Cultivation and Properties of Thiovulum majus Hinze 67 



added from a lagoon near Bergen op Zoom, Holland, in which 

 Thiovulum occurs. The cultures obtained were, however, much 

 less vigorous and more heavily contaminated than those grown at 

 the Pacific coast. Nevertheless, crude cultures, derived from this 

 material, could be maintained for a period of three months in the 

 above described small-scale continuous culture system, which 

 alleviated the strain on the limited seawater supply at Delft. 

 Cells from this culture did not survive a sufficient number of 

 purification steps to yield pure cultures but provided suitable 

 material for electron microscopy. 



Numerous experiments were carried out with cells from the 

 Pacific Grove enrichment cultures to develop methods suitable 

 for isolation and stationary cultivation. Since it proved easier to 

 maintain a concentration gradient rather than constant concen- 

 trations of sulfide and oxygen for a prolonged period of time, and 

 a gradient method had successfully been employed for cultivat- 

 ing Gollionella (10), the agar block method combined with lo- 

 calised aeration appeared promising in view of the strong chemo- 

 taxis of Thiovulum, which could be relied upon to lead the or- 

 ganisms to the area of optimal concentrations. The sterile media 

 were inoculated with Thiovulum suspensions purified by seven 

 to nine consecutive passages through sterile seawater; this seemed 

 adequate because it had been found that six such passages suf- 

 ficed to eliminate contaminants. 



After a few hours, the inoculated cells were usually observed 

 to have settled in a sharply defined zone above the agar. The 

 cells were crowded together in small agglomerates, apparently 

 creating an optimal micro-environment. Within the following 

 days the number of separate agglomerates increased until a com- 

 plete, sharply defined web was formed at about 1 cm above the 

 agar in the otherwise clear liquid; with lateral illumination indi- 

 vidual, sulfur-stuffed cells could be seen dancing around in the 

 web. On prolonged incubation the web rose slowly because of 

 increasing oxygen demand; when the available sulfide was nearly 

 consumed the web moved downward, but by this time signs of 

 decay appeared in the form of white strands hanging down the 

 sides of the flask, consisting of dead cells adhering to each other. 

 For three weeks after inoculation successful transfers to similar 

 flasks could be performed. Cultures failing to produce any growtli 



