Disinfectants during Clinical Use

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p Vol. 54, No /88/1158-$./ Copyright 1988, American Society for Microbiology A Suspension Method To Determine Reuse Life of Chemical Disinfectants during Clinical Use RICHARD A. ROBISON,'* HOWARD L. BODILY, DAENA F. ROBINSON,' AND RELLA P. CHRISTENSEN' Microbiology Section, Clinical Research Associates, Proovo, Utah 8464,1 and Department of Microbiology, Brigham Young University, Provo, Utah 846 Received 15 June 198/Accepted 8 September 198 In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for testing disinfectants, the Association of Official Analytical Chemists () use-dilution method, cannot be adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates () suspension method was developed to fill this need. It consists of adding.5 ml of a standard culture to 5. ml of test disinfectant and sampling the mixture after 1 min for surviving bacteria. When this test was compared with the use-dilution method under a simulated use situation, the two methods were generally equivalent in identifying disinfectant inactivation. In addition, the method was less time consuming, easier to perform, and less variable than the method. Use of the method in a clinical study demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing only. Reuse of chemical disinfectants for periods ranging from days to weeks has been a routine practice throughout the health care industry of the world. Many disinfectants, however, were never intended for prolonged, repeated use, and they lose their ability to inactivate microorganisms after a short time (5). Because clinicians have no way to assess the potency of a disinfectant, they unwittingly use solutions which may have little or no antimicrobial activity. This practice has important implications to the well being of large numbers of people who are treated daily in hospitals and outpatient medical and dental facilities, as well as the clinical personnel performing the treatment. In actual clinical use, chemical disinfectants are subjected to many conditions that challenge their antimicrobial stability. Some of these include dilution, age, and contamination with chemicals and organic matter (6, 9, 15). While many of the clinical challenges can be mimicked in laboratory testing, the true nature of many other potential inactivators can only be surmised. Dental office environments offer a particular problem owing to the broad range of possible contaminants contained in the large number of dental materials used routinely (i.e., resin polymers, glass filters, metal alloys, viscosity modifiers, etc.). These facts reinforce the need for reliable disinfectant efficacy testing. Many evaluation methods exist (4). Most of these, however, are designed to test only the initial activity of an unused product. Although new Environmental Protection Agency criteria now specify that documentation must be provided by vendors if reuse claims are made, an accepted, standardized protocol has not been specified. Test protocols used currently may not reflect clinical reality since stressing agents are limited to microorganisms and proteins (8, 11). A method is needed that will permit field testing of commercial disinfectants to establish reliable reuse recommendations and substantiate reuse claims of manufacturers (16). The only current standard test method, the Association of Official Analytical Chemists () use-dilution method, * Corresponding author. 158 cannot be used for this purpose because the volume of disinfectant required is too large to allow repeated testing over time without requiring an immense quantity of disinfectant to be dispensed initially. Such a large volume would give misleading results since the ratio of disinfectant to instruments would be unrealistic. The method is also qualitative, very time consuming, and difficult to standardize. Therefore, a quantitative suspension test (hereafter referred to as the Clinical Research Associates [] suspension method) was developed to circumvent these problems. In the development of such a method, an effort was made to adhere to the same culture-to-disinfectant ratio and exposure time used in the United States standards. The culture-to-disinfectant ratio used is identical with that of the phenol coefficient test, and the exposure time (1 min) is that specified in the use-dilution method. Other suspension-type tests such as the standard methods of France, Germany, The Netherlands, and the United Kingdom are methodologically more difficult. Some use washed bacterial suspensions, which have been shown to be less resistant than untreated broth cultures (13), or multiple exposure times which make interpretation of the results more complex (4, 13). The method was designed to be as simple as possible to perform and still give quantitative results. The purposes of this study were to compare results obtained with the suspension method with those of the use-dilution method to establish a point of reference between the two procedures and to use the suspension method in a clinical comparison study to determine its feasibility as a method for reuse testing. MATERIALS AND METHODS Media and chemicals. Tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) was used for the suspension method cultures. Tryptic soy agar (TSA) was made by the addition of 1.5% Bacto-Agar (Difco) to the TSB described above. Both the TSB and TSA used in the suspension method contained the neutralizers specified by

2 VOL. 54, 1988 REUSE LIFE OF CHIEMICAL DISINFECTANTS 159 the for letheen broth (). These were.% lecithin (Sigma Chemical Co., St. Louis, Mo.) and.5% Tween 8 (Fisher Scientific Co., Pittsburgh, Pa.). Nutrient broth used in the use-dilution method was prepared as specified by the (). It consisted of.5% beef extract (Difco),.5% NaCl (Mallinckrodt, Inc., St. Louis, Mo.), 1% peptone (Difco), and the same concentration of neutralizers as described above. Preparation of frozen stock cultures. Pseudomonas aeruginosa ATCC 1544, Salmonella cholerae-suis ATCC 18, and Staphylococcus aureus ATCC 6538, three standard strains used in disinfectant testing, were obtained directly from American Type Culture Collection (Rockville, Md.) for use in this study. Since S. choleraesuis and P. aeruginosa produce both smooth and rough colonies, mixtures of these colony forms were used to prepare TSB cultures which were grown at 3 C for 4 h without shaking. These cultures were mixed with equal volumes of sterile % glycerol (Sigma) and dispensed in 1.5-ml aliquots to sterile Nunc tubes (Vangard International, Neptune, N.J.). All tubes were stored in liquid nitrogen until used. Preparation of suspension method cultures. A thawed stock culture (1 ml) was added to 9 ml of TSB. This primary culture was incubated without shaking at 3 C for 3 h to generate log-phase cells. The secondary or test culture was prepared with a 1% inoculum from the primary culture and incubated for to 4 h at 3 C without shaking. Selection of disinfectants. The choice of which solutions to use in the methods comparison was based on what is currently being used in dentistry (5; Clinical Research Associates, Clin. Res. Assoc. Newsletter, 9:1, 1985). Environmental Protection Agency-registered disinfectants with various active ingredients were purchased for use in this study (see Table 1). Six solutions (acid glutaraldehyde, alkaline glutaraldehyde, glutaraldehyde-phenol, phenolic, and two solutions containing the same quaternary ammonium compound [quat]) were mixed and/or diluted by the directions of the manufacturer. Even though quats have lost the approval of the American Dental Association owing to their relatively low level of activity, at least 3% of practitioners in the United States continue to use quats because they are relatively nontoxic and inexpensive (5; Clinical Research Associates, Clin. Res. Assoc. Newsletter, 9:1, 1985). Therefore, some low-level disinfectants were included in this study. Disinfectant preparation, loading, and sampling for methods comparison. Care was taken to follow the directions of TABLE 1. the manufacturers in preparing these solutions. All glutaraldehyde-containing disinfectants were at a concentration of % before dilution. The acid glutaraldehyde was diluted 1:4, the alkaline glutaraldehyde was not diluted, and the glutaraldehyde-phenol was diluted 1:16. Only the alkaline glutaraldehyde and glutaraldehyde-phenol solutions required activation. The phenolic and quat preparations were diluted 1:3 and 1:64, respectively. The label on the quat suggested that distilled water be used as a diluent in areas of known water hardness. Therefore, to comply with label instructions and to provide a point of comparison with the other disinfectants, which were diluted with tap water, the same quat was included twice, once with tap water and once with deionized water as the diluents. A 3-liter batch of each solution was prepared according to the label instructions of each manufacturer. Table 1 shows the suggested reuse life, use-dilution, and final active ingredient concentration of each solution. All disinfectants were stored in covered plastic containers (Surgikos model 1) at room temperature (approximately C) for the duration of the study. The disinfectants were challenged by the addition of 1 ml of whole human blood at the end of each working day (5 days per week) for 4 weeks. Twice a week, in the morning, each disinfectant was sampled. This involved the removal of 1,8 ml for testing (3 organisms x 6 replicates x 1 ml per tube) and 45 ml for the method (3 organisms x 3 replicates x 5 ml per tube). use-dilution and suspension tests were performed on each sample. The disinfectant from the tubes was returned to the batch after testing (5); even so, approximately 15 ml of each disinfectant was lost each sampling day owing to the two assay procedures. This loss was taken into consideration when the percentage of blood in each disinfectant was computed for each sampling day. use-dilution method. The standard test employing organisms dried onto stainless-steel penicylinders was used (). Each disinfectant sample was tested against 6 carriers prepared from each of the three standard organisms listed above (18 total). The carriers were coated with suspensions prepared by daily transfers as specified by the. All transfers were performed in a C water bath. A secondary subculture was performed on each carrier (as recommended by the when disinfectant residual effects are suspected). Both subcultures were incubated at 3 C for 48 h. Growth in either tube was scored as a positive for that Reuse life,a use dilution, and active ingredient concentrations of the disinfecting solutions used in the methods comparison Disinfectant Sugested reuse Suggested Diluent Use concn of active ingredient(s) life (days) use dilution Acid glutaraldehyde 1 1:4 Tap water.5% Acid glutaraldehyde Alkaline glutaraldehyde 8 Undiluted.% Alkaline glutaraldehyde Glutaraldehyde-phenol 3 1:16 Tap water.44% Phenol.15% Sodium tetraborate.13% Glutaraldehyde.8% Sodium phenate Phenolic 1 1:3 Tap water.8% o-phenylphenol.3% o-benzyl-p-chlorophenol Quat-deionized water 1:64 Deionized water.% Isopropyl alcohol.1% Cetyldimethylethylammonium bromide.1% Benzalkonium chloride Quat-tap water 1:64 Tap water.% Isopropyl alcohol.1% Cetyldimethylethylammonium bromide.1% Benzalkonium chloride a Reuse life is defined as the length of time a disinfectant may be reused clinically, as opposed to shelf life, which is the stability of an unused solution.

3 16 ROBISON ET AL. carrier. Broth from all tubes showing growth was streaked on TSA to confirm the presence of the test organism and the absence of contamination. suspension method. A.5-ml sample of test culture was added to 5. ml of disinfectant in a C water bath. The suspension was mixed thoroughly and returned to the bath. After a 1-min exposure, a 1-ml sample was removed and diluted in TSB (with neutralizers). Samples of 1 ml from each dilution tube were assayed with duplicate pour plates in TSA (with neutralizers) poured at 45 C. The dilutions were extended as far as necessary to include the expected counts. The procedure was repeated three times for each standard organism, making a total of nine determinations on each disinfectant sample. A standard plate count employing serial 1:1 dilutions in TSB (with neutralizers) was performed on each test culture to establish a base-line concentration of viable organisms. All plates were incubated at 3 C for 48 h. Log reductions were calculated by the following formula: log reduction = log (number of organisms per milliliter before exposure) - log (number of organisms per milliliter after exposure). Disinfectant preparation, sampling, and testing for clinical comparison. Three commercially available glutaraldehydebased disinfectants (acid glutaraldehyde, diluted 1:4; alkaline glutaraldehyde, undiluted; and glutaraldehyde-phenol, diluted 1:16) were evaluated during actual clinical use. A latin-square study design was used. Three busy dental offices known to use liquid disinfectants instead of heat sterilization were sought as field-testing sites. Selection was based on the number of patients treated per day. The study was conducted in three 5-week periods. Each disinfectant was rotated randomly through the three offices, such that each agent was used in every office. A.5-liter batch of each solution was prepared according to the instructions of the manufacturer. A 5-ml sample was removed from each batch and placed in a sterile dark glass bottle which was kept in the laboratory at room temperature to serve as an unused control. The remaining,5 ml was dispensed to a clinical site for use. Each office used identical plastic containers with removable trays (Surgikos model 1). Twice a week, for 5 weeks, 3 ml was removed from each solution (and its unused control) for testing. The activity of each solution was assayed by the suspension method, using each of two separately prepared cultures of S. choleraesuis. With the means at our disposal, it was not possible to test each sample against all three standard organisms. Therefore, to provide replication, we decided to use one organism in duplicate. At the end of the study, the effectiveness of each solution at each time point was determined from the average of six log reduction values (two determinations at each of three sites). RESULTS Methods comparison. Figures 1 through compare the and methods in their ability to detect disinfectant inactivation over time. The activity of each disinfecting solution is represented by two plots. The upper graph demonstrates results obtained by the suspension method, and the lower graph shows results of the use-dilution method. The log reduction values ( method) and the number of negative tubes/6 ( method) were both plotted against time (and percentage of blood) to generate comparable curves. Each of the three APPL. ENVIRON. MICROBIOL. lines on a graph denotes a different test organism. A lower log reduction ( method) translates into a higher viable count and thus a lower level of disinfectant activity. Similarly, a lower number of negative tubes ( method) corresponds to an increase in the number of positive tubes and also represents a lower activity. A downward trend in any line represents a loss of antimicrobial activity for a particular disinfectant-method-organism combination. Figures 1 and demonstrate the antimicrobial activity of the acid glutaraldehyde (1:4) and alkaline glutaraldehyde (undiluted), respectively. Neither solution showed significant inactivation over time when subjected to the organic loading. Both the and methods predicted good disinfectant activity against all three organisms up through 4 days and showed that the two solutions were not affected substantially by the addition of blood (6.3%). The performance of the glutaraldehyde-phenol (1:16) is shown in Fig. 3. This preparation was affected noticeably by organic stress. Both the and methods indicated a sharp decline in effectiveness after the blood reached a concentration of about 3.%. However, some organismmethod differences were apparent. The method showed the day 1 solution to be effective against S. choleraesuis, whereas the method indicated that this same solution was almost completely ineffective against this organism. Conversely, the method indicated the day 1 solution to have some activity against S. aureus; however, the method showed little activity. > 8 FB 6 c.9 5 -g4 a: 3 -j n 6 5F (n Dg 4 I- *' 3 Z 1 I I I (.9) (1.5) (.5) (3.) (4.3) FIG. 1. Comparison of the and methods in monitoring the inactivation of a.5% acid glutaraldehyde solution during simulated clinical use. Three standard organisms were used. Each value is the mean of three separate determinations. Symbols:, S. aureus;, S. choleraesuis; A, P. aeruginosa.

4 VOL. 54, 1988 REUSE LIFE OF CHEMICAL DISINFECTANTS 161 C 6.Q 5 -S4 a: o 3 -j A o I I (.9) (1.5) (.5) (3.) (4.3) 1 4 FIG.. Comparison of the and methods in monitoring the inactivation of a % alkaline glutaraldehyde solution during simulated clinical use. Three standard organisms were used. Each value is the mean of three separate determinations. Symbols:, S. aureus;, S. choleraesuis; A, P. aeruginosa. Figure 4 shows the performance of the phenolic preparation. This solution lost activity against S. aureus only. The method showed an earlier inactivation which was more erratic than that depicted by the method; however, both identified the ineffectiveness of the solution against S. aureus after day 1 (4.3% blood). Adequate activity against the other two test organisms was detected equally by both methods. Figure 5 indicates the activity of the quat diluted in deionized water. This solution showed inactivation over time with respect to S. aureus and P. aeruginosa. Neither method indicated any substantial loss of activity against S. choleraesuis. The method depicted a more pronounced inactivation over time than the method, especially against S. aureus. The activity of the quat diluted with tap water is shown in Fig. 6. This preparation exhibited a pattern of effectiveness much different from that of the quat diluted with deionized water. The early lack of activity against P. aeruginosa was similarly detected by both the and methods; however, a steady decline in effectiveness against the other two organisms is more clearly represented by the data. A condensation of methods comparison data allows an easier interpretation of the results. When the data from each method were averaged across the three organisms used, eight values (one for each sample day) for each disinfectantmethod combination were obtained. A correlation analysis of this data produced a Pearson product moment of.91, indicating a strong agreement between the two methods. When the data were further collapsed across disinfectants, a single line for each method was produced (Fig. ). This overall comparison shows that disinfectant activity is predicted equivalently by both the and methods. Comparison of method variability. Table shows the coefficients of variation for each disinfectant-method combination. The coefficients were consistently smaller than those of the method. The coefficients for the acid and alkaline glutaraldehyde solutions could not be estimated reliably because of the small numbers of positive tubes obtained with these solutions. This did not prevent the assessment of the overall coefficient of variation for the method, which was almost six times greater than that of the method. Clinical comparison. Figure 8 shows how three glutaraldehyde-based disinfectants performed under actual clinical use, as monitored by the suspension method. The acid (1:4) and alkaline (undiluted) glutaraldchyde solutions remained effective for nearly the entire sampling period. The glutaraldehyde-phenol (1:16) preparation, however, steadily lost activity after the eighth day of use. The unused control solutions from all disinfectants showed full effectiveness throughout the 36-day sampling period. DISCUSSION The purpose of this study was not to suggest a replacement for the use-dilution method. Rather, it was to validate a procedure that could be used in cases that are (.9) (1.5) (.5) (3.) (4.3) 1 4 FIG. 3. Comparison of the and methods in monitoring the inactivation of a glutaraldehyde-phenol solution during simulated clinical use. Three standard organisms were used. Each value is the mean of three separate determinations. Symbols:, S. aureus; O, S. choleraesuis; A, P. aeruginosa.

5 16 ROBISON ET AL. c. C,)I ) CD 1_ I Q) z d (.9) (1.5) (.5) (3.) (4.3) FIG. 4. Comparison of the and methods in monitoring the inactivation of a phenolic solution during simulated clinical use. Three standard organisms were used. Each value is the mean of three separate determinations. Symbols:, S. aureus; O, S. choleraesuis; A, P. aeruginosa. inappropriate for the method, such as testing a disinfectant over time as it undergoes clinical use. This validation was accomplished by comparing the log reduction curve from the suspension method with the plot of negative tubes from the use-dilution method. The ideal disinfectant for comparing these two methods is one which exhibits a linear deterioration over time. The solution that most nearly fit this pattern was the glutaraldehyde-phenol (Fig. 3). This disinfectant had a high initial activity, lost activity through the course of the test period, and exhibited low activity at the end of the study. The shapes of the and curves for this solution are very similar, indicating a general agreement between the two methods. A closer inspection of these curves, however, reveals an inversion of the S. aureus and S. choleraesuis lines. This is presumed to be due to a fundamental difference between the methods. The method requires drying of organisms; the method does not. Our experience with environmental surface disinfection procedures, in which dried test organisms are used, has confirmed the well-established fact that most gram-positive organisms survive desiccation better than gram-negative organisms. The number of viable S. aureus organisms is almost 1, times greater than that of S. choleraesuis when these two organisms are dried under identical conditions (unpublished data, Clinical Research Associates). This disparity could be responsible for most of the organism-related differences observed between the two methods. Since these microorganism differences were method dependent it was necessary to average the data across organisms to compare the two methods more accurately. When this was done, the and methods were highly correlated (Pearson product moment =.91). A further compaction of the data across disinfectants produced the lines in Fig.. This graph shows the values to be slightly lower than those of the method, suggesting that the method is slightly more restrictive. It also shows that the two lines follow each other quite closely, indicating that disinfectant activity is equivalently predicted by both methods. Figure was constructed by equating a log reduction greater than or equal to 8 with 6 negative tubes. These numbers represent the maximum achievable values of each method. The concept of log reduction used here as a quantitative measure of disinfectant efficacy has been used by other investigators (14, 1). The minimum acceptable log reductions for several standard methods were summarized by Reybrouck (1). A value between 5 and 6 is representative of most suspension tests. A log reduction of 6 was the suggested minimum in a clinical study by Christensen et al. (3). However, a log reduction of 8 was established as the minimum acceptable level for the suspension method to come as close as possible to the pass-fail criterion of the use-dilution method (Fig. ). Therefore, any disin- C 5 )3 -g4,m 3 -j (.9) (1.5) (.5) (3.) (4.3) APPL. ENVIRON. MICROBIOL. 1 4 FIG. 5. Comparison of the and methods in monitoring the inactivation of quat diluted in deionized water. Three standard organisms were used. Each value is the mean of three separate determinations. Symbols:, S. aureus; O, S. choleraesuis; A, P. aeruginosa.

6 VOL. 54, (.9) (1.5) (.5) (3.) (4.3) FIG. 6. Comparison of the and methods in monitoring the inactivation of quat diluted in tap water. Three standard organisms were used. Each value is the mean of three separate determinations. Symbols:, S. aureus;, S. choleraesuis; A, P. aeruginosa. fectant which could not equal or exceed this value during use was considered to have questionable clinical efficacy. Although the and methods have similar predictive abilities, the use-dilution method has the major disadvantage of being highly variable (1, 8). Our a 8 * 6.Q 5 t5 ' _,-- s I I I I I I (.9) (1.5) (.5) (3) (4.3) FIG.. Overall comparison of the (log reduction) and (negative tubes) methods averaged across three standard organisms and the six disinfecting solutions used in the methods comparison. Symbols: *, log reduction; *, number of tubes showing negative growth ,,_z 15 REUSE LIFE OF CHEMICAL DISINFECTANTS 163 TABLE. Disinfectant Coefficients of variation of and methods Coefficient of variation Acid glutaraldehyde 3.4 a Alkaline glutaraldehyde 3.4 Glutaraldehyde-phenol Phenolic Quat-deionized water Quat-tap water Overall a-, The number of positive tubes/6 was so small that the coefficients could not be estimated reliably. findings confirmed this fact, with the overall coefficient of variation of the method being almost six times greater than the corresponding value. One contributing factor to the variability associated with the method is the fluctuating organism load on the penicylinders. The number of organisms attached to carriers is highly dependent on the bacterial species used. Differences up to 1-fold have been reported (E. C. Cole, W. A. Rutala, and J. L. Carson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, Q41, p. 64). Large differences in the bacterial load from carrier to carrier were also seen by Ascenzi et al. (1). In addition, they showed that most of the viable organisms were washed from the carrier into the disinfecting solution. This compounds the reproducibility problem, since the majority of the test organisms remain in the solution and are never subcultured. A comparison of disinfectants with respect to their inactivation over time was not the primary intent of this study. However, some interesting differences between solutions were apparent. The glutaraldehydes are known for their ability to continue to disinfect in the presence of high levels of organic contamination (, 1). Our study confirmed this point. Neither the acid (1:4) nor alkaline (undiluted) glutaraldehyde solution showed any significant inactivation owing to organic load during the study. Even at the minimal concentration of.5% active ingredient, the acid glutaraldehyde solution remained effective under the stress of 6.3% human blood and exposure to 1,6 organism-coated penicylinders. The other four solutions experienced some degree of inactivation. The phenolic solution lost activity against S. c 6.g 4 _? 3 1 ^- ^ ^ - A ^ A * v w \- _vo~ V~V V---VV \vv\ Use - Day FIG. 8. Log reductions for three glutaraldehyde-based disinfectants during actual clinical use. Each value is the mean of six determinations; two from each of three clinical sites, evaluated by separate cultures of S. choleraesuis. Symbols:, alkaline glutaraldehyde; *, acid glutaraldehyde; V, glutaraldehyde-phenol.

7 164 ROBISON ET AL. aureus only, and this did not occur until the blood concentration reached approximately 4.3%. The inactivation of the glutaraldehyde-phenol (1:16) solution was more extensive. A decline in effectiveness against all three test organisms was observed after the blood concentration reached approximately 4%. The relative ease with which some quats are inactivated is well known (5). The quat-deionized water solution showed a decrease in activity against S. aureus after only.9% added blood. This solution, however, was much more effective than the quat-tap water solution, which had a very low activity against P. aeruginosa before any blood was added. The type of water used to dilute this disinfectant caused log reduction differences of as much as 8 against P. aeruginosa. The second purpose of this study was to use the suspension method in field trials designed to evaluate reuse life claims of manufacturers. Since glutaraldehyde-based solutions are the current state of the art in liquid disinfectants, three of these agents were chosen for evaluation in a clinical comparison. The results of this study are shown in Fig. 8. This represents data from the clinically used solutions only. Results from the unused (control) portions showed no loss of activity throughout the 36-day sampling period. This is important since suggested reuse lives in general are not based on clinical use studies; rather, they are derived from tests on aged solutions which may have experienced simulated use only. It can be seen that the reuse life claims in Table 1 have no correlation with the performance of a disinfectant. The acid glutaraldehyde solution has a suggested reuse life of 1 days, but its activity remained high throughout the 36-day sampling period. Conversely, the glutaraldehyde-phenol is labeled with a 3-day reuse life, but under heavy use, it lost activity much sooner, probably owing to the high dilution (1:16) and thus a lower initial glutaraldehyde concentration. The alkaline glutaraldehyde had a reuse life claim more appropriate for its performance. This solution did not experience a loss of activity until 1 week after its labeled reuse life claim. These inconsistencies reinforce the need for a reliable method of clinical in-use testing which can establish proper reuse life claims. The suspension method fills this need. A modification of the suspension method is currently being tested in a field evaluation of disinfectants in which pour plates are replaced by membrane filtration. The number of test organisms has also been expanded to include Mycobacterium bovis and poliovirus type 1. Results of field testing with these additional organisms should provide an even more detailed picture of how disinfectants perform over time in clinical environments. ACKNOWLEDGMENTS We thank Melvin Carter of the Center for Statistical Research, Brigham Young University, for his help in the interpretation of the APPL. ENVIRON. MICROBIOL. data. We thank Kelly Lundeen and Debbie Cox for their technical assistance. This study was supported by Clinical Research Associates. LITERATURE CITED 1. Ascenzi, J. M., R. J. Ezzell, and T. M. Wendt Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists. Appl. Environ. Microbiol. 51: Association of Official Analytical Chemists Disinfectants, p In W. Horwitz (ed.), Official methods of analysis, 14th ed. Association of Official Analytical Chemists, Washington, D.C. 3. Christensen, E. A.,. B. Jepsen, H. Kristensen, and G. Steen In-use tests of disinfectants. Acta Pathol. Microbiol. Immunol. Scand. Sect. 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