Viability of Airborne Pasteurella pestis

WON, WILIAiM D. (University of Caifornia, Berkeley), ANm HAROLD Ross. Effect of diluent and relative humidity on apparent viability of airborne Pasteurella pestis. Appl. Microbiol. 14:742-745. 1966.-Airborne Pasteurella pestis (A-1122) at low humidities [20 to 50% relative humidity (RH)] exhibited exponential decay when either 1% peptone or Heart Infusion Broth (HIB) was used as the diluent in the viable assay system. At higher RH values (65 and 87%), however, the 1% peptone diluent adversely affected the viability assay. In contrast, HIB as diluent was remarkably effective in demonstrating a higher number of viable cells in aerosols held at high RH values. Similarly, with HIB as diluent, aerosols were shown to contain viable cells during 90 min of observation; with 1% peptone, viability was not detectable after 20 min in the airborne state. Relative humidity (RH) has striking biological effects on microorganisms. Dunklin and Puck (3) and Webb (5) reported that the death rate for aerosolized Serratia marcescens, Escherichia coli, Staphylococcus albus, Streptococcus hemolyticus, Bacillus subtilis and type 1 pneumococci was greatest at the intermediate RH zone. On the other hand, increased death rate due to exposure to high RH environments has also been observed for airborne Salmonella pullorum and smears of Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, and Salmonella derby (1, 4). This report describes results of experiments with Pasteurella pestis (A-1122), showing that diluents used in viable-cell assay of aerosol samples have an effect on the apparent viability of organisms aerosolized and maintained at various RH values. MATERIALS AND METHODS P. pestis cultures. The avirulent strain A-1 122 was grown as second-passage culture for 24 hr at 37 C in 200 ml of Heart Infusion Broth (HIB) contained in a 500-ml Erlenmeyer flask on a gyrotory shaker at 200 oscillations per min. Viability in these cultures, as determined by 10-fold serial dilution in 1.0% peptone broth, consistently assayed at approximately 2 X 101 organisms per milliliter. Stirred settling chamber. Description and procedure for the operation of the apparatus have been given (2). In lieu of the original cubical chamber, however, the unit used here was a cylinder measuring approximately 1.0 meter in diameter and 1.5 meters in height, with a contained volume of 1,200 liters. It was equipped with glove and viewing ports but did not have a temperature-regulatory mechanism. A motordriven fan, mounted on the bottom, provided stirred settling conditions within the tank. For creating and maintaining an atmosphere of a particular RH, water-saturated air was mixed with dry air and introduced into the chamber. The RH was checked by means of a self-circulating Weksler-type hygrometer within the unit. Bacterial aerosols. Cultures were centrifuged at 3,020 X g at 4 C for 10 min, after which period the cells were resuspended in 50 ml of the supernatant fluid. Such suspensions generally contained between 1010 and 2.0 X 1010 cells per milliliter. The suspension was held at 4 C until use. For each experiment, 25 ml of the concentrated suspension was used in a Wellstype atomizer operated with dry air at 20 psi for 10 min, giving a total fluid output of 2.3 ml. Aerosol sampling. At set intervals, samples were collected in glass impinger samplers, AGI-30 (7), containing 20 ml of HIB supplemented with 0.1% Dow Corning antifoam B. Sampling time was 1 min at a flow rate of 12.5 liters per min. The number of viable cells was determined by preparing 10-fold serial dilutions in 1% peptone (or other media; see Results) and plating 0.1 ml of the appropriate dilutions on Difco BloodAgar Base medium. In some instances,wherelow numbers of viable cells were expected, 0.25or 0.5-ml quantities were used for plating. Inoculated plates were incubated for 48 hr at 37 C.