erikson & Porteous (1955) have, however, emphasized the difficulties of detecting small numbers of contaminants within the mycelial colonies of A . israelii and pointed out that changes in the colony form in liquid or on surface media may not be apparent in relatively lightly contaminated cultures of A .…”

A Technique for Obtaining Uniform Inocula of Actinomyces israelii

“…Cultures were maintained as described by Erikson & Porteous (1953). (1953) were used in the first and second stages of the technique to be described.…”

“…diameter; the supernatant medium remains clear at all stages of growth. The colonies, variously described as ' cauliflower ' or ' bread-crumb ' in appearance (Erikson, 1940; Erikson & Porteous, 1953), do not disperse on mild agitation of the culture vessel although the colonies of certain contaminated cultures will disintegrate (Erikson & Porteous, 1955). The colonies of different pure strains in liquid culture vary in consistency, from those which can be fragmented into smaller colonies by the tip of a Pasteur pipette to those which are so tough as virtually to resist fragmentation by this means.…”