The ceils of Halobacterium sp., strain 5, contain a large number of highly refractile bodies of the type which PETTE~ (1932) suggested were gas-filled vacuoles. The present studies support PET~ER'S contention, but the evidence for the exact chemical nature of the vacuole content is still indirect. It is not carbon dioxide or oxygen, but might possibly be nitrogen. "Strain 5" loses spontaneously and with a high frequency the ability to make the vacuoles.
When vacuolated cells are subjected to pressure, the vacuoles disappear, but can recover upon aeration. Oxygen and the organic constituents of the growth medium stimulate the recovery, whereas 2.4-dinitrophenol inhibits it. A procedure is described for the isolation of the vacuoles. The vacuoles are bounded by a membrane which reveals itself in electron micrographs of thin sections as a i-layered structure about 30 ~ thick.
During a study in our laboratory of the bacterial infection of solar salt and salted fish, bacteria of the halobaeterium type containing strongly refractile bodies were often encountered. The refractile bodies of the halobacteria were first studied by PETT]~U (1932). She provided some evidence that they were gas-filled vacuoles, and that they in various respects resemble the gas vacuoles found in a few other types of bacteria and in some blue-green algae. StmuIw and PIJP]~R (1952) expressed doubt about the gaseous nature of the content of the vacuoles. Hocwr~K (1956) supported PETTER'S contention, mainly on the basis of the observations that cells containing vacuoles would collect at the surface in a liquid medium, and that the vacuoles would disappear if the cells were subjected to a pressure of a few atmospheres.
For the present investigation we have selected a strongly vacuolated strain, Halobacterium sp., strain 5, for a closer study of the conditions of vacuole formation and the properties of the vacuoles.
Methods
Culturing. Halobacterium sp., strain 5, and its non-vaeuolated mutant, strain 5 M, were grown on agar plates at 37 ~ C on the medium used by ~OHU and LAas~ (1963). Liquid cultures were grown in a medium of the following composition: crude solar salt (Trapani) 25o/0, MgSO 4 -7H20 1~ KC1 0.50/0, CaC12 -6HeO 0.020/0 . yeast autolysate (Mo~a and LAUSE~, 1963) 100/0 (V/V), tryptone (Oxoid) 0.50/0, in * Dedicated to Prof. C. B. VAN NIEL on the occasion of his 70th birthday.