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Bacteriostatic, germicidal, and sanitizing action of quaternary ammonium compounds on textiles
B A C T E R I O S T A T I C , G E R M I C I D A L , A N D S A N I T I Z I N G A C T I O N OF Q U A T E R N A R Y A M M O N I U M C O M P O U N D S ON T E X T I L E S I I . SANITIZATION OF COTTON FABRICS M . . ~ . LATLIEF, B.S., ~ iV[. T. GOLDSMITH, PIz.D., ':'~J . L . FRIEDL, B . S . , ~ : AND
L. S. STUART, M.S. ~:~ WASHINGTON, D. C.
monium salts does not reduce the total bacterial counts or counts of Escherin a r y a m m o n i u m salts against the test chia cell in laundry. A review of the literature reveals organism Proteus mirabilis were t h a t v e r y little scientific information clearly defined. I t seemed important, therefore, to extend the studies on is available concerning mechanical rethese compounds to determine their moval of microorganisms f r o m fabrics relative efficiency as sanitizing agents as opposed to the bactericidal effects of for cotton fabric and to clarify the soaps, bleaches, heat, etc., during existing information on their practi- laundering. Roberts, 5 using a standardized inoculum on fabric, recovered cal value f o r this use. Recent investigations on the effici- a total of 38 per cent of the original ency of q u a t e r n a r y a m m o n i u m com- inocu]um f r o m a series of five rinse waters as determined b y plate counts pounds as sanitizers a n d disinfectants She reported for l a u n d r y have employed bacterial of viable organisms. t h a t in procedures using one suds and plate counts on rinse waters to provide two rinses, only 20 to 50 per cent of an index to the sanitizing and germit h e original inoculum was eliminated cidal value of these products. Evif r o m the fabric. F o r this reason, it is dence of the sanitizing value of alkyl believed that sterile l a u n d r y rinse dimethyl benzyl a m m o n i u m chloride, used in a dilution of 1:5,000 ~at 40 ~ C., waters cannot be employed as an absolute index to sterile laundered fabwas advanced by Lawrence and r M u r p h y and reported by Lawrence. 2 i c s nor final evidence of the e]iminination f r o m the fabric itself of a speThe results of theii~ plate count studies on the first and second deep rinses in cific infectious agent. However, if sanitization can be demachines of Westinghouse Launderfined as a t r e a t m e n t which reduces the m a t type tend to s u p p o r t the contenbacterial count to a relatively safe tion of Bartlett, 3 t h a t q u a t e r n a r y amlevel, it might be conceded t h a t a remonium compounds can be used to asduction of the bacterial count in launsure s a n i t a r y finished laundry. They d r y rinse waters of 99.9 p e r cent or are, however, directly c o n t r a r y to the more could be accepted as .a practical conclusion drawn b y Mack 4 t h a t the p r e s u m p t i v e index to sanitizing efuse of bacteriostatic q u a t e r n a r y amficiency. I n actual practice a reduc*Bureau of Human Nutrition and Home Economics, U. S. Department of Agriculture. tion of this magnitude in the rinse **Production and Marketing Administramight even be employed as a presumption, U, S. Department of Agriculture. N A previous study, 1 the bacterio-
I static values of five selected quater-
324
LATLIEF ET AL. :
QUATERNARY AMMONIUM cOMPOUNDS AND TEXTILES
rive index for disinfection since the surviving 0.1 per cent might consist entirely of resistant sporeforming cells of nonpathogenic species against which the usual chemical germicides are known to be ineffective. In experimental rinse waters where resistant sporeforming bacteria have been eliminated from consideration, and the only bacteria present are cells of a selected nonsporeforming test organism, a reduction of 100 per cent in the rinse would be the minimum reduction that could be accepted as a presumptive evidence of disinfection. However, in test procedures of a similar type a lesser reduction of 99.9999 per cent has been accepted as an index to potential value with chemical sanitizers for dishes and eating utensils by the U. S. Public Health Service2 Acceptance of 99.9 per cent reduction of the inoeulum as determined by plate counts of viable cells in a rinse as a significant end point for sanitizing action in these studies seemed practical. For it would be reasonable to assume that a reduction of this magnitude might have practical significance in the day-to-day maintenance of sanitary conditions in home and commercial laundries where specific infectious agents were not known to be present. It should be pointed out also that t he selection of an end point at a higher percentage level would have required the use of a greater number of determinations in these studies than seemed feasible, if results were to be statistically reliable. PROCEDURE
As in previous investigations the following five quaternary ammonium compounds were employed: (A) para diisobatyl phenoxy ethoxy ethyl di-
325
methyl ~benzyl ammonium chloride; (B) alkyl (C8-Q8) dimethyl benzyl ammonium chloride; (C) alkyl (QC1~) naphthyl methyl pyridinium chloride; (D) dodecyl acetamido dimethyl benzyl ammonium chloride; and (E) cetyl trimethyl ammonium bromide. Henceforth these compounds will be referred to as quaternary A, quaternary B, etc. The dilutions tested ranged from 1:5,000 to 1:100,000, or from 1 oz. quaternary for 9.4 lb. of dry fabric to 1 Oz. quaternary for 187 lb. of dry fabric. Cotton, gauze-type diaper cloth was used as the carrier for the test organism. Two dry samples of this cloth, 4 by 5 inches, weighed 3 grams. Two test strips folded to fit conveniently into each Petri dish were autoclaved for twenty minutes at 250 ~ F., then dried in a hot air oven at 105 ~ C. The test organism was a typical strain of E . co li h a v i n g an average resistance of 1:90 to phenol, as determined by the method of the Association of Official Agricultural Chemists. 7 The stock culture was maintained at 5 ~ C. on Bacto-Tryptone Glucose Extract Agar slants ( T . G . E . ) - - t r y p t o n e 5 Gin., glucose 1 Gm., beef extract 3 Gm., agar 15 Gm., distilled water 1,000 c.c., with monthly transfer during the course of these studies. For the inoeulum 1 e.c. of a twentyfour-hour T.G.E. broth culture of the organism was used to seed Kolle flasks containing 60 c.e. of T.G.E. agar. After incubation at 37 ~ C. for twentyfour hours, the surface growth was removed with 50 e.e. of sterile distilled water and a homogeneous preparation obtained by filtration through coarse paper, Sehleicher and Schull, No. 588. The heavy suspension of cells was diluted to give a 50-60 reading on
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THE JOURNAL OF PEDIATRICS
Klett-Summerson photoelectric colorimeter using No. 42 blue filter. This suspension, on f u r t h e r dilution of 1:20, provided a s t a n d a r d inoculum of approximately 10,000,000 cells per cubic centimeter. Each two samples, or 3 grains of fabric, were inoculated with 4 c.c. of the standardized cell suspension and introduced into a pint j a r containing 96 e.c. of medieant. Determinations were made in duplicate for each dilution of each quaternary. F o r controls, two samples of fabric similarly inoculated were placed in a j a r containing sterile distilled water. On the assumption that approximately 50 per cent of the organisms m a y remain on the cloth, the inoeulum described above yielded a fabric-water p r e p a r a t i o n containing on the order of 300,000 'bacteria p e r cubic centimeter of extractable liquid, which is roughly equivalent to numbers reported to be found in final rinse waters of home laundries, s The jars were t r a n s f e r r e d to a launder-ometer where they were agitated at indicated t e m p e r a t u r e s f o r fifteen minutes. Each sample was twisted with sterile forceps to express the liquid, then introduced into a test tube containing 20 c.c. of 5 p e r cent distilled w a t e r solution of Tamol N2 A f t e r ninety m i n u t e s ' exposure in the Tamol N to assure complete neutralization of excess quaternary, the samples were discarded and d i l u t i o n plate counts on the rinse waters in the tubes were made using T.G.E. agar. Counts were made in duplicate f r o m two rinse tubes selected to yield data f r o m the duplicate q u a t e r n a r y treatments at each dilution tested. The counts on the distilled w a t e r controls were used as a base for deter-
mining the per cent of organisms surviving exposure to the various dilutions of quaternaries according to the following formula : Average n u m b e r of colonies per c.e. rinse a f t e r exposure to q u a t e r n a r y • I00. Average n u m b e r of colonies per c.c. rinse of control fabric.
The per cent reduction of viable organisms was obtained by deducting from ]00 the figures obtained as above. W h e n the experimentally contamin a t e d fabric was exposed to dilutions of 1:5,000 quate'rnary, 1 c.c. and 0.1 e.c. aliquots of the neutralizer rinse provided countable plates. Dilutions of 1:10, 1:100, and 1:1,000 of Tamo] N yielded, in most instances, plates with 30 to 300 colonies when the fabrie was treated with dilutions ranging f r o m 1:10,000 to 1:100,000 quaternary. F r o m the eontrol Tamol N tubes, dilution plates of 1:100, 1:1,000, and 1:10,000 were necessary to obtain similar data. Colonies were enumerated in a Quebec counter a f t e r forty-eight hours' incubation at 37 ~ C. RESULTS AND DISCUSSION The results show that, in genera], each q u a t e r n a r y at each t e m p e r a t u r e exhibits a n i n c r e a s i n g effectiveness with increasing concentration against the test organism E. coli, as determined by survival of viable cells in the Tamol neutralizer rinse. The five quaternaries were more efficient in reducing numbers of this organism at 45 ~ C. than they were at 20 ~ C. Sanitization, i.e., a 99.9 per cent reduction of the contaminating organisms, required for all five quaternaries 1:5,000 dilution, or 1 oz. q u a t e r n a r y to 9.4 lb. of cloth, when applied at 45 ~ C. for fifteen minutes. A t 20 ~ C., quaternaries B and D showed a similar activity.
LATLIEF ET AL. :
QUATERNARY AMMONIUM COMPOUNDS AND TEXTILES
Quaternaries A, C, and E approached this effectiveness with more than 99 per cent destruction. Quaternaries B, C, and D, when applied at 45 ~ C. in dilution of 1:10,000, or 1 oz. of q u a t e r n a r y to 18.7 lb. of cloth, also brought about more than 99 per cent reduction in numbers. It should be pointed out that the reductions above 99 per cent but less than the 99.9 per cent selected as an end point for this study cannot be considered as absolute evidence of failure to sanitize since TABLE I .
treatment with these same quaternaries for fifteen minutes at 45 ~ C. required a dilution of 1:5,000 to bring about sanitization. I n spite of the longer exposure period, five to twenty times the concentration of the quaternaries were required to sanitize the fabric than to render it bacteriostatic for sixteen hours. With high temperature applications it appears possible, therefore, to secure fabrics which have t e m p o r a r y residual b acteriostatic effects at lower concentrations than are
REDUCTION IN -'r CELLS OF E . O0l~ W!-IEN THE INOEULATED FABaIC EXPOSED FOR FIFTEEN ~aCJ:INUTES AT 20 ~ C. AND ArT 45 ~ C'.
Quaternary dilution Ounce q u a t e r n a r y : p o u n d s d r y f a b r i c .• Temperature
327
1:100,000
1:50,000
1:25,000
1:1%000
WAS
1:5,000
1:187 1:94 1:47 1:18.7 1:9.4 Quaternary P e r cent R e d u c t i o n A 19 13 53 71 99.3 ]3 0 28 49 91 99.9 20 ~ C. C 0 0 49 92 99.6 I) 41 50 60 88 99.9 E 33 66 74 91 99.6 A 39 49 67 91 99.9 ]3 33 67 78 99.4 99.9 45 ~ C. C 56 51 80 99.5 99~9 D 44 69 78 99.2 99.9 E 30 67 89 98 99.9 *The c h e m i c a l - d r y fabric r a t i o s a r e m a t h e m a t i c a l c a l c u l a t i o n s of the a c t u a l di l ut i ons used.
differences of this magnitude probably fall within the r a n g e of experimental error in the counting methods used. Tha~ reduction in count seems to depend on the concentration of quatern a r y and temperature of application is evident since there is, in generaI, a gradual diminution of ceils from no reduction in certain instances to:99,9 per cent. The eoneentration and tentperature relationship was noted also in studies relating to baeteriostatie effects of these compounds. ~ There it was shown that fabric treated with d i h t i o n s ranging from 1:25,000 to 1:100,000 for ten minutes at 45 ~ C. subsequently i n h i b i t e d the growth of P. mirabilis for sixteen hours, whereas fabric inoculated with N. coli prior to
required to secure a practical sanitizing benefit. A f u r t h e r comparison of the baeteriostatic and sanitizing properties of these same quaternaries reveals that 1:5,000 to 1:10,000 dilutions at 20? C. for ten minutes rendered the test fabric baeteriostatie for sixteen hours, whereas a 1:5,000 dilution applied at 20 ~ C. for fifteen minutes produced sanitization, according to the selected end point, only with quaternaries B and D, although the reductions observed with quaternaries A, C, and E closely approached the desired level. Such data indicate a fairly dose correlation between the~eoneentrations necessary to secure practical sanitizing action and residual bacteriostatic effects when a p
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THE JOURNAL OF PEDIATRIC S
plieations are made at relatively low temperatures. On the other hand, for fabric treated for ten minutes at 20 ~ C. and 45 ~ C. to remain bacteriostatie for twentyfour hours or more, dilutions ranging from 1:1,000 through 1:4,000 were found necessary. Thus, if prolonged baeteriostatie effects are desired, it would appear that concentrations considerably higher than those required for sanitizing would have to be employed in treating' fabrics. Insofar as the sanitizing' capacity of these quaternaries is concerned, these results seem to confirm the findings reported by Lawrence which showed that a 1:5,000 dilution of alkyl (C sCls) dimethyl benzyl ammonium ehlo~ ride was useful for this purpose. This sanitizing value seems, however, to be possessed by the other four compounds as well, provided time and temperature of treatment are adequate. Mack and associates, ~ on the other hand, concluded that the use of baeteriostatic agents does not reduce total bacterial counts of E. coll. The findings herein reported have demonstrated that quaternary ammonium compounds do reduce total counts of E. coli in fabrics provided they are employed a t suitable concentrations. Also, these reductions were considered to be of a magnitude sufficient to warrant claims of sanitizing value when the chemicals were properly applied, although at dilutions of 1:5,000 and above they did fall somewhat short of the end point which would be required to provide presumptive evidence of disinfection or a treatment that could be considered adequate for fabrics known to carry or strongly suspected of carrying infectious material.
CONCLUSIONS
As judged by their ability to reduce by 99.9 per cent the E. coli count in rinse waters of experimentally contalninated fabrics over control rinse waters, the five quaternaries studied appear to have potential value as sanitizing agents in ordinary laundry operations. The efficiency of all five quaternaries in reducing the E. ~oli count of fabrie rinse waters has been shown to depend upon the concentration of the chemical (related possibly to fabric-chemical ratio) and temperature of application. All five quaternaries, (A) para diisobutyl ph.enoxy ethoxy ethyl dimethyl benzyl ammonium chloride, (B) alkyl (Cs-C18) dimethyl benzyl ammonium chloride, (C) alkyl (C6-C12) naphthyl methyl pyridinium chloride, (D) dodeeyl acetamido dimethyl benzyl ammonium chloride, and (E) eetyl trimethyl ammonium bromide, when applied at 45 ~ C. for fifteen minutes in a dilution of 1:5,000 or at 1 oz. quaternary to 9.4 lb. of dry fabric, reduced by 99.9 per cent the E. coli count of the subsequent experimental neutralizer rinse over controls. At 20 ~ C. with fifteen minutes' exposure, all five quaternaries reduced the E. coli count of the experimental rinses by more than 99 per cent. A reduction of 100 per cent was not secured in the E. coli count of rinse. waters with any of the five quaternaries studied at dilutions of 1:5,000 and above at either 20 ~ or 45 ~ C. Thus, the potential sanitizing values noted cannot be considered as presumptive evidence of complete germicidal action which would be essential to indicate value for use in disinfection.
LATLIEF ET AL. :
QUATERkNARYAMMONIU1V[ COMPOUNDS AND TEXTILES 329
REFERENCES 1. Latlief, M. A., Goldsmith, M. T., Friedl, J. L., and Stuart, L. S.: Bacteriostatic, Germicidal, and Sanitizing Action 0s Quaternary Ammonium Compounds on Textries. Prevention of Ammonia Formation From Urea by Proteus mirabills, J. pEDIAT. 39: 730, 1951. 2. Lawrence, C. A.: Surface Active Quaternary Ammomnm Germieides, Academic Press, ]950, p. 188. 3. Bartlett, P. G.: Quaternaries in the Laundry Field, Soap & Sanit. Chem. 25: (3): 139, 1949. 4. Mack, P. B., ed.: Bacteriostatic Agents and Sterilization in the Diaper Laundry, Pennsylvania State College, Diaper Serv. Fellowship Res. Rpts. 32-33, 1949.
5. Roberts~ E. H.: The Bactericidal Effectiveness of I-Iome Laundering Methods for Silk and Rayon, Washington Agriculture Experimental Station Bull. 265, 1932. 6. Weber, G. R., and Black, L. A. : Laboratory Procedure for Evaluating Practical Performance of Quaternary Ammonium and Other Germicides Pro.posed ~or Sanitizing Food Utensils, Am. J. Pub. Health 38: 1405, 1948. 7. Association of Official Agricultural Chemists: Official Methods of Analysis, ed. 7, Washington, D. C., 1930, p. 88. 8. American Institute of Laundering: Home Washing Survey, Tech. Bull. No. 47. 9. Goetchius, G. R.: A Quaternary Inactivatot, Soap & Sanit. Chem. 25: (1): 131, 1949.
Erratum I n the a r t i c l e b y Dr. B. B. Breese, " A u r e o m y c i n i n the T r e a t m e n t of B e t a H e m o l y t i c Streptococcal I n f e c t i o n s , " i n the J a n u a r y , 1952, issue, the first sentence u n d e r A u r e o m y c i n Dosage on page 86 should r e a d : The i n i t i a l dose g i v e n was 20 rag. p e r k i l o g r a m of body weight in t w e n t y f o u r h o u r s a n d c a l c u l a t e d to the n e a r e s t 50 rag.
L. S. STUART, M.S. ~:~ WASHINGTON, D. C.
monium salts does not reduce the total bacterial counts or counts of Escherin a r y a m m o n i u m salts against the test chia cell in laundry. A review of the literature reveals organism Proteus mirabilis were t h a t v e r y little scientific information clearly defined. I t seemed important, therefore, to extend the studies on is available concerning mechanical rethese compounds to determine their moval of microorganisms f r o m fabrics relative efficiency as sanitizing agents as opposed to the bactericidal effects of for cotton fabric and to clarify the soaps, bleaches, heat, etc., during existing information on their practi- laundering. Roberts, 5 using a standardized inoculum on fabric, recovered cal value f o r this use. Recent investigations on the effici- a total of 38 per cent of the original ency of q u a t e r n a r y a m m o n i u m com- inocu]um f r o m a series of five rinse waters as determined b y plate counts pounds as sanitizers a n d disinfectants She reported for l a u n d r y have employed bacterial of viable organisms. t h a t in procedures using one suds and plate counts on rinse waters to provide two rinses, only 20 to 50 per cent of an index to the sanitizing and germit h e original inoculum was eliminated cidal value of these products. Evif r o m the fabric. F o r this reason, it is dence of the sanitizing value of alkyl believed that sterile l a u n d r y rinse dimethyl benzyl a m m o n i u m chloride, used in a dilution of 1:5,000 ~at 40 ~ C., waters cannot be employed as an absolute index to sterile laundered fabwas advanced by Lawrence and r M u r p h y and reported by Lawrence. 2 i c s nor final evidence of the e]iminination f r o m the fabric itself of a speThe results of theii~ plate count studies on the first and second deep rinses in cific infectious agent. However, if sanitization can be demachines of Westinghouse Launderfined as a t r e a t m e n t which reduces the m a t type tend to s u p p o r t the contenbacterial count to a relatively safe tion of Bartlett, 3 t h a t q u a t e r n a r y amlevel, it might be conceded t h a t a remonium compounds can be used to asduction of the bacterial count in launsure s a n i t a r y finished laundry. They d r y rinse waters of 99.9 p e r cent or are, however, directly c o n t r a r y to the more could be accepted as .a practical conclusion drawn b y Mack 4 t h a t the p r e s u m p t i v e index to sanitizing efuse of bacteriostatic q u a t e r n a r y amficiency. I n actual practice a reduc*Bureau of Human Nutrition and Home Economics, U. S. Department of Agriculture. tion of this magnitude in the rinse **Production and Marketing Administramight even be employed as a presumption, U, S. Department of Agriculture. N A previous study, 1 the bacterio-
I static values of five selected quater-
324
LATLIEF ET AL. :
QUATERNARY AMMONIUM cOMPOUNDS AND TEXTILES
rive index for disinfection since the surviving 0.1 per cent might consist entirely of resistant sporeforming cells of nonpathogenic species against which the usual chemical germicides are known to be ineffective. In experimental rinse waters where resistant sporeforming bacteria have been eliminated from consideration, and the only bacteria present are cells of a selected nonsporeforming test organism, a reduction of 100 per cent in the rinse would be the minimum reduction that could be accepted as a presumptive evidence of disinfection. However, in test procedures of a similar type a lesser reduction of 99.9999 per cent has been accepted as an index to potential value with chemical sanitizers for dishes and eating utensils by the U. S. Public Health Service2 Acceptance of 99.9 per cent reduction of the inoeulum as determined by plate counts of viable cells in a rinse as a significant end point for sanitizing action in these studies seemed practical. For it would be reasonable to assume that a reduction of this magnitude might have practical significance in the day-to-day maintenance of sanitary conditions in home and commercial laundries where specific infectious agents were not known to be present. It should be pointed out also that t he selection of an end point at a higher percentage level would have required the use of a greater number of determinations in these studies than seemed feasible, if results were to be statistically reliable. PROCEDURE
As in previous investigations the following five quaternary ammonium compounds were employed: (A) para diisobatyl phenoxy ethoxy ethyl di-
325
methyl ~benzyl ammonium chloride; (B) alkyl (C8-Q8) dimethyl benzyl ammonium chloride; (C) alkyl (QC1~) naphthyl methyl pyridinium chloride; (D) dodecyl acetamido dimethyl benzyl ammonium chloride; and (E) cetyl trimethyl ammonium bromide. Henceforth these compounds will be referred to as quaternary A, quaternary B, etc. The dilutions tested ranged from 1:5,000 to 1:100,000, or from 1 oz. quaternary for 9.4 lb. of dry fabric to 1 Oz. quaternary for 187 lb. of dry fabric. Cotton, gauze-type diaper cloth was used as the carrier for the test organism. Two dry samples of this cloth, 4 by 5 inches, weighed 3 grams. Two test strips folded to fit conveniently into each Petri dish were autoclaved for twenty minutes at 250 ~ F., then dried in a hot air oven at 105 ~ C. The test organism was a typical strain of E . co li h a v i n g an average resistance of 1:90 to phenol, as determined by the method of the Association of Official Agricultural Chemists. 7 The stock culture was maintained at 5 ~ C. on Bacto-Tryptone Glucose Extract Agar slants ( T . G . E . ) - - t r y p t o n e 5 Gin., glucose 1 Gm., beef extract 3 Gm., agar 15 Gm., distilled water 1,000 c.c., with monthly transfer during the course of these studies. For the inoeulum 1 e.c. of a twentyfour-hour T.G.E. broth culture of the organism was used to seed Kolle flasks containing 60 c.e. of T.G.E. agar. After incubation at 37 ~ C. for twentyfour hours, the surface growth was removed with 50 e.e. of sterile distilled water and a homogeneous preparation obtained by filtration through coarse paper, Sehleicher and Schull, No. 588. The heavy suspension of cells was diluted to give a 50-60 reading on
326
THE JOURNAL OF PEDIATRICS
Klett-Summerson photoelectric colorimeter using No. 42 blue filter. This suspension, on f u r t h e r dilution of 1:20, provided a s t a n d a r d inoculum of approximately 10,000,000 cells per cubic centimeter. Each two samples, or 3 grains of fabric, were inoculated with 4 c.c. of the standardized cell suspension and introduced into a pint j a r containing 96 e.c. of medieant. Determinations were made in duplicate for each dilution of each quaternary. F o r controls, two samples of fabric similarly inoculated were placed in a j a r containing sterile distilled water. On the assumption that approximately 50 per cent of the organisms m a y remain on the cloth, the inoeulum described above yielded a fabric-water p r e p a r a t i o n containing on the order of 300,000 'bacteria p e r cubic centimeter of extractable liquid, which is roughly equivalent to numbers reported to be found in final rinse waters of home laundries, s The jars were t r a n s f e r r e d to a launder-ometer where they were agitated at indicated t e m p e r a t u r e s f o r fifteen minutes. Each sample was twisted with sterile forceps to express the liquid, then introduced into a test tube containing 20 c.c. of 5 p e r cent distilled w a t e r solution of Tamol N2 A f t e r ninety m i n u t e s ' exposure in the Tamol N to assure complete neutralization of excess quaternary, the samples were discarded and d i l u t i o n plate counts on the rinse waters in the tubes were made using T.G.E. agar. Counts were made in duplicate f r o m two rinse tubes selected to yield data f r o m the duplicate q u a t e r n a r y treatments at each dilution tested. The counts on the distilled w a t e r controls were used as a base for deter-
mining the per cent of organisms surviving exposure to the various dilutions of quaternaries according to the following formula : Average n u m b e r of colonies per c.e. rinse a f t e r exposure to q u a t e r n a r y • I00. Average n u m b e r of colonies per c.c. rinse of control fabric.
The per cent reduction of viable organisms was obtained by deducting from ]00 the figures obtained as above. W h e n the experimentally contamin a t e d fabric was exposed to dilutions of 1:5,000 quate'rnary, 1 c.c. and 0.1 e.c. aliquots of the neutralizer rinse provided countable plates. Dilutions of 1:10, 1:100, and 1:1,000 of Tamo] N yielded, in most instances, plates with 30 to 300 colonies when the fabrie was treated with dilutions ranging f r o m 1:10,000 to 1:100,000 quaternary. F r o m the eontrol Tamol N tubes, dilution plates of 1:100, 1:1,000, and 1:10,000 were necessary to obtain similar data. Colonies were enumerated in a Quebec counter a f t e r forty-eight hours' incubation at 37 ~ C. RESULTS AND DISCUSSION The results show that, in genera], each q u a t e r n a r y at each t e m p e r a t u r e exhibits a n i n c r e a s i n g effectiveness with increasing concentration against the test organism E. coli, as determined by survival of viable cells in the Tamol neutralizer rinse. The five quaternaries were more efficient in reducing numbers of this organism at 45 ~ C. than they were at 20 ~ C. Sanitization, i.e., a 99.9 per cent reduction of the contaminating organisms, required for all five quaternaries 1:5,000 dilution, or 1 oz. q u a t e r n a r y to 9.4 lb. of cloth, when applied at 45 ~ C. for fifteen minutes. A t 20 ~ C., quaternaries B and D showed a similar activity.
LATLIEF ET AL. :
QUATERNARY AMMONIUM COMPOUNDS AND TEXTILES
Quaternaries A, C, and E approached this effectiveness with more than 99 per cent destruction. Quaternaries B, C, and D, when applied at 45 ~ C. in dilution of 1:10,000, or 1 oz. of q u a t e r n a r y to 18.7 lb. of cloth, also brought about more than 99 per cent reduction in numbers. It should be pointed out that the reductions above 99 per cent but less than the 99.9 per cent selected as an end point for this study cannot be considered as absolute evidence of failure to sanitize since TABLE I .
treatment with these same quaternaries for fifteen minutes at 45 ~ C. required a dilution of 1:5,000 to bring about sanitization. I n spite of the longer exposure period, five to twenty times the concentration of the quaternaries were required to sanitize the fabric than to render it bacteriostatic for sixteen hours. With high temperature applications it appears possible, therefore, to secure fabrics which have t e m p o r a r y residual b acteriostatic effects at lower concentrations than are
REDUCTION IN -'r CELLS OF E . O0l~ W!-IEN THE INOEULATED FABaIC EXPOSED FOR FIFTEEN ~aCJ:INUTES AT 20 ~ C. AND ArT 45 ~ C'.
Quaternary dilution Ounce q u a t e r n a r y : p o u n d s d r y f a b r i c .• Temperature
327
1:100,000
1:50,000
1:25,000
1:1%000
WAS
1:5,000
1:187 1:94 1:47 1:18.7 1:9.4 Quaternary P e r cent R e d u c t i o n A 19 13 53 71 99.3 ]3 0 28 49 91 99.9 20 ~ C. C 0 0 49 92 99.6 I) 41 50 60 88 99.9 E 33 66 74 91 99.6 A 39 49 67 91 99.9 ]3 33 67 78 99.4 99.9 45 ~ C. C 56 51 80 99.5 99~9 D 44 69 78 99.2 99.9 E 30 67 89 98 99.9 *The c h e m i c a l - d r y fabric r a t i o s a r e m a t h e m a t i c a l c a l c u l a t i o n s of the a c t u a l di l ut i ons used.
differences of this magnitude probably fall within the r a n g e of experimental error in the counting methods used. Tha~ reduction in count seems to depend on the concentration of quatern a r y and temperature of application is evident since there is, in generaI, a gradual diminution of ceils from no reduction in certain instances to:99,9 per cent. The eoneentration and tentperature relationship was noted also in studies relating to baeteriostatie effects of these compounds. ~ There it was shown that fabric treated with d i h t i o n s ranging from 1:25,000 to 1:100,000 for ten minutes at 45 ~ C. subsequently i n h i b i t e d the growth of P. mirabilis for sixteen hours, whereas fabric inoculated with N. coli prior to
required to secure a practical sanitizing benefit. A f u r t h e r comparison of the baeteriostatic and sanitizing properties of these same quaternaries reveals that 1:5,000 to 1:10,000 dilutions at 20? C. for ten minutes rendered the test fabric baeteriostatie for sixteen hours, whereas a 1:5,000 dilution applied at 20 ~ C. for fifteen minutes produced sanitization, according to the selected end point, only with quaternaries B and D, although the reductions observed with quaternaries A, C, and E closely approached the desired level. Such data indicate a fairly dose correlation between the~eoneentrations necessary to secure practical sanitizing action and residual bacteriostatic effects when a p
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plieations are made at relatively low temperatures. On the other hand, for fabric treated for ten minutes at 20 ~ C. and 45 ~ C. to remain bacteriostatie for twentyfour hours or more, dilutions ranging from 1:1,000 through 1:4,000 were found necessary. Thus, if prolonged baeteriostatie effects are desired, it would appear that concentrations considerably higher than those required for sanitizing would have to be employed in treating' fabrics. Insofar as the sanitizing' capacity of these quaternaries is concerned, these results seem to confirm the findings reported by Lawrence which showed that a 1:5,000 dilution of alkyl (C sCls) dimethyl benzyl ammonium ehlo~ ride was useful for this purpose. This sanitizing value seems, however, to be possessed by the other four compounds as well, provided time and temperature of treatment are adequate. Mack and associates, ~ on the other hand, concluded that the use of baeteriostatic agents does not reduce total bacterial counts of E. coll. The findings herein reported have demonstrated that quaternary ammonium compounds do reduce total counts of E. coli in fabrics provided they are employed a t suitable concentrations. Also, these reductions were considered to be of a magnitude sufficient to warrant claims of sanitizing value when the chemicals were properly applied, although at dilutions of 1:5,000 and above they did fall somewhat short of the end point which would be required to provide presumptive evidence of disinfection or a treatment that could be considered adequate for fabrics known to carry or strongly suspected of carrying infectious material.
CONCLUSIONS
As judged by their ability to reduce by 99.9 per cent the E. coli count in rinse waters of experimentally contalninated fabrics over control rinse waters, the five quaternaries studied appear to have potential value as sanitizing agents in ordinary laundry operations. The efficiency of all five quaternaries in reducing the E. ~oli count of fabrie rinse waters has been shown to depend upon the concentration of the chemical (related possibly to fabric-chemical ratio) and temperature of application. All five quaternaries, (A) para diisobutyl ph.enoxy ethoxy ethyl dimethyl benzyl ammonium chloride, (B) alkyl (Cs-C18) dimethyl benzyl ammonium chloride, (C) alkyl (C6-C12) naphthyl methyl pyridinium chloride, (D) dodeeyl acetamido dimethyl benzyl ammonium chloride, and (E) eetyl trimethyl ammonium bromide, when applied at 45 ~ C. for fifteen minutes in a dilution of 1:5,000 or at 1 oz. quaternary to 9.4 lb. of dry fabric, reduced by 99.9 per cent the E. coli count of the subsequent experimental neutralizer rinse over controls. At 20 ~ C. with fifteen minutes' exposure, all five quaternaries reduced the E. coli count of the experimental rinses by more than 99 per cent. A reduction of 100 per cent was not secured in the E. coli count of rinse. waters with any of the five quaternaries studied at dilutions of 1:5,000 and above at either 20 ~ or 45 ~ C. Thus, the potential sanitizing values noted cannot be considered as presumptive evidence of complete germicidal action which would be essential to indicate value for use in disinfection.
LATLIEF ET AL. :
QUATERkNARYAMMONIU1V[ COMPOUNDS AND TEXTILES 329
REFERENCES 1. Latlief, M. A., Goldsmith, M. T., Friedl, J. L., and Stuart, L. S.: Bacteriostatic, Germicidal, and Sanitizing Action 0s Quaternary Ammonium Compounds on Textries. Prevention of Ammonia Formation From Urea by Proteus mirabills, J. pEDIAT. 39: 730, 1951. 2. Lawrence, C. A.: Surface Active Quaternary Ammomnm Germieides, Academic Press, ]950, p. 188. 3. Bartlett, P. G.: Quaternaries in the Laundry Field, Soap & Sanit. Chem. 25: (3): 139, 1949. 4. Mack, P. B., ed.: Bacteriostatic Agents and Sterilization in the Diaper Laundry, Pennsylvania State College, Diaper Serv. Fellowship Res. Rpts. 32-33, 1949.
5. Roberts~ E. H.: The Bactericidal Effectiveness of I-Iome Laundering Methods for Silk and Rayon, Washington Agriculture Experimental Station Bull. 265, 1932. 6. Weber, G. R., and Black, L. A. : Laboratory Procedure for Evaluating Practical Performance of Quaternary Ammonium and Other Germicides Pro.posed ~or Sanitizing Food Utensils, Am. J. Pub. Health 38: 1405, 1948. 7. Association of Official Agricultural Chemists: Official Methods of Analysis, ed. 7, Washington, D. C., 1930, p. 88. 8. American Institute of Laundering: Home Washing Survey, Tech. Bull. No. 47. 9. Goetchius, G. R.: A Quaternary Inactivatot, Soap & Sanit. Chem. 25: (1): 131, 1949.
Erratum I n the a r t i c l e b y Dr. B. B. Breese, " A u r e o m y c i n i n the T r e a t m e n t of B e t a H e m o l y t i c Streptococcal I n f e c t i o n s , " i n the J a n u a r y , 1952, issue, the first sentence u n d e r A u r e o m y c i n Dosage on page 86 should r e a d : The i n i t i a l dose g i v e n was 20 rag. p e r k i l o g r a m of body weight in t w e n t y f o u r h o u r s a n d c a l c u l a t e d to the n e a r e s t 50 rag.