INDEPENDENT STUDY FINAL REPORT

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1 0 July University of Massachusetts Department of Microbiology Tsute C/zen MBL, Woods Hole 1993 Microbial Diversity Summer Course INDEPENDENT STUDY FINAL REPORT rt4- I

2 Enrichment of Chorochromathun aggregatum Paul Egland University of Iowa, Iowa City, Iowa C intensities of light. Methods diaphragm and positioning a large group of aggregates in different containing Chlorobium media at one-fifth concentration and a variety of cyanobacteria. Cultures were monitered microscopicaly at 100X to probes provided by Sandra Nierzwicki-Bauer. light source. The effect of light intensity was tested by closing the scope response to light, was tested by briefly sheilding the sample from the suffide levels with 2 ml of liquid from the sediment surface of natural using a plexiglass corer and transfered to flasks and jars which were samples. Incubation was continued in fluorescent light. Dichiorophenyl were done using video microscopy. Shock movement, or reversal in light. Secondary enrichment was acheived by inoculating Pfennig bottles obtained from the inlet to Oyster Pond. Thirteen samples were collected then sealed. Primary enrichment was done by incubation in fluorescent Samples used as inoculum for the enrichment cultures were Samples from successful enrichments were used for in situ Tests of phototactic activity of Chlorochromatium aggregatum observe results. oligonucleitide probing of 16S ribosomal RNA genes with the method and 1,1 Methylurea (DCMU) was added to some bottles to inhibit growth of sulfide. This consortium has never been isolated in pure culture. The goal of this research project was to develop enrichment reducing bacterium. In this association, the metabolism of these techniques for Chiorochromatium aggregatum and to take steps to identify the organisms that compose the consortium. Chlorochromatium aggregatum is a bacterial consortium composed of a green sulfur bacterium and an unidentified, motile sulfate organisms is closely linked, presumably by their cycling of sulfate and

3 The salinity of the water in this area varied between 0.4 and 1.4% NaC1 C pt-1-3 process. one field of view. Addition of DCMU seemed to kill some of the cyanobacteria in the possible to use light to disperse aggregates from areas of high because of morphology changes of the aggregates during the fixation when aggregates swam into areas of bright light. However, it was not organism of C. aggregatum. This result is slightly ambiguous only cases, no fewer than 20 Chlorochromatium aggregates were present in In tests for phototactic behavior C. aggregatum, no evidence of to be hybridization of the Proteobacteria delta probe to the central The results of in situ hybridization experiments show what seems examples. There did seem to be some evidence of reversal of direction green flocks which accumulated at the bottom of the bottles. In some increase this effect. The highest enrichment and longevity was obtained from cultures grown in one-fifth concentration Chiorobium medium with low sulfide shock movement in response to light exclusion was seen. This result was (depending on the level of the tides) as measured by refractometry. concentration. The concentration of Chlorochromatium was localized to culture. It may have been useful to add a higher concentration to consistent in trials with tethered aggregates as well as free swimming concentration. collected from the point where the inlet to Oyster Pond reaches the pond. The most successful primary enrichment was from a sample / Observations and Results

4 Abstract Heat Resistance of Myxococcus virescens myxospores Paul Egland University of Iowa, Iowa City, IA t fl, efficiency, the results obtained were not statistically meaningful. subject to heat treatment. The spores were then plated and cells, the spores were diluted in CU medium containing.4% agar and from an isolate of M. virescens. After sonication to kill vegetative Myxococcus virescens myxospores. Fruiting bodies were collected incubated at 32 degrees C. Due to poor plating and germination An attempt to was made to test the heat resistance of

5 Heat Resistance of Myxococcus virescens rwlyxospores Paul Egland University of Iowa, Iowa City, Iowa O insignificant. samples. Due to the low efficiency, the results are statistically fold lower than expected. No spores germinated in the heat treated myxobacteria colonieswere visible in the agar spots. The colonies were counted, however colony counts in the control plate were 50- After four or five days of incubation at 32 degrees C, Results pulses. Total spore counts were made using a Petroff-Hauser water bath for heat treatment. At five minute intervals, 100 p1 added to each spot. To maintain humidity, filter paper was placed in the lid of each petri plate and saturated with sterile distilled water. allowed to solidify. When cool, 100 p1 of uninoculated CTT agar was aliquots of spore suspension were spoiled in petri plates and which had been warmed to 40 C. Dilutions were then placed in a 70C counting chamber. Myxospores were diluted in CTT medium containing.4% agar U Vegetative cells were killed by sonication for three 30 second distilled water at a concentration of six fruiting bodies per ml. TPM medium and resulting fruiting bodies were suspended in sterile Nevarez-Moorillon. Myxospore production was induced by growth on An isolate of Myxococcus viresceris was obtained from Vicky Methods species. this project was to test the heat resistance of myxospores of other myxospores of other species has not yet been evaluated. The goal of resistant (Sudo and Dworkin 1969). However, heat resistance of It is known that spores of Myxococcus xanthus are heat