Simple Method for Killing Halophilic Bacteria in Contaminated Solar Salt.

Adding acid to brines or mixing acid phosphate with the salt used in preserving fish was recommended by Gibbons (Progr. Rep. Atlantic Fish. Exp. Sta., no. 14, p. 13, 1935), because it was observed that under acid conditions the halophilic bacteria that spoil salted fish cannot grow. The red halophile, Halobacterium cutirubrum, was found to be extremely sensitive to pH changes (Kushner and Bayley, Can. J. Microbiol. 9: 53, 1963). At pH 4, for example, the normally rodshaped bacteria form spheres which are no longer viable. This suggested that exposure to moderately low pH values might serve to prevent halophilic growth and also to sterilize solar salt. Two samples of solar salt, contaminated with red and white halophiles, were tested. One of these had been used for preserving fish; after drying, it still contained a large number of halophiles. The other sample contained a small number of halophiles, but counting was difficult. Saturated solutions were prepared by adding 60 ml of sterile distilled water, a few milliliters at a time, to 20 g of salt; 1-ml samples of these saturated solutions were placed in sterile petri dishes and treated with hydrochloric or acetic acid. The solutions were then dried overnight at 37 C, and the salt was taken up in 5.0 ml of sterile 20% NaCl. The number of viable cells in this solution was counted by the drop-plate method on 2% agar in a growth medium prepared as previously described (Kushner and Bayley), except that the NaCl concentration was reduced to 20%. Reducing the pH of the saturated "used" salt solutions to 5 greatly lowered the number of living organisms; at pH 2.7 or below, no viable halophilic bacteria remained (Table 1). The unused salt was also sterilized by lowering the pH, but it had a much lower buffering capacity than the used salt, presumably because of the presence in the latter of material extracted from fish. To reduce the pH of a saturated solution to 3.0 required 0.005 M HCl for the used salt, but only 0.001 M HCl for the unused salt.