The difficulties encountered in the surface growth of (CI.) of types B and D were investigated. Best results were obtained when nutrient agar was enriched with human blood (33 per cent.) and supplemented with (i) iron filings sprinkled on the surface of a seeded plate, or (ii) a development of Moore's cysteine-dithiothreitol system incorporated in the medium. The growth-enhancing effects of these two systems were not identical; different degrees of enhancement were obtained with the two systems when test cultures of different ages were used.

The results of quantitative studies with these culture procedures indicate that, although spores of of types A, B, C and D may be significantly viable particles involved in the growth of inocula in pour plates seeded from old cultures in fluid media, large proportions of the surface viable counts obtained from young cultures of type-D strains are regularly derived from vegetative cells. Viable counts obtained with young cultures of type D, the most demanding strains, on solid media greatly exceeded the maximum estimates for the spore content of the inocula used. There is clear evidence that a very large proportion of type-D spores does not proceed to germination or successful outgrowth under the test conditions.

In the course of this study, amendments to the recommended anaerobic jar procedure were developed and a standardised method is described.


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