Cultivation and Properties of Thiovulum majus Hinze 65 



ation tube. This was confirmed by adding some thionine to the 

 hquid phase. Without aeration the entire hquid remained color- 

 less. After aeration through an unprotected pipette, the redox 

 dye was oxidized to its colored form throughout the liquid. With 

 localized aeration a colorless zone of about 1 cm above the agar 

 surface could be obsened for several weeks. 



The culture flasks were allowed to equilibrate under local- 

 ized aeration overnight at ]2-L5 C before inoculation with 0.1-0.3 

 ml of a purified cell suspension. The purity of cultures obtained 

 in this manner was checked by inoculating 1 ml samples into the 

 following liquid media: (a) seawater, 0.1 per cent peptone; 

 ( b ) the medium of Baalsrud and Baalsrud ( 1 ) for the cultixation 

 of ThiohaciUus, made up with seawater; (c) seawater, 0.1 per 

 cent sodium acetate, 0.05% NH4CI, 0.05% K.HPO4; and (d) sea- 

 water, 0.1 per cent yeast autolysate, 0.1 per cent glucose. Dupli- 

 cates of these cultures were incubated aerobically at 12 C and at 

 room temperature. Anaerobic cultures were also prepared with 

 the glucose medium. 



X-ray Diffraction, Electron Microscopy, Photography 



The x-ray diffraction pattern of the sulfur inclusions of the 

 cells was determined on material taken from an enrichment cul- 

 ture. The cells were washed twice in seawater, centrifuged, and 

 distilled water added. After recentrifugation at low speed the 

 sediment was dried and used for the x-ray diffraction study. For 

 electronmicroscopy (3) cells from crude cultures were used after 

 a one-step purification and fixation with osmium tetroxide. The 

 preparations were shadowed with platinum and examined in a 

 modified Philips E.M. 100 electron microscope. Pictures of living, 

 motile cells were taken by means of a Zeiss photomicroscope with 

 a flash of 3 x 10-^ second (Fig. 2, 5-7). 



RESULTS 



The methods developed by Wijler for the cultivation of 

 Thiovulum were reproducible and dependable when repeated 

 at the Hopkins Marine Station; no specific inoculum was neces- 

 sary- to obtain good cultures. But similar experiments performed 

 in Holland with seawater from the Dutch coast, taken at Schev- 

 eningen or at den Helder, were unsuccessful unless some mud was 



