STUDIES ON THE ACTION OF WETTING AGENTS

STUDIES ON THE ACTION OF WETTING AGENTS ON MICROORGANISMS I. THE EFFECT OF ph AIM WETTING AGENTS ON THE GERMICIDAL ACTION OF PHENOLIC COMPOUNDS E. J. ORDAL, J. L. WILSON,1 ANi A. F. BORG Laboratories of Bacteriology and Oceanography, University of Washington, Seattle Received for publication December 19, 1940 Soaps have been extensively used, often with added alkali, to emulsify or make soluble phenolic compounds of limited solubility (Klarmann, Shternov and Von Wowern, 1929; McCulloch, 1936). There is, however, a good deal of controversial information in the literature dealing with the effect of soaps on the germicidal action of phenolic compounds. Frobisher (1927) reported that small amounts of sodium oleate delayed, larger amounts enhanced, and still larger amounts destroyed the germicidal activity of phenol. Hampil (1928) stated that pure soaps decreased the germicidal activity of phenol, meta-cresol, and secondary butyl phenol. Tilley and Schaffer (1925) showed that the bactericidal efficiency of cresol soap mixtures was greatly affected by the proportions of soap and cresol used. Schaffer and Tilley (1930) found that coconut oil soap, castor oil soap or linseed oil soap, when mixed with various phenolic compounds in the proportion of 1 part soap to 2 parts of phenol increased the germicidal efficiency of the phenols. Halvorson, Baylis, Ordal, and Wilson (1935), in a study of germicidal detergent compositions for simultaneous washing and disinfection, found that the addition of small amounts of various soaps and other wetting agents enhanced considerably the germicidal action of moderately alkaline detergent mixtures containing diphenols. Economics Laboratory, Inc., St. Paul, Minnesota. 117

118 E. J. ORDAL, J. L. WILSON AND A. F. BORG Tilley and Schaffer (1931) showed that the germicidal efficiency of phenol and ortho-cresol decreased coincidentally with an increase in percentage of sodium hydroxide added, until the percentage of sodium hydroxide equalled or slightly exceeded that required to neutralize the phenol. Since phenols are weak acids this indicates that phenolates are less efficient germicidal agents than the corresponding unionized phenols. Solutions containing soaps and phenols must necessarily be alkaline since the solubility of the higher fatty acids in water is extremely low. The degree of neutralization of the phenol which results, will depend upon the proportions and characteristics of the soap and phenol used. In order to evaluate the effects of soaps on phenolic compounds it is necessary, therefore, that the hydrogen ion concentration of a given solution be known, since only under these circumstances will the actual composition of the solution be known. From the dissociation constant it is then possible to determine the concentrations of undissociated phenol and of phenolate in a given solution. Many of the discrepancies in the literature can be accounted for by the neglect of control of the hydrogen ion concentration. This investigation deals with the effect of several soaps and of sodium lauryl sulfonate on the germicidal action of phenolic compounds in moderately alkaline solutions where the ph of the solution is rigidly controlled. METHODS Staphylococcus aureus (Insectide Board Strain) was used as test organism. This organism was selected because it is relatively resistant to the action of alkalis and because it possesses marked resistance to the action of soaps (Walker, 1924). Except in the experiment reported in table 5, germicidal tests were made according to the modified Reddish technique described by Cade and Halvorson (1934). Briefly this is as follows: Five ml. of the mixture to be tested was placed in a sterile test tube 25 by 150 mm., placed in a water bath at 250 or 37.50C. i 0.10C. When the solution in the test tube had reached the

ACTION OF WETTING AGENTS ON MICROORGANISMS 119 temperature of the water bath, 0.5 ml. of a 24-hour culture of Staphylococcus aureus was added, and the tube agitated thoroughly. At five minutes and ten minutes from the time of addition of the culture, standard loopfuls of the mixture were transferred to tubes of sterile, molten, nutrient agar at 42-44TC. The tubes were rotated between the palms of the hands to insure mixing, flamed, and the contents poured into sterile Petri dishes. After solidifying, the plates were inverted and placed in an incubator at 37.50C. At 48 hours the colonies were counted and recorded. Plates with 0:500 colonies were counted completely, proportional sections were counted on plates with 500:10,000 colonies, and plates with a number of colonies greater than 10,000 were arbitrarily designated "10,000+." This procedure gave a wider latitude in comparing germicidal solutions than the standard Reddish technique using tubes of broth. The solutions tested were made up from stock solutions adjusted to the desired ph. Except in the experiment reported in table 4, where the ph was determined calorimetrically, the ph of buffers and phenols was determined at 25TC. with Bailey hydrogen electrodes, and the ph of solutions containing wetting agents was determined with glass electrodes calibrated against buffer solutions at the desired ph and with similar sodium ion concentration. A Leeds and Northrup type K potentiometer was used in conjunction with a Leeds and Northrup thermionic amplifier. In the experiment reported in table 5, one ml. of a 24-hour culture of Staphylococcus aureus was added to 49 ml. of the solution to be tested in a 125 ml. Erlenmeyer flask at 37.50C. After 10 minutes exposure a sample was withdrawn and plated. Dilutions were made using measured quantities of sterile M/50 phosphate buffer at ph 7 in sterile 250 ml. Erlenmeyers as dilution media. Three or five plates were made from each dilution. EXPERIMENTAL RESULTS In table 1 the germicidal action of phenol at ph 10 is compared with the action of unbuffered phenol at 25TC. and 37.50C. It is apparent that at 25TC. a 1: 35 solution of phenol at ph 10

120 E. J. ORDAL, J. L. WILSON AND A. F. BORG has very nearly the same activity as a 1:70 solution of unbuffered phenol. The dissociation constant of phenol at 250C. is 1.06 X 10-1 (International Critical Tables). On the assumption that the concentrations of undissociated phenol and of phenolate are equal to the activities, the actual concentration of the undissociated phenol in the 1:35 solution at ph 10 is 1.38 per cent, whereas the concentration of undissociated phenol in the 1:70 control is 1.43 per cent. This result suggests that the undissociated phenol is the active germicidal agent. However, a solution TABLE 1 The germicidal action of phenol in I per cent carbonate buffer at ph 10 NBACTURIAL COUNT DIUTION OF PRUNOL 25C. 37.50C. 5 Mpi. 10mi. 5min. 10 min. ph 10 1:35 35 2 0 0 1:40 750 110 0 0 1:50 600 1400 80 0 1:60 10,000+ 10,000+ 1300 190 Control (unbuffered) 1:70 16 0 0 0 1:80 410 75 0 0 1:90 3500 800 33 0 1:100 10,000+ 10,000+ 900 151 K. (phenol) = 1.06 X 10-10 at 250C. Staphylococcus aureus (24 hour culture). of 1:50 phenol at ph 10 is somewhat more active than a 1:100 solution of unbuffered phenol, although the concentrations of undissociated phenol in the two solutions are very nearly the same. Since no appreciable germicidal activity against S. aureus can be ascribed to a 1 per cent carbonate buffer at ph 10, this must indicate that the phenolate ion has some toxicity. At 37.50C. a 1:90 solution of unbuffered phenol is nearly equivalent to a 1:50 solution at ph 10. Here, the actual concentration of undissociated phenol can not be determined since the dissociation constant of phenol at 37.50C. is not available.

ACTION OF WETTING AGENTS ON MICROORGANISMS 121 The effect of the addition of several soaps and sodium lauryl sulfonate to buffered phenol solutions at ph 10 is shown in table 2. Clearly, the addition of the wetting agents increases the germicidal action of the solutions containing phenol. There is, however, a distinct difference in the effects of the several wetting agents. The solutions containing sodium palmitate and the solution containing sodium myristate but no phenol were gels. Improper mixing with the culture may explain in part the inferior germicidal action of these solutions. TABLE 2 The effect of specific wetting agents on the germicidal action of phenol in 1 per cent carbonate buffer at ph 10 x vdacteza COUN Wetting agent t Sodium Sodium Sodium SOdiUM Sodium N z oleate palmitate myristate laurate ricinolate sulf None 5 10 5 10 a 10 5 10 5 10 5 10 5f 10. in. 10. *i. 1in. Ain. mi. A. in. min. min. mi. n. mman 1:80 3 0 308 31 0 0 0 0 17 0 0 0 104+104+ 1:90 7 0 1200 217 0 0 0 0 28 0 0 0 10'+ 10'+ 1:100 11 0 1800 470 0 0 4 0 182 6 0 0 10'+ 10'+ 1:110 12 0 3300 90 0 0 14 0 92 6 0 0 10'+ 10'+ 1:120 67 6 10'+ 10'+35 0 113 5 650 97 0 0 104+ 10+ None 10'+ 10'+ 104+ 10'+ 10'+ 10'+ 10'+ 10'+ 10+ l0'+ 104+ 1l0'+ 10'+ 10'+ Wettingagents presentin 0.1 percentconcentration. Staphylococcus aureus (24 hour culture). Temperature of experiment 37.50C. A comparison of sodium oleate and sodium laurate over a range of concentrations from 0.006 to 1.6 per cent is shown in table 3. There is a distinct difference in the behavior of the two soaps on dilution. In the stronger concentrations sodium laurate is more effective than sodium oleate, but on dilution there is a sharp loss of activity with sodium laurate, while sodium oleate remains relatively effective over a wider range of concentrations. No attempt has been made as yet to correlate this behavior with the surface tension or interfacial tension of the solutions. The decrease in germicidal activity of several phenolic compounds with increase in alkalinity and the effect of adding 0.1 per cent sodium laurate to each solution is shown in table 4. At each

122 E. J. ORDALt, J. L. WILSON AND A. F. BORG given hydrogen ion concentration, the addition of the soap enhances the germicidal activity of the solutions containing phenolic compounds. The information obtained from the experiments shown in tables 1 to 4 is limited by the method. The data present show TABLE 3 The effect of concentration of soaps on the germicidal action of phenol in I per cent carbonate buffer at ph 10 DILnTION OF PRUNOL DaCTUg COUN CONCENTRATION 0F1AP olesteoapdium laumte 5 min. 10 5 min. 10 min. 1:80 1.6 0 0 0 0 1:100 1.6 15 0 0 0 1:120 1.6 66 2 0 0 1:80 0.4 2 0 0 0 1:100 0.4 2 0 0 0 1:120 0.4 23 0 0 0 1:80 0.1 0 0 0 0 1:100 0.1 10 1 0 0 1:120 0.1 23 4 47 1 1:80 0.025 2 0 900 48 1:100 0.025 5 0 5,000 900 1:120 0.025 17 4 10'+ 7,000 1:80 0.006 6 0 3,300 605 1:100 0.006 175 10 104+ 8,00 1:120 0.006 614 85 104+ 10'+ Staphylococcus aureus (24 hour culture). Temperature of experiment 37.50C. that the addition of wetting agents to solutions of phenols in buffered alkaline solutions does in general increase the germicidal action of such solutions. The decrease in germicidal activity of phenol at ph 10 due to partial neutralization may be more than compensated for by the addition of some wetting agents. This is shown by a comparison of the action of undissociated phenol

ACTION OF WETTING AGENTS ON MICROORGAMSMS 123 (table 1) with the action of solutions to which sodium lauryl sulfonate, sodium laurate, sodium myristate, and sodium oleate have been added (table 2). It is shown in table 3 that the increase in activity of the phenol soap solutions is a function of TABLE 4 The effect of sodium laurate on phenolic compounds in I per cent carbonate buffers BACTERIAL COUNT SODIUM LAU- PHUNOUC COMPOUND ph 9 ph 10 ph 11.2 5min. 10mim. 5min. 10min. 5 mn. 10min. None None 104+ 104+ 104+ 10'+ 104+ 104+ 0. 1% None 10+ 10'+ 104+ 104+ 8500 1500 None 0.045% thymol 100 8 6000 600 10'+ 104+ 0.1% 0.045% thymol 0 0 0 0 40 0 None 0.10% o-phenyl- 0 0 150 14 104+ 104+ phenol 0.1% 0.10% o-phenyl- 0 0 0 0 150 5 phenol None 0.07% o-phenyl- 85 0 104+ 104+ 104+ 104+ phenol 0.1% 0.07% o-phenyl- 0 0 0 0 500 55 phenol None 0.04% 2-chloro-o- 0 0 2,200 600 104+ 104+ phenylphenol 0.1% 0.04% 2-chloro-o- 0 0 0 0 1200 60 phenylphenol None 0.017% 2-chloro-o- 600 300 104+ 104+ 104+ 104+ phenylphenol 0.1% 0.017% 2-chloro-o- 0 0 1000 220 2000 250 phenylphenol Staphylococcus aureus (24 hour culture). Temperature of experiment 37.50C. the concentration as well as the character of the wetting agent. From table 4 it appears that a particular soap, sodium laurate, increases the germicidal action of phenolic compounds other than phenol, and that this effect occurs at ph 9 and ph 11.2 as well as at ph 10. However, the data do not provide an explanation

124 E. J. ORDAL, J. L. WILSON AND A. F. BORG for the increased germicidal activity obtained on the addition of the wetting agents to the solutions containing phenols. Further information on the germicidal behavior of buffered solutions, with or without phenol, and wetting agents was sought by counting the number of colonies from organisms surviving exposure to these solutions for a fixed period of time. The procedure used is described in the section on methods. The results are shown in table 5. TABLE 5 The effect of wetting agents on the germicidal action of phenol Buffer, 1 per cent DLmUON TTAL MLAT COUNTS PAR ML. AlTMR 10 WnITH ZXPOBUIM OF WRIrNGO AGeN PENOL ph 7 ph 9 ph 10 ph 11 15,600,000 14,000,000 17,000,000 16,000,000 1:200 3,100,000 3, 900, 000 1 1, 000, 000 7,000,000 0.1% sodium 4,700,000 1,400,000 420,000 laurate 1:200 0.1% sodium 85 17,000 23,300 laurate 0.1% sodium oleate 2,800,000 830,000 93,300 1:200 0.1% sodium oleate 520 5400 11,700 0.1% sodium lauryl 3,200,000 3,300,000 980,000 110,000 sulfonate 1:200 0.1% sodium lauryl 1000 400 2800 12,900 sulfonate Staphylococcus aureus (24 hour culture). Temperature of experiment 37.50C. Carbonate buffers were used except at ph 7 where a phosphate buffer was used. No appreciable germicidal activity can be ascribed to the buffer systems tested. The differences in plate count after exposure to the several buffers must be ascribed to experimental error and to the error of random sampling. It is apparent that 1: 200 buffered solutions of phenol do exhibit germicidal action and that there is a progressive decrease in the germicidal action between ph 7 and ph 10. This coincides with a decrease in the concentration of undissociated phenol as indicated in table 6.

ACTION OF WETTING AGENTS ON MICROORGANISMS 125 Buffered solutions of sodium laurate, sodium oleate, and sodium lauryl sulfonate exhibit germicidal action which becomes progressively greater with increase in alkalinity from ph 9 toph 11. Solutions containing buffer, phenol, and wetting agent are definitely more germicidal than solutions containing buffer and phenol, or buffer and wetting agents, although the effectiveness of such solutions decreases with increase in alkalinity from ph 9 to ph 11. Since the germicidal action of solutions containing buffer, phenol and wetting agents decreases, whereas the action of the solutions with buffer and wetting agents becomes greater when the alkalinity is increased from ph 9 to ph 11 it seems logical to conclude that here, as with the solutions of phenol without TABLE 6 Ratio of concentrations of phenolate and undisaociated phenol ph 7 9 10 11 C.H6OJ/C6H5OH...1.06 X 10-' 0.106 1.06 10.6 wetting agents, the undissociated phenol is more toxic than the phenolate. One might consider the increased germicidal action of solutions contaming buffer, phenol, and wetting agents to be due to a sensitization of the cells by the wetting agent making it more susceptible to the action of the undissociated phenol. However, further investigation is required before any final answers can be given to questions concerning the mechanism by which the wetting agents increase the toxicity of solutions containing phenols. SUMMARY 1. Undissociated phenol is more toxic to Staphylococcus aureus than phenolate. 2. The addition of wetting agents to buffered solutions of phenolic compounds does in general increase the germicidal

126 E. J. ORDAL, J. L. WILSON AND A. F. BORG activity of such solutions. The specific effect is a function of the character as well as concentration of the wetting agent employed. 3. The germicidal action of solutions containing buffer, phenol, and wetting agent is greater than that of solutions containing buffer and phenol or buffer and wetting agent at a given hydrogen ion concentration. 4. The germicidal action of solutions containing buffer, phenol and wetting agent decreases with increase in alkalinity from ph 9 to ph 11 although the germicidal action of solutions of buffer and wetting agents increases in the same range. This indicates that the presence of the wetting agent enhances the action of the undissociated phenol more than that of the phenolate. REFERENCES CADE, A. R., AND HALVORSON, H. 0. 1934 The synergistic action of soaps on the germicidal efficiency of alkalies. Soap, 10, 17-19. FROBISHER, M. 1927 Studies upon the relationship between surface tension and the action of disinfectants with special reference to hexyl resorcinol. J. Bact., 13, 163-182. HALVORBoN, H. O., BAYLISS, M., ORDAL, E. J., AND WILSON, J. L. 1935 Germicidal detergents. Soap, 11, 25-28. HAMPIL, B. 1928 The effect of pure soaps on the bactericidal properties of phenolic germicides. J. Bact., 16, 287-300. International Critical Tables of Numerical Data, Physics, Chemistry, and Technology; prepared by the National Research Council of the U. S. A., 1929. KLAP.ANNY, E., STCERNOV, V. A., ANi VON WowuAN, J. 1929 The germicidal action of halogen derivatives of phenol and resorcinol and its impairment by organic matter. J. Bact., 17, 423-442. MCCULLOCH, E. C. 1936 Disinfection and Sterilization. Lea and Febiger, Philadelphia. 526 pp. ScEmVzER, J. M., AND TILLEY, F. W. 1930 Germicidal efficiency of soaps and of mixtures of soaps with sodium hydroxide or with phenols. J. Agr. Research, 41, 737-747. TILLEY, F. W., AN SCHAub, J. M. 1925 Germicidal efficiency of coconut oil and linseed oil soaps and of their mixtures with cresol. J. Infectious Diseases, 37, 369-367. TIuLr, F. W., AND SCHAnFER, J. M. 1931 Germicidal efficiency of mixtures of phenols with sodium hydroxide, with glycerine and with ethyl alcohol. J. Agr. Research, 43, 611417. WALKER, J. E. 1924 The germicidal properties of chemically pure soaps. J. Infectious Diseases, 35, 667-6.