68 Marine Microbiology 



in the media for detection of contaminants, were considered pure. 

 These cultures were also characterized by the absence of tur- 

 bidity outside tlie zone of growth of Thiovulum. Isolation and 

 subsequent culti\'ation were repeated successfully several times 

 with material from different enrichment cultures. Cultivation on 

 a somewhat larger scale in a 3 L Fernbach flask, provided with 

 an agar-layer and localised aeration proved entirely satisfactory. 

 As incubation temperature, 12-15 C proved most suitable, but it 

 has not yet been detemiined whether this corresponds to the 

 optimum temperature for the organisms or renders the culture 

 conditions more stable. 



Addition of 0.005 per cent yeast autolysate to the agar block 

 did not appreciably affect growth of pure cultures. When special 

 Agar (Noble, Difco) was substituted for Bacto Agar in the nor- 

 mal medium, development was somewhat slower. No growth oc- 

 curred in the medium suitable for ThiohaciUus, with thiosulfate 

 as hydrogen donor. Artificial seawater prepared according to 

 Pringsheim (17) and Lyman and Fleming (11) could not re- 

 place natural seawater, the cells dying off within three days. 



The moi-phology of the organisms studied in California and 

 in Holland was similar. In accordance with the results of Faure- 

 Fremiet and Rouiller (5) the sulfur inclusions were found to be 

 birefringent when examined in the polarizing microscope; ma- 

 terial derived from cells subjected to osmotic shock by immersion 

 in distilled water furnished an x-ray diffraction pattern similar to 

 that given for orthorhombic sulfur by Hanawalt et al. (7). 



Organisms from the Pacific as well as from the North Sea 

 proved to be gram-negative and lacked catalase, which may ex- 

 plain their sensitivity to high oxygen concentrations. 



The puzzling failure to detect locomotor organs in so motile 

 an organism, reported by Faure-Fremiet and Rouiller (5), in- 

 vited reinvestigation; but no flagella could be observed by stain- 

 ing or dark field and phase-contrast microscopy. Electron micros- 

 copy also failed to reveal flagellation when specimens were pre- 

 pared by allowing drops of cell suspensions to evaporate on the 

 membranes followed by washing. However, cells previously fixed 

 by means of osmium tetroxide invariably showed a large number 



