Development of an Aerosol Surface Inoculation Method for Bacillus Spores

Abstract

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10 7 CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.In response to the 2001 Bacillus anthracis spore incidents in the United States, many studies have investigated various technologies to sample media, detect biological agents, and decontaminate materials (3,9,10,(17)(18)(19)21). These laboratory studies typically use small coupons and laboratory inoculants to simulate the materials and biological agents that occur in field events. In these studies, test coupons should have reproducible numbers of viable spores on varied surface types. In addition, it is critical that the coupons are prepared so that the inoculation is representative of contamination as it occurs in the field. Liquid inoculation protocols that use suspended spores in aqueous buffers have been the primary methods used to prepare test surfaces for biological agent decontamination and detection studies (15,16,20). Liquid inoculation methods offer advantages in that they allow relatively easy control of the number of spores and the contaminated area. However, it is unclear whether surfaces contaminated by liquid inoculation methods are representative of surfaces contaminated with aerosolized Bacillus anthracis spores. Therefore, it is necessary to investigate the impact of various spore deposition methods on Bacillus anthracis decontamination and detection studies.The conventional particle deposition method via aerosolization is comprised of an aerosol generating system, such as a particle nebulizer, to introduce the spores into a chamber and a second chamber to allow the spores to settle onto the target material surfaces. The spore surface concentrations are controlled by varying settling time and the initial aerosolized spore concentration in the chamber. tested various methods of collecting ...

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies

“…In recent years, various methods have been developed in order to generate reproducible spore monolayers (42)(43)(44). However, the lack of detail concerning the individual instruments installed in the particular setup or the precise operating principle (42,43), as well as elaborate and costly technology (44), makes it difficult to recreate the reported spraying devices for sample preparation.…”

“…However, the lack of detail concerning the individual instruments installed in the particular setup or the precise operating principle (42,43), as well as elaborate and costly technology (44), makes it difficult to recreate the reported spraying devices for sample preparation. In this study, we present a method for easy sample preparation by spray deposition, which was successfully employed for the production of homogeneous B. subtilis spore monolayers.…”

Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores

“…Accordingly, the current study utilized an aerosol-based inoculation method, as to more accurately represent previous bioterror surface contamination. The current MDI-based method was previously shown to generate particles in which the majority (~85%) contained one or two spores (Lee et al, 2011, Carrera et al, 2005). …”

“…These spores were prepared specifically for use as a B. anthracis surrogate during surface sampling studies (Brown et al, 2007a). Coupon surfaces were inoculated via metered dose inhaler (MDI) according to the aerosol deposition-based inoculation methods described previously (Calfee et al, 2013; Lee et al, 2011), yielding ~10 5 spores cm −2 over the 929 cm 2 coupon surface. A single MDI was utilized across the entire trial for each material type.…”

Effect of inoculation method on the determination of decontamination efficacy against Bacillus spores

“…The Bacillus subtilis spores (ATCC 19659, Manassas VA) were prepared as previously reported by Rastogi et al (2009), Rogers et al (2005) and further described in Lee et al (2011). Briefly, a seed culture was initiated in tryptic soy broth (TSB) and then sporulated on sporulation media at 37 °C for 10 ± 4 days.…”