Development of a dehydrogenase activity measurement test for Streptomyces venezuelae using triphenyl tetrazolium chloride

Streptomyces venezuelae is an aerobic bacterium that produces jadomycin. Jadomycin has shown activities against bacteria, yeasts, and fungi, as well as cytotoxic properties to cancer cells. Because of the wide range of its inhibitory actions, jadomycin shows promise as a novel antibiotic and cancer treatment drug. This study investigated the differences in activity and structure of Streptomyces venezuelae under different environmental conditions and during different stages of growth, sporulation, and jadomycin production. The study was carried out in three phases.;The second phase involved investigating activity differences of S. venezuelae under different environmental conditions using the TIC technique to measure dehydrogenase activity. Results indicated that the transition from live cells to spores was not be 100% efficient. TF yield (mumol/CFU) increased during the first 32 h of jadomycin B production, after which a gradual decrease was noted. The increase in dehydrogenase activity directly relates to the production of jadomycin B. The maximum yield of jadomycin B was reached approximately 25 hours after ethanol shock. The TTC test for measuring dehydrogenase activity of S. venezuelae should be developed in the production media.;The final phase investigated structural changes of S. venezuelae during different stages of growth and sporulation. Samples from each stage (growth, ethanol shock, and jadomycin B production) were viewed with scanning and transmission electron microscopy. Notable changes in structure were decreasing cell diameter, increasing cell length, and increasing branching and budding with the four consecutive stages. Numerous large vesicles were noted during the growth phase, while fewer, smaller ones could be seen during acclimatization and ethanol shock. Cells were dividing during the acclimatization and ethanol phases but branching and budding, indicating spore formation, were observed during the production of jadomycin B.;The first phase consisted of investigating the potential use of a dehydrogenase activity measurement test using trlphenyltetrazolium chloride (TTC) to measure the dehydrogenase activity of S. venezuelae during growth in maltose-yeast extract-malt extract (MYM) broth. The testing conditions, including extraction solvent, number of extractions, assay incubation time, assay incubation temperature, and medium pH, were examined. It was found that the test can be applied to S. venezuelae and the best conditions included three extractions using methanol, an incubation temperature of 22 or 30°C, a medium pH of 6, and an incubation time between 1 and 4 h. An incubation temperature of 30°C was used in place of 22°C because available equipment could more easily maintain a constant temperature at 30°C and an incubation time of 1 h was chosen. A standard curve of triphenyl formazan (TF) versus colony forming units (CFU) showed that the test could be used to quantify the number of live S. venezuelae cells.