Possible Overestimation of Surface Disinfection Efficiency by Assessment Methods Based on Liquid Sampling Procedures as Demonstrated by In Situ Quantification of Spore Viability

Abstract

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ؎ 5% of viable cells were detected among liquid-sampled cells compared to 79% ؎ 5% or 47% ؎ 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.The contamination by bacterial spores of liquids and surfaces is a major source of problems in numerous settings (e.g., the food industry or hospitals) and also in the context of potential bioterrorism attacks.

Into the well—A close look at the complex structures of a microtiter biofilm and the crystal violet assay

“…Our general observation was that the heterogeneity of the biomass in the wells increased dramatically over time as each developed seemingly stochastically differently, depending on the microenvironment in the individual well. This heterogeneous growth may contribute to the deviations often found in this model as the local microenvironment in each well may influence growth, strength, and resilience [ [23] , [24] , [25] ].…”

Critical review on biofilm methods

“…First, only a fraction of the sessile community can be suspended by sonication, typically between 5% and 90% of the community (Edmonds et al, 2009;Muller et al, 2011). Second, the physiological properties of the detached population may not reflect the physiology of sessile cells, as different populations could exhibit different adhesive and detachment properties on the material (Grand et al, 2011). In both microtiter tray based assays, the pegs or wells can further be coated with different molecules to investigate or promote biofilm formation to different biotic and abiotic supports.…”

Persistence and Inactivation ofBurkholderia malleiChina 7 Deposited on Nonporous Laboratory Materials

“…It has been suggested that for instances in which the recovery of microorganisms from test materials is <100%, the disinfection efficacy could be overestimated. 11 In the present study, recovery of B mallei after 1 hour of drying ranged from 79% to 86% for all 3 test materials. Therefore, it is possible that a similar overestimation may occur when assessing persistence with an extraction-plating procedure.…”