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

Abstract

f Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 ؋ 10 7 spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques. Microbial contamination on surfaces is a recurring problem within health, pharmaceutical, and food industry sectors (1, 2, 3). Thus, decontamination is a crucial step to ensure the sterility of food processing equipment, minimize spread of pathogens, and prevent the transmission of nosocomial infections (4). Common decontamination and disinfection procedures that are widely used for microbial inactivation include high temperatures, chemicals, or ionizing radiation (reviewed in reference 5). In order to ensure the efficiency and to validate the continuous functionality of a disinfection or sterilization procedure, biological testing standards are required. Bacterial spores are frequently used as a biological indicator (BI) of sterility, primarily because bacterial spores exhibit elevated resistance to chemical and physical methods of sterilization (6-11). Hence, a process that achieves full spore inactivation ensures complete elimination of other contaminating microorganisms.Variations in the performance of a BI have been reported repeatedly (12, 13). Besides variations in the intrinsic resistance properties of the microorganisms conferred, for instance, by variations in genetic traits or alteration of sporulation conditions (14), extrinsic factors also may affect the performance of BIs and, subsequently, the accurate determination of spore resistance and inactivation. For example, the sterilization results may be altered by poor choices of the carrier material for spore deposition (13,15) and, in particular, the BI manufacturing procedure (16). The method by which spores are mounted on carriers also is extremely impo...

The cluster model of ultraviolet disinfection explains tailing kinetics

“…Increased photoprotection from ultraviolet rays can be conferred on cells due to aggregation (Coohill 1986;Mamane-Gravetz and Linden 2005;Raguse et al 2016;Gerba and Betancourt 2017). This protective effect can be explained in terms of shadowing at the microscopic level by Mie scattering effects in which ultraviolet radiation is scattered away locally at the surface of cells (Kowalski 2009).…”

“…Since then, many other micro‐organisms have been noted to aggregate and it has long been suspected that this tendency confers on them a degree of protection from biocidal factors. Clustering, also referred to as clumping, and, in water disinfection, as agglomeration or aggregation, provides increased resistance to all biocidal factors except ionizing radiation (Kowalski ; Raguse et al ). Clustering occurs when micro‐organisms aggregate as a result of hydrophobicity, ionic surface charge and/or physical surface features (Mamane‐Gravetz and Linden ; Raguse et al ; Gerba and Betancourt ).…”

“…Cerf (1977) cited clustering as a possible cause of tailing for general models of disinfection. Recently, several researchers have provided evidence and concluded more definitively that clustering causes tailing in ultraviolet survival curves (Blatchley et al 2005;Coohill and Sagripanti 2008;Raguse et al 2016).…”

From patent to product? 50 years of low‐pressure plasma sterilization

“…The drastic consequences of this effect were demonstrated by Raguse et al by comparing survival curves of 5.10 7 B. subtilis spores using spot and spray deposition in an ICP setup at 10 Pa in Ar (cf. Figure ) . Due to the stacking by standard spot deposition, the inactivation after 90 s is less than one log increment, whereas in the same time more than 99.99% of the sprayed spores are inactivated.…”

“…For example, the sample carriers should be investigated for survivors using scanning electron microscopy or DPA release of the spores during germination. Furthermore, the recovery can be improved by covering the sample carrier with a 10% aqueous polyvinyl alcohol (PVA) solution to pull the spores from the substrate together with the dried PVA . (A comment on possible errors during spore handling was recently given by Peter Setlow).…”