Project objectives were to develop an improved plating medium to achieve reproducible colony counts and accurate quantification for growth or survival of Spirillum volutans; and to develop an improved method for selection of S. volutans from nature. Growth enhancement studies began with casein hydrolysate-succinate-salts medium (CHSS). Casein hydrolysate concentration, type of casein hydrolysate, pH, and phosphate concentration optima were determined. Growth of S. volutans doubled after 24 h with the addition of potassium phosphate buffer (1 mM final concentration) to CHSS medium (CHSS-P). Addition of supplements to CHSS-P medium, and modified Bordetella and Brucella media failed to increase the cell yields of S. volutans. Punctiform colonies did form in CHSS-P medium (0.7% agar) pour-plates, yet at a recovery rate much lower than reported by Padgett et at. (1982) for surface growth on spread plates. The role of amino compounds as possible growth factors was investigated. Thin-layer chromatography, gas chromatography, and amino acid analysis were employed to identify those amino compounds (if any) that decreased or disappeared in the medium after 48 h of growth. None could be found. Numbers of viable cells were not greatly increased, however, viability was prolonged by growing S. volutans in dialysis sacs suspended in CHSS-P medium. The tolerances of S. volutans to several antimicrobial compounds were determined. Attempts at using hay infusion, Pringsheim, and CHSS-P media supplemented with various compounds as selective agents did not result in predominance of S. volutans. / M.S.

The relationships of the fourteen (14) species of Campylobacter were derived by comparison of the partial 16S ribosomal RNA sequences determined using reverse transcriptase and oligonucleotide primers specific for regions in the 16S rRNA molecule. These species formed three distinct RNA sequence homology groups. We propose that the following species remain in the genus Campylobacter (homology group I): C. coli, C. jejuni, C. laridis, C. fetus, C. hyointestinalis, C. concisus, C. mucosalis, C. sputorum and the "catalase-negative to weakly-positive" (CNW) strains. C. pylori, "C. cinaedi" and "C. fennelliae'' were not related to the true campylobacters at the genus level. However, they were related to each other and to Wolinella succinogenes at the genus level and constituted homology group II. These four species should be reclassified and placed within a single genus based on 16S rRNA sequence similarity. C. cryaerophila and C. nitrofigilis also exhibited a high level of RNA sequence homology with each other but not with any other species tested; they constituted homology group III and should be considered as being a single genus. A comparison of the 165 rRNA sequence data from the three homology groups with the same sequences from representatives of the three major phylogenetic branches of the purple bacteria indicated that these genera form a single phylogenetic branch which is only distantly related to the purple bacteria. / Master of Science

A comparative study of the microaerophilic <u>Campylobacter jejuni</u> strain H840 with an aerotolerant mutant, MC711-01, revealed that catalase and, to a lesser extent, SOD activity is correlated with the aerotolerance and enhanced resistance of MC7ll-0l to H₂O₂. When cells were cultured under 6% oxygen, the specific activity of catalase was significantly higher in crude extracts of MC711-01 than of H840. The catalase activity of MC711-01 more than doubled when cells were cultured under 21% oxygen, and this activity was 2.6 times greater than that of H84O; no corresponding increase was observed in strain H840. There was no significant difference in the mean SOD activity of the two strains when cultured under 6% oxygen or in H840 cells cultured under either 6% or 21% oxygen; however, the SOD activity of MC711-01 increased 1.5 times when cells were cultured under 21% oxygen. Survival studies revealed that MC711-01 was significantly more E resistant to H₂O₂ when cultured under either 6% or 21% oxygen. However, both MC711-01 and H84O were more susceptible to H₂O₂ when grown under 21% oxygen, indicating that both strains might be more highly stressed when the cells are grown at this oxygen tension. The present study suggests that in <u>C. jejuni</u>, the level of catalase activity may influence the degree of susceptibility to H₂O₂ and consequently, the degree of aerotolerance. / Master of Science