possible overestimation of surface disinfection efﬁciency ... · the method utilized to assess...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2011, p. 6208–6214 Vol. 77, No. 170099-2240/11/$12.00 doi:10.1128/AEM.00649-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Possible Overestimation of Surface Disinfection Efficiency byAssessment Methods Based on Liquid Sampling

Procedures as Demonstrated by In SituQuantification of Spore Viability�

I. Grand,1,2,3 M.-N. Bellon-Fontaine,1,2 J.-M. Herry,1,2 D. Hilaire,3 F.-X. Moriconi,3 and M. Naïtali1,2*AgroParisTech, UMR Micalis, Equipe B2HM, 91300 Massy, France1; INRA, UMR 1319 Micalis, Equipe B2HM,

78350 Jouy en Josas, France2; and DGA, Maîtrise NRBC, 91710 Vert Le Petit, France3

Received 22 March 2011/Accepted 27 June 2011

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often basedon counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldomachieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells arerepresentative of the levels of survivors in whole populations. The present study was thus designed to determinethe “damaged/undamaged” status induced by a peracetic acid disinfection for Bacillus atrophaeus sporesdeposited on glass coupons directly on this substrate and to compare it to the status of spores collected inliquid by a sampling procedure. The method utilized to assess the viability of both surface-associated andliquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyesand quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principalresult of the study was that the viability of spores sampled in the liquid was found to be poorer than that ofsurface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% � 5% of viablecells were detected among liquid-sampled cells compared to 79% � 5% or 47% � 4%, respectively, when theviability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of thesurvivors 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 canthus overestimate the efficiency of surface disinfection procedures.

The contamination by bacterial spores of liquids and sur-faces is a major source of problems in numerous settings (e.g.,the food industry or hospitals) and also in the context of po-tential bioterrorism attacks. A variety of chemical and physicaldecontamination methods are available. Whichever procedureis used, it is a critical point to determine the level of decon-tamination of the treated object, particularly when the contam-ination of inert surfaces is considered. Indeed, most of thestandard test methods used to assess the efficiency of a disin-fectant applied to surfaces (1, 3, 7) are based on the countingof microbial survivors sampled in a liquid (19). Studies havebeen performed to optimize sampling efficiency as a functionof the type of material and the mode of contamination (6, 11,19, 22), and Sagripanti and Bonifacino (22) developed a three-step method that, according to the authors, allowed to ob-tained near 100% of sampling efficiency for some materials.However, most of the time, total recovery is hard to beachieved and typical sampling efficiencies range from 20 to90% for bacterial spores (11, 13, 19). Efficiencies inferior to10% have sometimes been obtained for porous substrates (21).One might therefore wonder whether evaluations of microbialsurvivor numbers in liquid-sampled cells are representative of

the levels of survivors in whole populations or otherwise if the“damaged/undamaged” status of cells remaining on solid sub-strate after the sampling procedure is the same as that ofliquid-sampled cells.

Fluorescent dyes associated with microscopic observationsenable the assessment of cell viability, even for surface-associ-ated cells. Such methods were recently utilized to provideinformation on the penetration of antimicrobial agents intobiofilms (5, 10). It is difficult to label bacterial spores becauseof the impermeability of their envelopes (18). Nevertheless,assessments of spore viability with fluorescent dyes have beenperformed successfully (17). The utilization of selected dyeshas enabled the detection of heterogeneity in the response ofbacterial endospores to injuries (9) and investigation of themechanisms of spore killing by various treatments (18, 27).

In this context, the present study was thus designed to de-termine the “damaged/undamaged” status induced by a per-acetic acid (PAA) disinfection for Bacillus atrophaeus spores(selected as a model [3]) deposited on glass using a methodthat did not include a liquid sampling step and to compare thisstatus to that of cells removed in liquid by the sampling pro-cedure. We thus developed a microscopic method to quantifythe spore viability in situ on material. The same method wasalso applied to spore suspensions. A schematic representationof the experiment is given in Fig. 1. After selection of suitablefluorescent dyes, calibration curves between viable (i.e., thatcan initiate germination and recover a metabolic activity) andcultivable (i.e., that can germinate and multiply) spores were

* Corresponding author. Mailing address: AgroParisTech, INRA,UMR Micalis, Bioadhesion, Biofilm et Hygiene des Materiaux, 25Avenue de la Republique, 91300 Massy, France. Phone: (33) 1 69 53 6473. Fax: (33) 1 69 93 51 44. E-mail: [email protected].

� Published ahead of print on 8 July 2011.

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established for both liquid-suspended and surface-associatedcells. The percentage of viable spores was then compared tothe percentage of cultivable spores for the cells sampled inliquid after PAA disinfection. The percentage of viability ofliquid-sampled spores was also compared to that of spores onsolid substrates.

MATERIALS AND METHODS

Strain, preparation of spore stocks and contamination of inert surfaces.Three independent stock of spores of B. atrophaeus CIP 7718 (Institut Pasteur,France) were prepared as previously described (13). Briefly, after growth on asporulation medium, the spores were collected by scraping the surface of the agarand then washed four times in 0.15 mol of NaCl liter�1. A thermal treatment (30min, 90°C) was performed on an aliquot of each suspension in order to killvegetative cells and to determine the percentage of spores. When it was �95%,the suspension (at a concentration of between 2 � 109 and 1 � 1010 cultivablespores per ml, depending on the stock) was stored at 4°C.

Glass (Erie Scientific) was used as the solid substrate. Glass coupons (14 by 30mm) were autoclaved (20 min, 121°C), naturally dried under aseptic conditions(i.e., within a class II biological safety cabinet), and stored in the dark beforeutilization. They were inoculated by spores deposited as small droplets (5 drop-lets of 10 �l of stock suspensions, leading to a inoculation level that was between1 � 108 and 5 � 108 spores per coupon) and then naturally dried for 90 min, justbefore disinfection assays.

Disinfection assays on contaminated surfaces. Peracetic acid (PAA; 32% indilute acid acetic; Sigma, Germany), an oxidative agent, was used as the disin-fectant. Its efficacy against spores was evaluated according to adaptations of theEuropean standard EN 14561 (7). Briefly, the inoculated solid substrates wereimmersed in 10 ml of 0.1% PAA solution in 0.15 mol of NaCl liter�1 at 20°C forincreasing contact times (from 30 s to 5 min). The action of the biocide wasstopped by transfer of the disinfected substrate into a glass test tube (diameter,15 mm) containing 10 ml of a neutralization solution (3 g of L-�-phosphatidyl-choline, 30 g of Tween 80, 5 g of sodium thiosulfate, 1 g of L-histidine, and 30 gof saponin/liter), after which the survivors were evaluated by plate counts and byusing a microscopic method.

Plate counts of cultivable spores. After disinfection, for plate counts, sporeswere first removed from solid substrates and collected into the 10 ml of neutral-ization solution by vortex-shaking (30 s) with 5-g glass beads (diameter, 2 mm).Cultivable cells among liquid-sampled cells (Fig. 1) were then evaluated by the

drop counting method (8), during which six 10-�l drops of 1/10 serial dilutions in0.15 mol of NaCl liter�1 of neutralized suspensions were placed on the surfacesof Trypticase soy agar plates (bioMerieux, France). The two more concentratedsuspensions were also directly plated (two times, using 1 ml each time) to lowerthe limit of detection, which was equal to 5 CFU per solid substrate. The plateswere incubated at 30°C for up to 72 h. The results were mean numbers ofsurvivors obtained during three or four experiments performed on independentstocks.

Microscopic method for viable spores. The microscopic method was appliedafter the sampling procedure on cells found in the neutralization solution (liquid-sampled cells [Fig. 1]). The sampling procedure was performed as for the platecount, except that the spores from five glass coupons were successively detachedin the same neutralization solution to obtain sufficiently concentrated suspen-sions for microscopic observation. The microscopic method was also applied tosolid substrates directly, without the sampling procedure (direct in situ cells [Fig.1]) and also after the sampling procedure on cells remaining on the solid sub-strate (nonremoved cells [Fig. 1]). The microscopic method included the fluo-rescent labeling of spores, image acquisition, and image analyses.

Fluorescent labeling of spores. Spores were labeled with Syto 61 (Invitrogen,France) at a final concentration of 5 � 10�6 mol liter�1 and Chemchrome V6(AES-Chemunex, France) at a final dilution of 1/100 from a commercial stocksolution (catalog no. 200-R1007-03). Syto 61, a DNA-binding molecule, enabledthe red staining of the whole population of cells. The Chemchrome V6, anesterase activity marker, green labeled the viable cells.

To label surface-associated spores, the substrates were transferred from theneutralization solution to Trypticase soy broth (TSB; bioMerieux) and incubated(40 min, 37°C) in order to initiate the germination of spores. The TSB was thencarefully removed, and the substrate was covered with 250 �l of a labeling mixcontaining Syto 61 and Chemchrome V6. After a period of incubation to activateChemchrome V6 (40 min, 37°C), the substrates were placed on glass coverslipsfor microscopic observations. For liquid-sampled cells, the steps were identical.Suspension aliquots (100 �l) were diluted in TSB (100 �l), incubated (40 min,37°C), labeled (20 �l of the labeling mix), and then incubated again (40 min,37°C). Labeled suspensions were deposited between two glass coverslips formicroscopic observations.

Image acquisition by confocal laser scanning microscopy (CLSM). Micro-scopic observations were performed at the MIMA2 microscopy platform using aLeica SPX2 AOBS CLSM (Leica Microsystems, France) with a �63 waterimmersion objective lens (HCX Apo, numerical aperture 1.2). The scan speedwas 400 Hz. Fluorophore excitation was performed with a 488-nm argon laserand a 633-nm helium laser. Emitted fluorescence was recorded within the ranges

FIG. 1. Schematic representation of the experiments described in the present study.

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of 500 to 550 nm and 640 to 660 nm to visualize Chemchrome V6 and Syto 61fluorescence, respectively. Ten Z-stacks (1-�m step) were acquired for eachcoupon of solid substrate for surface-associated spores, and 10 images wereacquired for each sample of spores in liquid.

Images analyses. For surface-associated spores, three-dimensional projections(Easy 3-D) of deposited structures were reconstructed using IMARIS software(Bitplane, Switzerland). A quantitative structural parameter for biovolume wascalculated using PHLIP (26), a freely available Matlab-based image analysis tool.Biovolume is defined as the number of foreground pixels in an image stackmultiplied by the voxel volume (e.g., the product of the squared pixel size and thescanning step size) (16). The biovolume (�m3) was calculated for each fluores-cence channel. The “green biovolume/red biovolume” ratio was determined foreach Z-stack, and the percentage of viable spores for a given glass coupon wasthe mean of the ratios found for the 10 Z-stacks acquired for this coupon. Undereach experimental condition, mean values were then calculated from the analysesof between two and five coupons from independent studies.

For cells in suspensions, the percentage of viable spores in each sample wasdetermined by the independent counting of green- and red-labeled cells summedfrom the 10 acquired images. Mean values were then calculated from betweenthree and five independent experiments.

Calibration curves between the percentages of cultivable and viable spores.Mixtures of spores containing a known percentage of cultivable cells were ob-tained by vortex-shaking stock suspensions and dead spore suspensions (vol/vol).The stock suspensions were considered to contain spores that were all cultivable.Dead spore suspensions were obtained by the application of PAA (0.2%, 20 min)which enabled a �4-fold logarithmic reduction in the spore population (evalu-ated by plating). They thus contained �0.01% of cultivable spores. The mixtureswere deposited on glass coupons, or not, before labeling and analysis to obtaincalibration curves between the percentage of cultivable (noted N) and viable(noted V) spores for deposited and liquid-suspended spores, respectively.

Statistical analysis. Analyses of variance (ANOVA) were performed usingStatgraphics software (Manugistic). The P values tested the statistical signifi-cance of each of the factors through F tests. When P values were �0.05, thesefactors had a statistically significant effect at a 95% confidence level.

RESULTS

Combination of Syto 61 and Chemchrome V6 dyes enabledquantification of the viability of liquid-suspended and surface-associated spores after a PAA disinfection procedure. Syto 61and Chemchrome V6 dyes were tested in pairs to allow thenumbering of all Bacillus atrophaeus spores on the one handand viable spores on the other hand. In contrast to Syto 61labeling, Chemchrome V6 labeling was only achieved whenspores were incubated in TSB to allow germination (data notshown). As described in Materials and Methods, we selected atime of 2 � 40 min to first initiate germination and thenactivate the dyes, without spore outgrowth. Under these con-ditions, �95% of the spores detected by transmission micros-copy were found to be red stained (data not shown), and73% � 6% of the spores displayed green fluorescence in non-disinfected stock suspensions (Fig. 2). In contrast to liquid-suspended spores, 92% � 6% of nondisinfected depositedspores were detected as being viable (Fig. 2).

Calibration curves between cultivable and viable spores wereestablished not only for liquid-suspended spores but also forspores deposited and dried on glass, using mixtures of PAAdisinfected and nondisinfected spore suspensions. Spore repar-tition on the material was found to be heterogeneous. Thespores were aggregated in clusters, probably because of thedrying step. The height of the clusters varied, with a mean of 20to 30 �m. As illustrated in Fig. 2, the calibration curves differedfor liquid-suspended and deposited cells (P � 0.05), mainlybecause of the difference in the Chemchrom V6 labeling ofuntreated cells. The following relationships were obtained bylinear regression (Microsoft Excel):

N � �1.47 � V � 3.78, R2 � 0.990 (1)

N � �1.12 � V � 0.02, R2 � 0.996 (2)

for liquid-suspended cells (equation 1) and deposited cells(equation 2), respectively, where N is the percentage of culti-vable spores, and V is the percentage of viable spores.

In both cases, a good linear relationship (R2 � 0.990) wasobserved between the experimental percentages of viablespores obtained by labeling and the theoretical percentages ofcultivable spores expected for the range of mixtures tested,taking account of the fact that 27 and 8% of the cells were notstained green in nondisinfected mixtures of liquid-suspendedand deposited cells, respectively. In mixtures containing�0.01% of the cultivable cells, esterase activity was still ob-served (3 � 3 and 2% � 2% for liquid-suspended and depos-ited spores, respectively).

A good correlation was found between liquid-sampled sur-vivors evaluated by classical plate counts or by using themicroscopic method. The inactivation of deposited spores wasperformed by exposure to 0.1% PAA for increasing contacttimes. After neutralization, the cells were sampled in the liquidas described in Materials and Methods. The sampling effi-ciency (number of liquid-sampled cells/number of depositedcells, both evaluated by plating) calculated on blanks (materi-als immersed for 5 min in NaCl at 0.15 mol liter�1 instead ofPAA) was found to equal 70% � 18%, which is in agreementwith the literature (21). The “damaged/undamaged” status ofliquid-sampled spores was evaluated by both plating (Nl) andthe microscopic method (Vl).

The results (Table 1) showed an increase in the effectivenessof the disinfectant that was correlated with the gradual in-crease in contact time, whichever technique was used to eval-uate the survivors. Applying the calibration curve previouslydetermined (equation 1) to the data on viable spores (Vl)enabled calculation of the percentage of cultivable cells usingthe microscopic method (Ncl). A good linear correlation (R2 0.930) was observed between the assessment of survivors inliquid-sampled cells by the microscopic method (Ncl) and the

FIG. 2. Calibration curves between the percentages of viablespores and cultivable spores for liquid-suspended (f) and glass-depos-ited (F) cells.

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standard plate counting technique (Nl) (Table 1 and Fig. 3).ANOVA performed on the results of Nl and Ncl did not dem-onstrate any statistical difference between the two evaluations(P � 0.05).

After disinfection, viability differed for spores observed di-rectly in situ on glass, remaining on glass after a samplingprocedure, and found in the sampling liquid. In addition toprevious experiments in which the evaluation of microbial sur-vivors was carried out on liquid-sampled spores and under thesame disinfection conditions (0.1% PAA for increasing contacttimes), the microscopic method was applied to solid substratesafter or without the liquid sampling procedure. This made itpossible to determine the viability of nonremoved and direct insitu spores, respectively. As found previously, the spores ag-gregated into clusters that were not found to be different inshape and height for both nonremoved and direct in situspores. The contact time with PAA had an impact on thepercentage of viable spores with both types of glass-associatedcells: the percentage of viable cells declined more over time fordirect in situ spores (Vd) than for nonremoved spores (Vn)(Table 2) (P � 0.05). The difference in labeling between direct

in situ and nonremoved spores is illustrated in Fig. 4 for adisinfection time of 2 min.

The viability of surface-associated spores was also comparedto that of spores sampled in the liquid. This could not be doneby directly considering the experimental values for the viabilityof liquid-sampled spores (Vl) because, in a given population,the fluorescent response differed for spores analyzed as liquid-suspended or deposited on glass (Fig. 2). However, using thecorrelation curves previously determined, we were able to cal-culate an equivalent viability for liquid-sampled spores (Vcel,Table 2) corresponding to the response that they would havedisplayed if they had been analyzed as deposited rather thanliquid-suspended spores. Three homogeneous groups were ob-tained by ANOVA performed on the data for Vn, Vd, and Vcel

(P � 0.05). After disinfection, surface-associated spores werefound to be more active than liquid-sampled spores.

Finally, for each duration of disinfection, the values of Vd

(Table 2), i.e., the percentages of viable cells obtained exper-imentally for direct in situ spores, were compared to the valueof Vcd (Table 2), i.e., the percentages calculated from theviability of both nonremoved and liquid-sampled spores usingthe sampling efficiency previously determined (i.e., 70%, asreferred to above). The linear correlation between Vd and Vcd

is illustrated in Fig. 5 (R2 0.813).

DISCUSSION

The goal of the present study was to assess the “damaged/undamaged” status of spores deposited on a solid substrateafter PAA disinfection using a method that avoided a liquidsampling step and to compare this status with that of cellsfound in liquid after the sampling procedure. To achieve this,we utilized a microscopic method that includes the fluorescentlabeling of spores and quantification by image analyses. Wefirst selected dyes that could assess spore viability after PAAdisinfection. They were selected as a function of the mecha-nisms of action of PAA. No drop in optical density values (4)was observed for PAA-killed spores placed under germinationconditions, unlike nondisinfected spores (data not shown), sug-

TABLE 1. Comparison of survivors among liquid-sampled sporesevaluated by plate counts and by the microscopic method, after the

application of 0.1% PAA to glass-deposited spores

PAA contacttime (min)

Mean % � SDa

Nl Vl Ncl

0 100b 77 � 8 109 � 110.5 101 � 25 61 � 14 86 � 201 59 � 32 33 � 7 45 � 112 5 � 5 15 � 4 18 � 65 0.3 � 0.2 2 � 2 -1 � 3

a Values are expressed as follows: Nl, percent cultivable spores; Vl, percentviable spores; and Ncl, calculated percentage of cultivable spores. Ncl was calcu-lated from Vl, the percentage of viable spores determined experimentally, andusing the calibration curve previously determined, i.e., see equation 1 in the text:Ncl (1.47 � Vl) � 3.78.

b Nondisinfected suspensions were considered to be 100% cultivable.

FIG. 3. Correlation between the assessment of surviving liquid-sampled spores by a plate count and those calculated from the micro-scopic method using the calibration curve.

TABLE 2. Survivors among surface-associated spores evaluated bythe microscopic method, after the application of 0.1% PAA to glass-

deposited spores, and compared to liquid-sampled spores

PAA Contacttime (min)

Mean % � SDa

Vd Vn Vcel Vcdb

0 94 � 6 98 � 31 98 � 10 97 � 120.5 69 � 26 83 � 13 76 � 18 78 � 131 71 � 15 80 � 13 40 � 10 52 � 82 47 � 4 79 � 5 17 � 5 34 � 45 19 � 5 60 � 14 –1 � 3 17 � 5

a Values are expressed as follows: Vd, percent viable direct in situ spores; Vn,percent viable nonremoved spores; Vcel, calculated equivalent percentage ofviable liquid-sampled spores; and Vcd, calculated percentage of viable in situspores. Vcel is the equivalent viability of liquid-sampled spores, i.e., their percentviability if they were analyzed as deposited spores rather than liquid-suspendedspores. Vcel (Ncl � 0.02)/1.12 (see equation 2 in the text), with Ncl as thecalculated percentage of cultivable liquid-sampled spores: Ncl (1.47 � Vl) �3.78 (see equation 1 in the text).

b Calculated from the percentage of viable liquid-sampled and nonremovedspores, taking into account a sampling efficiency of 70%. Vcd (0.7 � Vcel) �(0.3 � Vn).



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gesting that PAA-killed spores did not enter the early phase ofgermination. It was thus necessary to select a dye that labeledspores that were unable to initiate germination in order tostain the whole population of spores. DNA-binding dyes areoften used to label whole populations of vegetative cells, butthe literature contains contrasting observations concerning thelabeling of dormant spores with such dyes. The impermeabilityof the envelopes of dormant spores, especially the inner mem-brane, is supposed to limit the penetration of dyes into the coreand their binding to nucleic acids (18). According to Cronin

and Wilkinson (9), dormant endospores were found to bemoderately permeable to Syto 9, and an increase in fluores-cence was observed during outgrowth and germination. In con-trast, Laflamme et al. (17) obtained significant labeling withDAPI (4�,6�-diamidino-2-phenylindole) and Syto 9 dyes, in thesame way as we managed with Syto 61. This dye was thus foundto be a good marker for the whole population of spores.

Because of the limited enzymatic activity of spores (23),Chemchrom V6 did not label dormant spores. It was, however,found to be efficient in labeling viable—but not PAA-dam-aged—spores, insofar as a germination step was added. Fluo-rogenic esterase substrates measure both enzyme activity(which is required to activate probe fluorescence) and cellmembrane integrity (necessary for the intracellular retentionof their fluorescent products) (14). Esterase activity is widelyused to assess the viability of vegetative cells. It can also beused as a practical parameter to quantify spore germinationsince it is an early event that occurs close to or during stage IIof germination when spore hydration and cortex hydrolysisstart and silent enzymes are activated (12). The loss of observ-able esterase activity in PAA-treated spores is most likely in-dicative of germination obstruction before the enzyme activa-tion stage, as referred to above.

After labeling, the second important step was to quantify thepercentage of spore viability. It was performed on both liquid-suspended and deposited spores. We were able here, for thefirst time to our knowledge, to demonstrate the possibility ofquantifying the viability of spores within multilayered clusters.An in situ visualization of disinfection treatment on three-dimensional microbiological structures has recently beenachieved, but the studies were performed on vegetative cells

FIG. 4. Three-dimensional projections of CLSM observations of B. atrophaeus spores subjected to PAA disinfection (2 min, 0.1% PAA).(A) Nonremoved spores. (B) Direct in situ spores. (A1 and B1) Syto 61-labeled spores. (A2 and B2) Chemchrom V6-labeled spores. (A3 and B3)Overlays of both staining techniques. In these images, the percentages of active esterase cells are 77 and 45% for nonremoved and direct in situspores, respectively.

FIG. 5. Correlation between the assessment of viable direct in situspores determined experimentally and the assessment calculated fromthe data of viability of liquid-sampled and nonremoved spores.



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organized in a biofilm (10, 25). In the case of Bacillus spores,DNA-binding molecules and esterase activity markers wereformerly used to analyze their responses to inactivation treat-ments using flow cytometry (9), so that the spores were ana-lyzed as liquid-suspended cells. CLSM observations have alsobeen made of membrane-filtrate spores labeled by DNA-bind-ing molecules (17), in which case quantification was performedby the counting of individual spores and not on large deposits,as was performed during our study.

Several observations validate the quantification, includingthe linearity of the calibration curves between cultivable andviable spores, not only liquid-suspended but also deposited(Fig. 2). Calibration curves were obtained by analyses of mix-tures of nondisinfected and PAA-disinfected spore suspen-sions. Nearly 3% of spores were still stained green in thePAA-disinfected suspension, when less than 0.01% was foundto be cultivable. The capacity to initiate germination, esteraseactivity, and membrane integrity was thus still present in somedisinfected spores incapable of division under the experimentalconditions. It is widely acknowledged that conventional culturetechniques are highly selective and underestimate the real vi-able cell count, which accounts for both cultivable and viablebut noncultivable cells (15, 20). Furthermore, not all (73% �6%) of the red-stained spores were green stained among non-disinfected spore suspensions. That indicates the presence ofnonviable spores in these suspensions, a situation that thecultivability cannot reveal. The higher percentage of viabilityobserved for nondisinfected spores on materials (92% � 6%)may have resulted from an overestimation of green stainingcompared to red staining in deposited clusters. In that context,calibration curves were indispensable in order to have correctand comparable data. The use of these calibration curves al-lowed us to obtain other results that also validated the quan-tification: we showed that the conventional plating method andmicroscopic method produced similar results regarding PAAefficiency in cells sampled in liquid (Table 1 and Fig. 3); therewas a good correlation between the survivors determined byexperimentations directly on a solid substrate and those calcu-lated from data of liquid-sampled and nonremoved survivors(Table 2 and Fig. 5).

Since the microscopic method provided quantitative infor-mation on the viability of both surface-associated and liquid-suspended spores, it was possible to demonstrate that the per-centage of viable cells sampled in the liquid was smaller thanthe percentage of viable cells that were still surface associatedafter the sampling procedure and also smaller than the percentviability of surface-associated cells that were not subjected toliquid sampling. There are few data in the literature concern-ing such comparative evaluations. Allion (2) observed thatafter a disinfection procedure, the number of cultivable cells ofPseudomonas, Staphylococcus, and Listeria evaluated by theplating of liquid-sampled cells was much lower than the num-ber of viable cells observed directly on a solid substrate byfluorescent labeling. Mention should also be made of the re-sults obtained by Sagripanti and Bonifacino (22), who showedthat Bacillus subtilis spores dislodged from different types ofmaterials by sonication appeared to be more resistant to dis-infection than those that were loosely attached. These authorsconcluded that disinfection efficiency was overestimated whena one-step sampling method was used, as we also concluded

here. The existing data suggest that dead spores are easier toremove from surfaces, as previously observed (M.-N. Bellon-Fontaine, unpublished data). The easier release of dead cellsmay be due to the abolition of active interactions between cellsand inert surfaces, a hypothesis that is more applicable tovegetative cells than to dormant spores. One can also supposethat, in the case of disinfection, dead spores may be preferen-tially localized on the outside of deposits and thus be easier toremove from the surface.

In conclusion, the evaluation of survivors only among cellssampled in liquid overestimated the efficiency of surface dis-infection procedures in our experimental conditions, highlight-ing the limitations of assessment methods based on a liquidsampling procedure. Nevertheless, it should be noted that themicroscopic method cannot be used alone to determine theefficiency of a disinfectant: it cannot estimate more than 2 to 3logarithmic reductions, and it evaluates viability and not culti-vability, which is also important to determine. The microscopicmethod can be used to test disinfection results obtained usingstandard assessments on liquid-sampled cells. For example, wepreviously showed by such a standard assessment that the na-ture of the solid substrate has an influence on disinfectionefficiency (13). We arrived at the same conclusion by applyingthe microscopic method on both glass and painted stainlesssteel (data not shown). A combination of several complemen-tary techniques thus appears suitable to assess results of dis-infection. An additional interesting possibility that is to beconsidered is to evaluate survivors on the substrate withoutliquid sampling, through direct culture in the liquid medium.This test will produce an “all or nothing” (growth or non-growth) answer, but quantitative values can be obtained bymultiplying the assays and using the fraction-negative ap-proach of the Holcomb-Spearman-Karber procedure (24).

ACKNOWLEDGMENTS

This study was supported by doctoral grants from the French Min-istry of Defense (DGA/MRIS) and the French National ScientificResearch Centre (CNRS).

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