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A technique was developed for measuring 14CO2uptake by chemolithotrophic bacteria directly in pyritic materials associated with coal and coal refuse. There was good correlation between 14CO2uptake, as determined by this technique, and the most probable number of iron-oxidizing bacteria. Maximal 14CO2uptake occurred in coal refuse material 2 to 3 years old, and only slight incorporation was demonstrated in fresh material or material 40 years old. Samples taken from the surface of the coal refuse pile always demonstrated maximal 14CO2uptake, and in most samples, only slight activity was demonstrated at depths below 8 to 10 cm. Optimal uptake of 14CO2by natural samples occurred at 20 to 30 C and at a moisture content of between 23 and 35%. In addition to chemolithotrophic bacteria, heterotrophic fungi and yeasts were also routinely isolated in high numbers from acidic coal refuse. In contrast, acidophilic, heterotrophic bacteria were either absent or present in low numbers in such acidic samples.

Specific immunofluorescence staining was applied to the study of the localization, distribution, and growth of Thermoplasma acidophilumin its natural habitat, the coal refuse pile. Different antigenic groups of T. acidophilumcould be isolated from the same refuse pile, and the same antigenic groups were isolated from piles from different geographical areas. No correlation could be established between the antigenic groups and the pH or temperature of the habitats. Brightly fluorescing cells of T. acidophilumwere detected on microscope slides buried in contact with the coal refuse material or immersed in the water in the stream draining a refuse pile. T. acidophilumgrew when inoculated into either coal refuse material and/or an aqueous extract of coal refuse when incubated at its optimal temperature of 55 C, but not when incubated at room temperature or 37 C. The coal refuse pile appears to be a primary habitat for T. acidophilum.

Turnover times for water passing through several Sulfolobus acidocaldarius-containing springs were determined by measuring the dilution rates of small amounts of sodium chloride that were added to the springs. Chloride was diluted out exponentially, while concentrations of the bacteria remained constant. Additionally, temperature, pH, and chemical composition of the springs also remained constant during the time that the chloride was being diluted. The springs are thus steady-state systems, and since the rates of bacterial growth must be at least equal to the chloride dilution rates, minimal doubling times for the bacterial populations can be calculated. Half-times for chloride dilution, equivalent to bacterial doubling times, were on the order of 10 to 20 h for springs ranging in volume from about 20 to 2,000 liters, but approximately 30 days for two larger springs of about 1 million liters. Formaldehyde-fixed cells of a serologically distinguishable strain of S. acidocaldariuswere also added as markers to four of the smaller springs, and the dilution rates of these bacteria were compared with the chloride dilution rates. The rates agreed reasonably well, thus verifying the growth rates obtained from the chloride dilution rates. In three springs, exponential growth was studied by draining the springs and allowing them to refill with bacteria-free water. Exponential doubling times were on the order of a few hours, much more rapid than steady-state doubling times. The methods used in this work may have wider utility in aquatic environments.

Conti, S. F. (Dartmouth Medical School, Hanover, N.H.), and T. D. Brock. Electron microscopy of cell fusion in conjugating Hansenula wingei. J. Bacteriol. 90:524–533. 1965.—The heterothallic yeast Hansenula wingeiis a favorable organism for the study of the process of cell fusion, since strong agglutination of cells of the two mating types ensures a high percentage of cell fusions. The initial agglutination reaction results in cell-wall deformation, so that the walls in the region of contact are tightly appressed over an extensive area. The fusion process is initiated when the walls of two cells elongate, and this elongation seems to be restricted to the region where the cells touch. Occasionally, one cell is seen to push in the wall of the other, but in many cases both cells elongate equally, as would be expected in an isogamous organism. The precise disposition of the elongating wall probably reflects the manner in which the cells initially become associated in the agglutinated cell clump. Soon after wall elongation begins, cell-wall fusion occurs along the margin of contact. Only after fusion is complete is the wall separating the two cells dissolved away. If wall dissolution begins at one edge of the conjugation tube, a flap is formed in which can be seen the remnants of the fused walls. Alternatively, dissolution can begin at the center of the conjugation tube, proceeding towards the outside. Conjugating cells are uninucleate, and the nuclei are large and frequently lobed or elongated. After the conjugation tube is formed, the nuclei migrate towards the center, and fusion occurs only over a small region where the nuclear membranes come in contact. After nuclear fusion, the first diploid bud forms from the conjugation tube and at right angles to the tube axis. The diploid nucleus then migrates into this bud. Frequently, in the later stages of conjugation, a large vacuole develops in each of the original cells. All of the above events will occur in a medium devoid of a nitrogen source and in which vegetative budding will not occur.

The physiology of the bacteria living in Boulder Spring (Yellowstone National Park) at 90 to 93 C was studied with radioactive isotope techniques under conditions approximating natural ones. Cover slips were immersed in the spring; after a fairly even, dense coating of bacteria had developed, these cover slips were incubated with radioactive isotopes under various conditions and then counted in a gas flow or liquid scintillation counter. Uptake of labeled compounds was virtually completely inhibited by formaldehyde, hydrochloric acid, and mercuric bichloride, and inhibition was also found with streptomycin and sodium azide. The water of Boulder Spring contains about 3 μg of sulfide per ml. Uptake of labeled compounds occurs only if sulfide or another reduced sulfur compound is present during incubation. The pH optimum for uptake of radioactive compounds by Boulder Spring bacteria is 9.2, a value near that of the natural spring water (8.9). Many experiments with a variety of compounds were performed to determine the temperature optimum for uptake of labeled compounds. The results with all the compounds were generally similar, with broad temperature optima between 80 and 90 C, and with significant uptake in boiling (93 C) but not in superheated water (97 C). The results show that the bacteria of Boulder Spring are able to function at the temperature of their environment, although they function better at temperatures somewhat lower. The fine structure of these bacteria has been studied by allowing bacteria in the spring to colonize glass slides or Mylar strips which were immediately fixed, and the bacteria were then embedded and sectioned. The cell envelope structure of these bacteria is quite different from that of other mesophilic or thermophilic bacteria. There is a very distinct plasma membrane, but no morphologically distinct peptidoglycan layer was seen outside of the plasma membrane. Instead, a rather thick diffuse layer was seen, within which a subunit structure was often distinctly visible, and connections frequently occurred between this outer layer and the plasma membrane. The thick outer layer usually consisted of two parts, the outer part of which was sometimes missing. Within the cells, structures resembling ribosomes were seen, and regions lacking electron density which probably contained deoxyribonucleic acid were also visible.