Growth of Thioploca Ingrica in a Mixed Culture System

Previously, Thioploca ingrica had not been grown in the laboratory and observation of the organism was limited to specimens taken directly from natural mud samples. The method described here makes laboratory cultivation of T. ingrica in mixed culture possible. The medium consists of low concentrations of extracted hay, water, and mud with its natural bacterial flora. Four weeks after the hay and mud were mixed, the medium was inoculated with T. ingrica washed from Lake Erie mud. It was found that the higher the concentration of hay, the lower the success rate and the longer the elapse of time before the organism established itself. Conversely, longevity of a successful culture improved with higher concentrations of hay. The growth of the organism in the flasks was observed in situ with an inverted microscope. OHIO J. SCI. 80(1): 30, 1980 From its discovery, Thioploca has been compared to Beggiatoa (Lauterborn 1907). Both are filamentous and gliding, contain sulfur inclusions, and share ultrastructural similarities (Lauterborn 1907 and Maier et al 1965). However, Kolkwitz (1912) noticed that the free filaments of Beggiatoa tolerated higher levels of oxygen (O2) and hydrogen sulfide (H2S) than the sheathed bundles of Thioploca filaments. The Bass Island area of Western Lake Erie was found to possess a rich mud population of T. ingrica (1960, Randies, personal communication). Such mud samples, kept in glass jars in the laboratory, supplied living organisms for years. In recent years, the population in Lake Erie has declined to the point where active bundles are no longer detectable. The hay enrichment method, successfully used for Beggiatoa (Faust et al 1961), was modified in this study to provide lower levels of H2S by varying the concentration of finely ground (60 mesh) extracted hay from 0.2 to 0.5 g/60 ml tap water in 125 ml Erlenmeyer flasks. Estimated with lead acetate paper, the level of H2S rose to a maximum in about 10 days after inoculation with 2 or 4 ml of Thioploca-free mud from Western Lake 'Manuscript received 15 August 1978 (#78-46). Erie and then declined slowly. Inoculation of the mud-hay flasks with active bundles of Thioploca ingrica (washed from aliquots of native Lake Erie mud maintained in jars in the laboratory) was delayed for 2 to 6 weeks in order to avoid the period of maximum H2S levels. The flasks were incubated at room temperature up to 52 weeks and examined at 4 week intervals for the presence of healthy filament bundles with an inverted microscope through the bottom of the flasks. When preliminary examination indicated that 4 and 5 week delays seemed best, the 4 week delay was used routinely. Table 1 shows that the lower the concentration of extracted hay, the higher the percentage of positive cultures when inoculation with T. ingrica was delayed for 4 weeks. Hay concentrations of 0.2 and 0.3 g were about equally successful with 93 and 92% respectively. Simultaneous inoculation with mud and T. ingrica was 67% successful in the lowest concentration of hay, but failed in all higher concentrations. Since lower concentrations of hay produced lower H2S levels, the data were in agreement with previous observations of the lower H2S tolerance of Thioploca (Kolkwitz 1912). Beggiatoa was simultaneously inoculated with its associated flora (Faust et al 1961) and thrived in the maximum FLS levels