Cultivation of hyperthermophilic archaea in capillary tubes resulting in improved preservation of fine structures

Abstract

A method for cultivating hyperthermophilic archaea that results in very high cell densities and in improved structural preservation of the cells is described. Cellulose capillary tubes, originally introduced as containers for embedding for electron microscopy, were filled with cells, closed at both ends, and put into sterile culture medium. Within these capillaries, which serve as ultrafiltration chambers, cells could be cultivated to much higher cell densities than in regular cultures. The capillaries containing cells were processed for ultrathin-sectioning by fixation, freeze-substitution, and embedding. Using this cultivation procedure, centrifugation, which may destroy sensitive structural components, could be avoided, and the cells of hyperthermophilic archaea were well-preserved. These undisturbed cells revealed the following new structural features: (1) a high number of tubules in ultrathin-sections, indicating a well-preserved network of Pyrodictium cells and tubules; (2) “ultraflat areas” of Pyrodictium cells, with the two membranes being in direct contact and, at some places, bulging out, forming evaginations; (3) novel cell-to-cell connections between Thermoproteus cells and, similarly, between Pyrobaculum cells; and (4) a surface coat on Pyrobaculum aerophilum cells. The cultivation procedure offers distinct advantages over conventional techniques and might be applicable for improved electron microscopy of other sensitive microorganisms.