Quantitative Neutralization Assay of Fungicidal Activity of Disinfectants

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, My 1987, p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 31, No. 5 Quntittive Neutrliztion Assy of Fungicidl Activity of Disinfectnts BOHDAN TERLECKYJ AND DAVID A. AXLER* Deprtment of Microbiology, Immunology, nd Pthology, Pennsylvni College of Poditric Medicine, Phildelphi, Pennsylvni Received 23 My 1986/Accepted 26 Februry 1987 A quntittive ssy using neutrliztion medium ws developed to evlute fungicidl ctivity of disinfectnts. Concentrted Dey-Engley neutrlizing broth ws used in this study nd ws demonstrted to inctivte vrious chemicl gents within 5 min when disinfectnt concentrtions were reduced to specific levels. Addition of this Dey-Engley broth to test tubes contining fungl cells nd disinfectnts permitted control of the vrious interction times. Subsequent concentrtion of the disinfectnt-treted cells to 1-ml finl volume lso permitted exmintion of lrger popultion for the presence of resistnt cells. After timed exposures of 15, 30, nd 60 min, only three of seven disinfectnt solutions were found to be lethl for quntittive popultions of 11 fungi tested. The recommended use dilution formultions of quternry mmonium product nd n iodophor product were the lest effective gents. Vrious fungi, including Trichophyton mentgrophytes, Epidermophyton floccosum, nd AspergiUus niger, survived 30- to 60-min interctions with these disinfectnt solutions. The most resistnt orgnism encountered ws AspergiUus fumigtus, which survived 60 nd even 90 min of exposure to most disinfectnts. Only use dilutions of chlorine dioxide formultion, glutrldehyde formultion, nd n ethyl lcohol product were effective ginst this species nd ll of the other fungi fter 15-min interction. In clinicl settings, queous quternry mmonium compounds nd other chemicl gents hve been frequently used nd misused s instrument disinfectnts (6, 14, 16). Lrge ptient volume, limited instruments, nd time constrints re invribly encountered in such prctices. Under these conditions, cliniclly useful disinfectnts my not be effective becuse the ctul exposure of instruments to vrious solutions is usully less thn tht required for proper disinfection. Even though clinicl personnel re quite stringent in prepring proper disinfectnt concentrtions, the recommended interction time between instruments nd disinfectnts is frequently ignored nd forgotten (3). In ddition, the use of certin chemicl gents my be questionble considering the mny reports implicting disinfectnt solutions s sources of nosocomil infections (4, 7, 10, 13-15). However, becuse of their efficcy ginst grm-positive bcteri nd fungi, these gents my still be used in some busy clinics, especilly those involved in dermtologic tretments (16). The bctericidl efficcy of mny commercilly vilble disinfectnts hs been well documented, but very little informtion exists on their fungicidl properties (5, 11). A disinfectnt cn be clssified s fungicidl bsed on the Assocition of Officil Anlyticl Chemists suspension test, which specifies tht only Trichophyton mentgrophytes vr. interdigitle be utilized s the test orgnism (5). Although mny disinfectnts re lso tested ginst the yest Cndid lbicns, there is no generl consensus s to whether one or more thn one test orgnism should be used to determine the fungicidl efficcy of the vrious gents (18). Furthermore, very little quntittive dt re vilble on their efficcy ginst mny other common fungi. Consequently, fungicidl gents s defined by the Assocition of Officil Anlyticl Chemists method re ssumed to be effective ginst ll of the fungi usully encountered in cutneous nd opportunistic infections, s well s environmentl contminnts. * Corresponding uthor. 794 The im of this investigtion ws to devise quntittive ssy with neutrliztion system tht could be used to evlute the fungicidl ctivities of disinfectnts nd exmine severl disinfectnt products s to their efficcies in killing vriety of fungi fter timed interctions. MATERIALS AND METHODS Microorgnisms. Clinicl isoltes of C. lbicns, T. rubrum, nd T. mentgrophytes were used in this study, s well s lb strins of Epidermophyton floccosum (ATCC 18397), Microsporum gypseum, Microsporum cnis (ATCC 10214), Scopulriopsis breviculis, Aspergillus fumigtus, nd Aspergillus niger. Environmentl isoltes of Mucor species nd Fusrium species were lso included s test microorgnisms. All fungi were routinely grown on Sbourud dextrose gr slnts for 1 to 3 weeks t 30 C under erobic conditions. Fungl suspensions. Turbid suspensions were prepred when chrcteristic growth nd sporultion of ech fungus ws observed by microscopic exmintion. The fungl growth on Sbourud slnts ws scrped off (with sterile glss pipette) nd suspended in sterile sline solutions. The crude suspensions were trnsferred to sterile test tubes nd vigorously gitted for 2 to 3 min with Vortex mixer so tht dditionl spores nd hyphl cells could be dislodged from the visible culture ggregtes. Some suspensions, notbly E. floccosum nd M. cnis cultures, were chilled in ice wter nd homogenized in het-sterilized Wring blender by exposure to 2 to 3 pulses of 15 s ech. Any remining fungl clumps were then removed by filtrtion through sterile guze sponges. The turbid suspensions were then concentrted by centrifugtion t 5 C nd suspended in finl volume of 8 ml with sterile sline. Vibility counts of ech suspension were performed on the dy of their preprtion. CFU were determined by plting 1-ml smples of seril 10-fold dilutions on Sbourud dextrose gr pltes. These smples were then spred evenly

2 VOL. 31, 1987 ASSAY OF FUNGICIDAL ACTIVITY OF DISINFECTANTS 795 TABLE 1. Disinfectnt solutions Disinfectnt Distributor(s) Chemicl gent Use dilution Amphyl Lehn nd Fink; Sterling drug Phenolic mixture (15% o-phenylphenol nd 6.3% 1:50 dilution (2% solution) p-tert-mylphenol) Goldicide Pedinol Phrmcl Quternry mmonium mixture (6.5% benzlko- 1:85 dilution (1.18% solution) nium chloride nd 6.5% cetyldimethylethylmmonium bromide) Wescodyne West Chemicl Products Co. Iodophor mixture (17.8% iodophor compounds 1:212 dilution (0.47% solution) providing 1.6% minimum titrtble iodine) Alcide-LD Alcide Corp. Chlorine dioxide formultion (1.52% sodium 1:6 dilution chlorite nd 9.5% orgnic cid ctivtor) Soncide Wve Energy Systems Inc. 2% Glutrldehyde formultion Undiluted (100%) Sporicidin Sporicidin Co. Glutrldehyde-phente mixture (2% glutrl- 1:16 dilution dehyde, 7.05% phenol, 1.2% sodium phente) Ethyl lcohol Fisher Scientific Alcohol mixture (88% ethnol, 5% methnol) Undiluted (93%) Product dilution (concentrtion) recommended for instrument disinfection. throughout the gr surfce by tilting the pltes from side to side. All dilution pltes were incubted upright t 30 C nd exmined dily for up to 10 dys. Visible colonies were usully observed fter 3 to 7 dys of growth nd counted to determine the CFU/ml of ech suspension. During these vibility determintions, ll suspensions were stored t 4 C but for no longer thn 1 to 2 weeks before disinfectnt evlution experiments were done. Disinfectnts. The disinfectnt solutions exmined in this study included n queous quternry mmonium formultion (Goldicide), n iodophor formultion (Wescodyne), phenolic formultion (Amphyl), chlorine dioxide formultion (Alcide-LD), n cid glutrldehyde product (Soncide), glutrldehyde-phente formultion (Sporicidin), nd dentured ethyl lcohol product. The ctive chemicl gents of these products nd the use dilutions recommended by the mnufcturers for instrument disinfection re shown in Tble 1. All solutions were prepred to mnufcturer specifictions on the dy of the experiments. Inctivtion of disinfectnts. Dey-Engley (D/E) neutrlizing medium ws used in this study to inctivte the vrious disinfectnts fter timed exposures to fungl cells (8). The composition of this medium nd the identifiction of the vrious neutrlizing components re shown in Tble 2. This medium ws initilly devised to inctivte residul quntities of disinfecting gents nd lso to permit growth of bcteri surviving exposure to vrious disinfectnts (8). In this investigtion, concentrted form (1.43x) of D/E medium ws prepred nd used so tht it could be dded directly to vessels contining fungi nd disinfectnts. Addition of this 1.43x neutrlizing broth served to reduce the disinfectnt concentrtion immeditely, nd it lso lowered the neutrlizing components to their norml (lx) concentrtion. Norml strength D/E broth ws lso prepred, but it ws used primrily to wsh fungl cells. To determine the neutrlizing cpcity of the D/E medium, series of vibility experiments ws devised using the yest C. lbicns s the test microorgnism. The procedure involved dding 4.5 ml of concentrted (1.43 x) D/E medium to 2.0 ml of the vrious disinfectnt solutions nd llowing 5 min for inctivtion (4.5 ml times 1.43X concentrtion divided by 6.5-ml totl volume equls 0.99x concentrtion of D/E medium, nd 2 ml of disinfectnt divided by 6.5-ml totl volume equls 3.25-fold dilution of the recommended use dilutions). Exctly 0.2 ml of the C. lbicns cell suspension ws then dded to these disinfectnt-neutrlizing medium solutions. After 15-min exposure to these solutions, the yest cells were pelleted by centrifugtion t 5 C nd suspended in 6.5 ml of norml strength D/E medium. Vibility counts were then scertined to determine whether ny disinfectnt ws still ctive in the presence of the neutrlizing components. Controls in these experiments included exposing yest cells to disinfectnt solutions diluted with sterile distilled wter (2 ml of disinfectnt nd 4.5 ml of H20) nd to concentrted D/E medium diluted with sterile distilled wter (4.5 ml of concentrted medium nd 2 ml of H20). Fungicidl disinfectnt ssy. The quntittive ssy s developed for fungicidl evlution of vrious disinfectnts in timed experiments ws s follows. (i) Add 0.25 ml of cell suspension (known concentrtion) to 3.75 ml of disinfectnt solution yielding 4 ml of the use dilution concentrtion contining vible cells. For controls, 0.25 ml of the cell suspension ws dded to 3.75 ml of sterile distilled wter. The use dilutions of the disinfectnts were s recommended for instrument disinfection, except for Soncide, which ws prepred s 50% solution (Tble 1). (ii) Vortex for 10 s nd let stnd t 25 C for pproprite interction time (15, 30, or 60 min). (iii) Add 9 ml of concentrted (1.43x) D/E medium TABLE 2. D/E neutrlizing medium (norml strength, ph 7.6) Componentb (concn [%]) Function Crbohydrte source (nutrient) Glucose (1)... Tryptone (0.5)...Amino cid source (nutrient) Yest extrct (0.25)...Vitmin nd minerl source Lecithin (0.7)... Neutrlizes 1:750 quternry compoundsc Sodium thiosulfte (0.6)...Neutrlizes 2% chlorine nd 2% iodinec Tween 80 (0.5)... Neutrlizes 2% phenolic compoundsc Sodium bisulfite (0.25)... Neutrlizes 2% formldehyde nd 1% glutrldehydec Sodium thioglycolte (0.1)... Neutrlizes 1:1,000 mercuril compoundsc Bromcresol purple (0.002)...Growth indictor Approximtely 10 to 13 ml of 2 N NOH ws dded per liter to djust the ph to 7.6 ± 0.1. The medium ws het sterilized in n utoclve t 121'C with 15 lb/in2 for 20 min. Concentrted (1.43x) D/E medium ws prepred by mixing the sme ingredient quntities to finl volume of 700 (rther thn 1,000) ml with distilled wter. b Glucose, tryptone, sodium thioglycolte, nd bromcresol purple were obtined from Difco Lbortories, Detroit, Mich., yest extrct ws obtined from BBL Microbiology Systems, Cockeysville, Md., nd ll other components were obtined from Fisher Scientific Co., Pittsburgh, P. c Neutrlizing components inctivte these concentrtions of ntimicrobil gents in 10 ml of medium finl dilution.

3 796 TERLECKYJ AND AXLER to tubes yielding finl volume of 13 ml (for 3.25-fold dilution of disinfectnt concentrtions). An dditionl 19 ml of norml strength D/E medium ws dded to the Soncide nd ethyl lcohol disinfectnts to obtin the required eightfold dilution necessry for inctivtion of these products. (iv) Centrifuge (3,000 rpm for 15 min t 5 C) nd wsh pellet of cells with 8 ml of norml strength D/E medium. (v) Centrifuge nd suspend pellet of cells with 1 ml of norml strength D/E medium. (vi) Inoculte 0.15 ml into ech of three Sbourud slnts nd incubte t 30 C for severl weeks. These slnts were exmined t lest twice week for the presence of growth. Appernce of fungl colonies on Sbourud slnts ws interpreted s survivl of the fungus of the timed exposure to the disinfectnt. In ech experiment, vibility counts of the controls were scertined to determine the number of vible cells tht were exposed to the disinfectnt solutions. With this ssy, s few s 10 vible cells could be detected per milliliter of the finl suspended volume. RESULTS Inctivtion of disinfectnts. To test the neutrlizing cpbility of D/E medium, vibility studies were performed with C. lbicns. All of the disinfectnts were diluted with either concentrted D/E neutrlizing broth or sterile distilled wter. After 5-min interction with disinfectnt nd the D/E broth components, vible numbers of C. lbicns cells were exposed for 15 min to these solutions s well s to the wter-diluted disinfectnts (Tble 3). At the concentrtions indicted, most of the wter-diluted disinfectnts were effective in killing 6.6 x 106 CFU of C. lbicns in 15 min, except for the Sporicidin product, with which 145 CFU survived (<0.01%). At its norml recommended strength (1:16 dilution), the Sporicidin product ws effective in killing ll of the yest cells in the 15-min exposure time. However, when these disinfectnts were diluted with concentrted D/E medium, most of these chemicl gents were rpidly inctivted within 5 min becuse the yest cells survived the subsequent 15-min exposure. A 100% vibility of C. lbicns ws obtined in D/E brothdiluted solutions of the Amphyl, Goldicide, Wescodyne, Alcide-LD, nd Sporicidin products (Tble 3). With the Soncide solution, only 0.15% of the initil inoculum survived, indicting tht neutrliztion of this glutrldehyde formultion ws incomplete. Neutrliztion of the ethyl lcohol product did not occur since no yest cells survived in the D/E broth-diluted solution of lcohol. This ltter result TABLE 3. Vibility of C. lbicns fter 15 min of exposure to disinfectnts diluted with D/E neutrlizing medium or distilled wter Vibility (CFU/ml) in Disinfectnt disinfectnt diluted with: % Survivl (concn or dilution) Wter D/E medium Amphyl (0.62%) x Goldicide (0.36%) x Wescodyne (0.145%) x Alcide-LD (1:19.5) x Sporicidin (1:52) x Soncide (17.5%) x Alcohol (28.6%) Controls 6.6 x x These represent 3.25-fold dilutions of the norml recommended use dilution of ll disinfectnts except for the Soncide product. ANTIMICROB. AGENTS CHEMOTHER. TABLE 4. Vibility of C. lbicns fter 15 min of exposure to Soncide nd lcohol solutions diluted with D/E neutrlizing medium or distilled wter Diluted with D/E medium Diluted with wter Disinfectnt nd concn (%) Vibility % Survivl Vibility % Survivl (CFU/ml) (CFU/ml) %Sri Soncide x x x x x x x Alcohol x x x x Controls 6.2 x x ws not unexpected since D/E medium contins no effective neutrlizing gent ginst lcohols (Tble 2). The results (Tble 3) demonstrte tht five of the seven disinfectnts were totlly inctivted when concentrted D/E broth ws dded to these chemicl solutions. The concentrtions of these five disinfectnts were mximl for the neutrlizing cpcity of the D/E medium becuse the survivl rte of C. lbicns (6.4 x 106 CFU) t higher concentrtions ws less thn 100% for these disinfectnts (dt not shown). In ddition, the results lso indicte tht D/E medium ws not toxic to the yest cells since the 6.4 x 106 CFU in the D/E medium control compred fvorbly to the 6.6 x 106 CFU in the sterile wter control. Complete inctivtion of the Soncide nd ethyl lcohol products ws obtined when these disinfectnts were diluted further to lower concentrtions. Soncide (6.3%) in the presence of D/E broth ws totlly inctivted within 5 min, nd 100% of the yest cells survived the subsequent 15-min exposure (Tble 4). In the bsence of neutrlizing medium, however, this 6.3% concentrtion killed bout 89% of the vible C. lbicns cells. Higher concentrtions of the Soncide product were only prtilly inctivted when diluted with D/E medium (Tble 4). Neutrliztion of Soncide thus required tht the concentrtion be reduced to 6.3% with D/E broth. This represents n eightfold dilution of the initil 50% solution. In reference to the ethyl lcohol product, some neutrliztion ppered to occur when the concentrtion ws reduced to 11.6% (Tble 4). Wheres 11.6% lcohol did kill 47% of the yest inoculum fter the 15-min exposure, this sme concentrtion diluted with D/E broth yielded 100% survivl of 6.2 x 106 CFU of C. lbicns. This pprent inctivtion of the lcohol product ws probbly due to the presence of orgnic mtter in the D/E broth, the inoculum, or both becuse no specific neutrlizing component ws present in the medium for lcohol inctivtion. Fungicidl evlution of disinfectnts. The quntittive fungicidl ssy developed s described bove ws used to test vrious fungi ginst disinfectnts t their recommended use dilution concentrtions in timed experiments. After 15-, 30-, nd 60-min exposures of fungl cells to these gents, the fungus-disinfectnt solutions were diluted 3.25-fold (8-fold with the Soncide nd lcohol products) with D/E neutrlizing medium to inctivte the vrious disinfectnts rpidly. The results of these timed experiments re presented in Tbles 5 nd 6.

4 VOL. 31, 1987 ASSAY OF FUNGICIDAL ACTIVITY OF DISINFECTANTS 797 TABLE 5. Survivl nd growth of fungi fter 15- nd 30-min interctions with vrious disinfectnts Growth with the following disinfectnts nd interction times (min)b: Fungus CFU Goldicide Wescodyne Sporicidin Amphyl Alcide-LD Soncide Alcohol Cndid lbicns 6.6 x Trichophyton rubrum 2.7 x Trichophyton mentgrophytes 1.3 x Epidermophytonfloccosum 2.8 x Microsporum cnis 5.3 x NPc NP - - Microsporum gypseum 4.0 x NP NP - - Scopulriopsis breviculis 2.4 x _ +d - Aspergillus niger 8.0 x ± - _ - + Aspergillus fumigtus 1.6 x Mucor sp. 3.8 x Fusrium sp. 2.5 x Number of CFU exposed per ml of disinfectnt t the use dilution. b -, No growth of fungl cells in the three inoculted Sbourud slnts fter exposure to disinfectnt; +, growth of fungl cells in ll three inoculted Sbourud slnts. c NP, Disinfectnt ws not tested with this fungus. d +, Vrible results such s growth in only one of the three inoculted Sbourud slnts. Most of the fungi tested were not susceptible to the Goldicide product, which ws the lest effective disinfectnt in this investigtion (Tble 5). This quternry mmonium formultion ws unble to kill the resistnt members of mny fungl cell popultions, even fter 60-min exposure to this gent (Tble 6). The Wescodyne product ws lso ineffective ginst mny fungi in the 15-min interction time but improved somewht fter 30-min exposure (Tble 5). Nevertheless, E. floccosum, A. niger, nd A. fumigtus ll survived 60-min exposure to Wescodyne (Tble 6). Other disinfectnts, such s the Amphyl nd Sporicidin products, were more effective in their fungicidl ctivities fter 15- to 30-min interction times but, once gin, resistnce to these gents ws encountered in cell popultions of A. fumigtus nd A. niger (Tbles 5 nd 6). In contrst to these results, ll of the fungi tested were effectively killed by the Alcide-LD product nd the lcohol product fter 15-min exposure. Vible cell popultions of 104 to 106 CFU were found to be susceptible to these highly effective disinfectnts (Tble 5). The 50% Soncide solution could lso be included in this group becuse it ws lethl for ll fungi, except for A. niger, which displyed vrible results t the 15-min time period. The undiluted Soncide product ws, however, effective in killing this A. niger popultion fter 15-min exposure (dt not shown). DISCUSSION Development of quntittive ssy tht uses neutrlizing medium hs provided severl dvntges in the fungicidl evlution of disinfectnts. Fungl suspensions hrvested from in vitro growth on Sbourud dextrose gr consistently yielded lrge numbers of chrcteristic conidi with some hyphl cells, hyphl frgments, nd chlmydospores, which re the resistnt spores produced by mny fungi. Both susceptible nd resistnt fungl cells were thus present in these suspensions. Addition of D/E neutrlizing medium directly into vessels contining disinfectnts nd fungl cell popultions permitted control of the vrious interction times becuse the neutrlizing components hve been shown to inctivte the vrious chemicl gents within 5 min (Tbles 3 nd 4). Furthermore, greter percentge of the disinfectnt-treted popultion ws subsequently surveyed for the presence of vible resistnt cells. Addition of ml inoculum from finl suspended volume of 1 ml to ech Sbourud slnt permitted exmintion of 15% of the popultion s compred with 1% by other methods (5, 18). This quntittive ssy ws thus more sensitive in detecting survivors of disinfectnt-treted popultions thn re other systems. The quntittive neutrliztion ssy ws used to determine whether or not the recommended disinfectnt concen- Fungus TABLE 6. Survivl nd growth of fungi fter 60 min of exposure to vrious disinfectnts CFU Growth with the following disinfectntb: Goldicide Wescodyne Sporicidin Amphyl Alcide-LD Soncide Alcohol Cndid lbicns 6.6 x Trichophyton rubrum 2.7 x 106 Trichophyton mentgrophytes 1.3 x Epidermophyton floccosum 2.8 x Microsporum cnis 5.3 x _ NPc Microsporum gypseum 4.0 x NP Scopulriopsis breviculis 2.4 x Aspergillus niger 8.0 x Aspergillus fumigtus 1.6 x Mucor sp. 3.8 x Fusrium sp. 2.5 x 106 Number of CFU exposed per ml of disinfectnt t the use dilution concentrtion. b -, No growth of fungl cells in the three inoculted Sbourud slnts; +, growth of fungl cells in ll three inoculted Sbourud slnts. c NP, Disinfectnt ws not tested with this fungus.

5 798 TERLECKYJ AND AXLER trtions could kill fungl popultions in cliniclly useful interction times. The results presented in Tbles 5 nd 6 illustrte severl obvious nd interesting points. The use of queous qutemry mmonium formultions nd iodophor formultions for instrument disinfection is questionble if 15- to 30- min interction time is desired, especilly in busy clinicl prctices (Tble 5). These chemicl gents require longer thn 60 min of interction to kill vible fungl cell popultions of 105 to 106 CFU/ml (Tble 6). Moreover, both queous qutemry mmonium formultions nd iodophors hve been implicted s sources of nosocomil infections in hospitl settings (4, 7, 10, 13). Grm-negtive microorgnisms such s Serrti mrcescens, Pseudomons cepci, nd Pseudomons eruginos hve been reported to survive in these nd other disinfectnt solutions (2, 6, 12, 14, 15). Such opportunistic bcteri, long with vrious fungi, re invribly involved in cutneous infections (1, 17). They could esily contminte instruments used for collecting skin nd nil specimens, s well s for debridement of cutneous ulcertions or wounds. Disinfection of such instruments before reuse would thus be reltively equivocl in queous quternry mmonium formultions nd iodophors, especilly considering indequte exposures nd lck of good physicl clensing (3, 9). Other chemicl gents such s the Amphyl nd the Sporicidin products would seem to be more pproprite for this tsk, but the fungicidl efficcy of even these disinfectnts hs to be questioned considering the results of this investigtion. Furthermore, since Mycobcterium tuberculosis is significntly more resistnt to chemicl gents thn re fungi (9), it is lso questionble whether these tuberculocidl gents (Amphyl nd Sporicidin) cn effectively kill mycobcteril popultions of 105 CFU in 15 min of exposure. The most resistnt fungi encountered in this study were cell popultions of A. fumigtus nd A. niger. These two fungi survived not only 60 min of exposure to the Goldicide, Wescodyne, nd Sporicidin products but lso 90-min interction with these disinfectnt formultions (dt not shown). A. fumigtus lso demonstrted resistnce to the ction of the Amphyl product fter 60- nd 90-minute exposures. The only disinfectnts tht were effective ginst these Aspergillus species in the 15- to 30-min time period were the Alcide-LD, Soncide, nd ethyl lcohol products. Therefore, even though ll of the disinfectnts used in this investigtion re considered to be fungicidl by the Assocition of Officil Anlyticl Chemists suspension test, only the ltter three were unequivoclly shown to be effective ginst the vrious fungi tested. The resistnce displyed by A. fumigtus strongly emphsizes the need to use this fungus s test microorgnism in evluting disinfectnts. Chemicl gents should not be clssified s fungicidl bsed solely on their efficcy ginst one test orgnism, nmely T. mentgrophytes, s is currently required (5). This microorgnism is not the most resistnt fungus ccording to our quntittive findings nd the results of nother study (18). It thus seems pproprite to suggest tht A. fumigtus should be primrily used s the test orgnism, possibly long with C. lbicns nd T. mentgrophytes s other test fungi in the evlution of disinfectnts. A fungicidl gent should be ble to kill stndrd quntittive popultion of the most resistnt fungus fter resonble exposure to the recommended use dilution. Otherwise, questionble disinfectnts will continue to be used nd bused in clinicl settings where their fungicidl clssifiction will be ssumed to men effectiveness ginst ll fungi tht contminte instrument surfces. ANTIMICROB. AGENTS CHEMOTHER. ACKNOWLEDGMENTS We thnk Frederic C. Jewett nd Lynnmrie Ctpno for their technicl ssistnce in this study nd Terri E. Wilson for preprtion of the mnuscript. This work ws supported by reserch funds from the Pennsylvni College of Poditric Medicine. LITERATURE CITED 1. Amonette, R. A., nd E. W. 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