Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media 



The chemically defined broth was compared to the semisynthetic broth for maintaining 

 growth in five sequential transfers of 10 strains of the LDB: Philadelphia 1. Philadelphia 2, 

 Philadelphia 3, Philadelphia 4, Pontiac, Flint 1, Bellingham. Miami Beach. Detroit 1, and Togus 1. 

 The defined broth (with no starch) supported growth of the tested strains equally well. Indeed, in 

 one or more passages, the cell yields were higher than those observed in the casein hydrolysate 

 broth -suggesting that some amino acids of the defined mixture were at levels preferable to those 

 in the casein hydrolysate broth. The various strains of the organism generally adapted readily to 

 the two media. Although results obtained with the different strains varied to some degree, in 

 general we found that the defined medium containing 0.05'"' starch was superior lo the casein 

 hydrolysate medium. 



D. Physiology 



In most of the experiments with the various solid and liquid media, cells were examined for 

 morphology, catalase production, and hydrolysis of starch. Cells were also stained with the 

 serogroup-specific fluorescent antibody (FA) rabbit-anti LDB conjugate, and the presence of 

 surface antigen was compared with that of cells grown on the F-G medium. Catalase production 

 was extremely limited or absent, and no hydrolysis of starch was observed using any of these 

 media. In all cases, cells grown on either the semisynthetic or chemically defined medium stained 

 by direct FA as well as or better than control cells grown on F-G medium. 



Morphology of the LDB varies widely under different growth conditions (Fig. 7). In general, 

 growth covers the entire surface of an agar plate because of the liquid present; growth apparently 

 starts sooner and is more rapid in higli humidity. However, we did not observe spreading or 

 crawling motility. When plates were incubated between NaOH pellets (to absorb CO, ). the pellets 

 also functioned as dessicants, and the surfaces were dried-producing discrete colonies with entire 

 edges; results were similar when plates were incubated 7 to 10 days, and the colonies were typical 

 of those grown on CYE medium. The slower growth observed under these conditions may have 

 been the result of depleted CO2 or a dried surface or both. Cellular morphology changed progres- 

 sively in broth, with large masses of filaments or chains of bacilli in the logarithmic phase 

 breaking into shorter filaments and ultimately forming single and double cigar-shaped cells. With 

 prolonged incubation or limited substrate, cells became coccal shaped (Fig. 7). The organism 

 grew at 25°C, 30°C, and 37°C. The cellular morphology was the same at 30°C and 37°C, but at 

 25° C cells appeared as tine, small bacilli (Fig. 7). We did not observe refractile cells or cells which 

 miglit suggest spores or microcysts but did see swollen cell terminals which were probably 

 spheroplasts. 



SUMMARY AND DISCUSSION 



In reviewing the characteristics of the LDB, we see a set of apparently contlicting growth 

 requirements and physiological responses, which, according to the limited data presently avail- 

 able, may be more apparent than real. We have an aerobic organism which sometimes seems 

 sensitive to excesses of oxygen, which can grow under the microaerophilic condition of the 

 candle jar, which produces limited amounts of-if any— catalase, and which has a specific require- 

 ment of cysteine as an -SH amino acid. With the lability and extreme reactivity of the -SH of 

 cysteine, particularly in alkaline solutions and in the presence of metals, it would not be difficult 

 to visualize this as a factor limiting growth in nature or in laboratory media. Thus the limited 

 aerobic requirements are supportive and not in conflict with this cysteine requirement. Further- 

 more, a preference for acid pH further supports a stable -SH form of cysteine because the 

 molecule is higlily reactive in alkaline conditions and is rapidly oxidized to cystine in the pres- 

 ence of iron. At neutral or alkaline pHs, cysteine spontaneously forms addition compounds with 

 aldehydes, ketones, and unsaturated molecules (5, 7, <*?). 



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