The microbiology of saturated salt solutions and other harsh environments, I. Growth of a salt-dependent bacterial form in LiCL-saturated nutrient broth.

Abstract

The growth of bacteria in sodium chloride solutions has long been a subject of interest both as an example of stress tolerance and as a practical problem in saltpreservation of food products.1 More interesting than NaCl tolerance per se is extreme halophilism, as manifested by some species of Halobacterium that grow in saturated NaCl brines, and cease to do so at about half-saturation (ca. 15%). Halobacterium cutirubrum transferred to water apparently disintegrates into lipoprotein droplets by a disaggregation process rather than by osmotic bursting.2 NaCl-adapted organisms are not under the most extreme osmotic stress because the effect of NaCl on osmotic pressure is not great in comparison with many other salts. Water activity, A,, is a convenient index for comparison of various salt solutions.3 A, = p/po, where p = vapor pressure over the salt solution at temperature T and po = vapor pressure over pure water at the same temperature. For saturated NaCl at 25?C, A,, = 0.78, whereas CaCl2 and MgCl2 = ca. 0.3, and LiCl = 0.11. Osmotic pressure (P) is a function in ln po/p,4 hence P a (-log A,,). From these relationships, the water activity of saturated LiCl is approximately sevenfold lower and the osmotic pressure ninefold higher than NaCl at saturation. During a 6-month period of incubation, two of three LiCl broth cultures which had initially contained only conidiospores of Penicillium notatum became contaminated with a motile bacilloid form, perhaps as a result of exposure during periodic samplings. This organism was not observed at 3 months, but was present and active after 6, and appears to be the most extreme halophile yet observed. Experimental.-Broth cultures were of the following composition: glucose, 5 gm; Bactopeptone, 1 gm; Bacto-yeast extract, 1 gm; LiCl, 450 gm; and H20, 1000 gm, pH ca. 6. The spore inoculum was obtained from a soil isolate grown on saturated Ca(OAc)2-nutrient medium. Cultures had been incubated at 25?C in room light of about 50 ft-c. Bacterial counts were made by hemacytometer after thoroughly suspending sedimented materials. When examined after 3 months, LiCl broth cultures were clear except for spores, both suspended in the solution and at the bottom of the vessels. By the 6th month, a readily visible graywhite sediment had formed in one vessel, and a trace of light sediment in another. Under the microscope the heavier sediment consisted of gelatinous debris, spores, and a bacilluslike form. This organism ranged from solitary rods to extended streptobacilloid chains of 3-30 cells in length. Individual cells measured ca. 4-5 ;k X ca. 1-15 u. Cell walls were heavy and distinct. The cytoplasm, unstained, appeared dense and granular. Chains of cells resembled Bacillus megatherium (Fig. 1), but were uniformly Gram-negative. In the most abundant sample, the bacterial population averaged ca. 105 per cm3 counting each cell in chains as a unit, and no other microbial form was observed. In ordinary nutrient medium (without LiCl) inoculated from the same conidiospore supply used in the LiCl broth, there is no indication of these organisms. Of three additional cultures constituted and handled similarly but saturated with LiOAc, one also contained an organism that could not be distinguished from those in LiCl media. Four other LiCl cultures which had been sealed for 6 months were devoid of bacteria.