Infection of mice by aerosols of Klebsiella pneumoniae under hyperbaric conditions.

Both the physical behavior of aerosols and survival of airborne Serratia marcescens in hyperbaric chambers with a helium-air mixture at 20 atm of pressure was approximately the same as in the system at ambient pressures. Exposure of mice to aerosols of Klebsiella pneumoniae at 1-, 2-, and 17-atm (ca. 101-, 203-, and 1,722-kPa) pressures of helium-oxygen mixture showed that the number of viable organisms constituting a 50% lethal dose was not significantly affected by the hyperbaric conditions.

Evidence for more than one division of bacteria within airborne particles.

When the protocol that we had used to demonstrate a single division of bacterial cells in airborne particles was changed to one that increased the glycerol content of the atomizer fluid from 1 to 5% (vol/vol), thus producing larger particles, more than two (and nearly three) divisions of bacteria occurred within 6 h of aerosol time.

Evidence that bacteria can form new cells in airborne particles.

Serratia marcescens incubated for 8 h at 31 degrees C in a chemically defined medium contained in shake flasks was aerosolized into rotating-drum aerosol chambers at 30 degrees C and saturated humidity. Cells furnished tryptone (Difco) and glycerol just before aerosolization increased (in viable numbers and countable cells) almost twofold within 1 to 2 h after becoming airborne, whereas cells not furnished additional tryptone decreased in viable numbers at a faster rate than the number of particles removed by gravitational settling. Limited tests with a Coulter Counter showed that cell volume changes occurred in growing cells that did not occur in the nongrowing population.

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.

Evidence for propagation of aerobic bacteria in particles suspended in gaseous atmospheres.

One factor involved in the possibility that airborne microbes might contaminate the Jovian atmosphere, is whether microbes have the capacity to propagate in air. Prior to these studies, the evidence was that the airborne state was lethal to microbes. By mixing an aerosol of aerobic bacteria with another containing 14C glucose, we were able to detect the presence of 14CO2, showing that the airborne cells were metabolically active. In the same type of experiment, we were able to show that thymidine was incorporated into the acid-insoluble fraction of samples, indicating the formation of DNA. Finally, we were able to show, both by an increase in numbers of viable cells and a parallel increase in particle numbers, that at least two new generations of cells could occur. As of this writing, evidence for propagation of anaerobic bacteria has been negative.

Correlations between free radical production and viability of lyophilized bacteria.

When Serratia marcescens cultures were treated with dilute solutions of phenol or hydrogen peroxide before drying or by lyophilization at suboptimal pH, the log of the number of cells surviving lyophilization was correlated with subsequent free radical production by the dried cells. Since the rate of free radical production and rate of death were similarly affected by temperature, the log of the number of cells surviving after 6 days was inversely related to the free radical concentration at that time. Free radicals were produced in proportion to the log of oxygen pressure, and viability was inversely related to oxygen tension; again, free radical concentration appeared to be correlated with the death of organisms.

Studies on survival of bacteria: rhythmic response of microorganisms to freeze-drying additives.

Various materials were mixed with suspensions of Serratia marcescens and other organisms. Samples were removed and frozen at intervals after mixing; the number of cells that survived both freeze-drying and exposure to air varied rhythmically as a function of time between mixing and freezing. When assayed before or immediately after drying there were essentially no fluctuations. The response was evident only when these dried samples were exposed to air. In a typical experiment, the number of cells surviving in the sample frozen 30 sec after adding propyl gallate was at least 10 times that in samples frozen either 20 sec earlier or 20 sec later. Other "peaks" in survival were observed at approximately 125 and 450 sec, but the times at which the peaks were observed were not consistent from one experiment to the next. Although we have been unable to control or predict the time at which maxima in resistance occur, we have shown that the phenomenon does occur with Escherichia coli and Saccharomyces cerevisiae as well as with S. marcescens. Furthermore, a rhythmic response also was obtained after a change in pH or cell concentration. It appears that microorganisms respond physiologically and synchronously to changes in their environment, and some of these responses have survival value.

Effect of 3 percent hydrogen peroxide on the viability of Serratia marcescens.

Campbell, Jack E. (University of California, Berkeley), and R. L. Dimmick. Effect of 3% hydrogen peroxide on the viability of Serratia marcescens. J. Bacteriol. 91:925-929. 1966.-Populations of Serratia marcescens were exposed to 3% H(2)O(2) at temperatures from 0 to 20 C. The reaction appeared to follow an Arrhenius plot, but variable numbers of diminutive colonies were found after cell numbers started to decrease. Colony numbers varied on different sampling media and increased when additional incubation was imposed. The overall reaction was sensitive to age of culture, and growth capabilities of treated samples varied with time of treatment, especially during times when no loss of viability was noted. Catalase activity per cell did not correlate with changes in sensitivity; iron added to growth medium increased catalase activity and decreased sensitivity, but not in the same manner. Although the fundamental reaction is presumably molecular in nature, present methods of viability assay measure more than single events and are not suitable for these studies.