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INDEPENDENT STUDY FINAL REPORT

Microbial Diversity Summer CourseMBL, Woods Hole 1993

Tsute C/zen

Department ofMicrobiologyUniversity of Massachusetts

July30. 1993

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Microbial Diversity 1993

Paul Egland University of Iowa, Iowa City, Iowa 52242

Enrichment of “Chorochromathun aggregatum”

“Chlorochromatium aggregatum” is a bacterial consortiumcomposed of a green sulfur bacterium and an unidentified, motile sulfatereducing bacterium. In this association, the metabolism of theseorganisms is closely linked, presumably by their cycling of sulfate andsulfide. This consortium has never been isolated in pure culture.

The goal of this research project was to develop enrichmenttechniques for “Chiorochromatium aggregatum” and to take steps toidentify the organisms that compose the consortium.

MethodsSamples used as inoculum for the enrichment cultures were

obtained from the inlet to Oyster Pond. Thirteen samples were collectedusing a plexiglass corer and transfered to flasks and jars which werethen sealed. Primary enrichment was done by incubation in fluorescentlight. Secondary enrichment was acheived by inoculating Pfennig bottlescontaining Chlorobium media at one-fifth concentration and a variety ofsuffide levels with 2 ml of liquid from the sediment surface of naturalsamples. Incubation was continued in fluorescent light. Dichiorophenyl1,1 Methylurea (DCMU) was added to some bottles to inhibit growth ofcyanobacteria. Cultures were monitered microscopicaly at 100X toobserve results.

Samples from successful enrichments were used for in situoligonucleitide probing of 16S ribosomal RNA genes with the method andprobes provided by Sandra Nierzwicki-Bauer.

Tests of phototactic activity of “Chlorochromatium aggregatum”were done using video microscopy. Shock movement, or reversal inresponse to light, was tested by briefly sheilding the sample from thelight source. The effect of light intensity was tested by closing the scopediaphragm and positioning a large group of aggregates in differentC intensities of light.

— / Observations and ResultsThe most successful primary enrichment was from a sample

collected from the point where the inlet to Oyster Pond reaches the pond.The salinity of the water in this area varied between 0.4 and 1.4% NaC1(depending on the level of the tides) as measured by refractometry.

The highest enrichment and longevity was obtained from culturesgrown in one-fifth concentration Chiorobium medium with low sulfideconcentration. The concentration of Chlorochromatium was localized togreen flocks which accumulated at the bottom of the bottles. In somecases, no fewer than 20 Chlorochromatium aggregates were present inone field of view.

Addition of DCMU seemed to kill some of the cyanobacteria in theculture. It may have been useful to add a higher concentration toincrease this effect.

In tests for phototactic behavior” C. aggregatum”, no evidence ofshock movement in response to light exclusion was seen. This result wasconsistent in trials with tethered aggregates as well as free swimmingexamples. There did seem to be some evidence of reversal of directionwhen aggregates swam into areas of bright light. However, it was notpossible to use light to disperse aggregates from areas of highconcentration.

• The results of in situ hybridization experiments show what seemsto be hybridization of the Proteobacteria delta probe to the centralorganism of “C. aggregatum.” This result is slightly ambiguous onlybecause of morphology changes of the aggregates during the fixationprocess.

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Microbial Diversity 1993

Paul Egland University of Iowa, Iowa City, IA 52242

Heat Resistance of Myxococcus virescens myxospores

Abstract

An attempt to was made to test the heat resistance ofMyxococcus virescens myxospores. Fruiting bodies were collectedfrom an isolate of M. virescens. After sonication to kill vegetativecells, the spores were diluted in CU medium containing .4% agar andsubject to heat treatment. The spores were then plated andincubated at 32 degrees C. Due to poor plating and germinationefficiency, the results obtained were not statistically meaningful.

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Microbial Diversity 1993

Paul Egland University of Iowa, Iowa City, Iowa 52242

Heat Resistance of Myxococcus virescens rwlyxospores

It is known that spores of Myxococcus xanthus are heatresistant (Sudo and Dworkin 1969). However, heat resistance ofmyxospores of other species has not yet been evaluated. The goal ofthis project was to test the heat resistance of myxospores of otherspecies.

Methods

An isolate of Myxococcus viresceris was obtained from VickyNevarez-Moorillon. Myxospore production was induced by growth onTPM medium and resulting fruiting bodies were suspended in steriledistilled water at a concentration of six fruiting bodies per ml.

U Vegetative cells were killed by sonication for three 30 secondpulses. Total spore counts were made using a Petroff-Hausercounting chamber.

Myxospores were diluted in CTT medium containing .4% agarwhich had been warmed to 40 C. Dilutions were then placed in a 70Cwater bath for heat treatment. At five minute intervals, 100 p1aliquots of spore suspension were spoiled in petri plates andallowed to solidify. When cool, 100 p1 of uninoculated CTT agar wasadded to each spot. To maintain humidity, filter paper was placed inthe lid of each petri plate and saturated with sterile distilled water.

ResultsAfter four or five days of incubation at 32 degrees C,

myxobacteria colonieswere visible in the agar spots. The colonieswere counted, however colony counts in the control plate were 50-fold lower than expected. No spores germinated in the heat treated

O samples. Due to the low efficiency, the results are statisticallyinsignificant.