Biohazards of investigations on the transmissible spongiform encephalopathies.

There is considerable current interest in the agents that cause the spongiform encephalopathies: scrapie, transmissible mink encephalopathy, kuru, and Creutzfeldt-Jakob disease (CJD). The unusual properties of these agents, their elusiveness, and their pathogenicity for humans (in the cases of kuru and CJD) make these agents interesting subjects of investigation but also make imperative a consideration of their potential biohazards in the laboratory. In view of both the potential pathogenicity of these agents and the potential hazards of many laboratory procedures, a series of physical containment levels, each of which corresponds to a range of composite risk factors, are suggested. The estimated composite risk factor used is a function of the potential pathogenicity or relative risk factor of the agent and the potential hazard of a laboratory procedure. The lowest risk factors (1 to 2+) correspond to levels of containment similar to those recommended by the Center for Disease Control for class II microorganisms, while the highest risk factors (5 to 8+) correspond to levels similar to those for class III microorganisms. The use of such a biohazard ranking system aids in a rational approach to selection of equipment and procedures.

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.

Evaluation of a class III biological safety cabinet for enclosure of an ultracentrifuge.

An evaluation of a special safety cabinet housing a high-speed centrifuge was made. The cabinet enclosed both the top access port and the drive and pumping machinery of the centrifuge. A titanium rotor was loaded with tubes containing a bacterial culture, weakened, and driven until rotor rupture occurred. There were several bent and broken components in the centrifuge, and bacteria leaked from the vacuum chamber. Although the forces were sufficient to displace the cabinet, none of the test bacteria were found outside the cabinet.

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.

Evaluation of Moloney murine sarcoma and leukemia virus complex as a model for airborne oncogenic virus biohazards: survival of airborne virus and exposure of mice.

Aerosols of the Moloney murine sarcoma virus (MuSV-M) and leukemia virus (MuLV-M) complex (MuSV-M/MuLV-M) were generated from refluxing atomizers and then aged in rotating drums at 21 degrees C holding temperature with relative humidities ranging from 25 to 76%. The MuSV-M and MuLV-M aerosolized from the same tumor extract preparation survived almost equally at the four humidity levels. Both viruses remained viable in the airborne state for at least 2 hours after aerosolization. When mice were exposed to airborne MuSV-M/MuLV-M, no macroscopic lesions were observed in lungs or other tissues examined during the 2-month postexposure period. On the basis of this study, MuSV-M was determined unsuitable as a "model system" in which a simple aerosol dose response could be used for biohazard evaluation of oncogenic virus aerosols.