MAY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
The characterization factor and the point representing this stock on the rectangular portion of Figure 5 may be located from either the viscosity a t 210" F. and the gravity, or from the gravity and boiling point. Two slightly different points, 1 and 2, result. For estimation of viscosities it is preferable t o use the index point based on a viscosity measurement. This fixes the characterization factor at 10.32 and the index point a t 3. The viscosity a t 900" F. is then estimated by projecting a line from 900 on the temperature scale through t h e index point to the viscosity scale where it intersects a t a value of 0.2 centistoke. From Figures 4 and 5 the viscosity of any liquid stock a t temperatures u p to the critical may be estimated from ordinary laboratory inspection data. Pressure up to 500 pounds per square inch has little effect on these relationships. A complete viscosity-temperature curve for a heavy stock requires combination of the high-temperature data estimated from Figure 5 with actual laboratory measurements a t temperatures below 210" F. The complete curve thus derived may or may not be a straight line below 210" F. when plotted
609
on Figure 4, depending on the width of boiling range and other factors such as wax content.
Literature Cited (1) Andrade, E. N. da C., Nature, 125,309-10 (1930). (2) Coats, H . B . , Brown, G. G., Dept. Eng. Research Univ. Michigan, Circ. Series 2 (Dec., 1928). (3) Cragoe, C. S., Proc. World Petroleum Congress, London, 1933, 529. (4)Fortsch, A. P., and Wilson, R. E., IND.ENG.CHEM.,17, 291 (1925). (5) Generau, R. P . , Ibid,,22, 1382 (1930). (6) Hatschek, E., "Viscosity of Liquids," 1 s t ed., 1928. (7) Herschel, W. H., IRD.ENQ.CHEM.,14, 718-19 (1922). (8) Lederer, E. L.,Kolloid-Beihefte, 34, 270-338 (1931). (9) Watson, K. M., and Nelson, E. F., IND.ENG.CHEM.,25,880-7 (1933). (10) Watson, K. M., Nelson, E . F.,Murphy, G. B.,Ibid., 27, 1460-4 (1935). (11) Watson, K. M . , and Wirth, C., IKD. ENQ.CREM.,Anal. E d . , 7, 72 (1935). RECEIVED December 4, 1935. Presented before the Division of Petroleum Chemistry a t the 90th Meeting of the American Chemical Society, San Fiancisco, Calif., August 19 t o 23, 1935.
Germicidal Properties of Phenolic Compounds CECIL G. DUNN Massachusetts Institute of Technology, Cambridge, Mass.
HE research presented in this paper was undertaken to determine the germicidal efficacy, under various conditions, of different solutions of sec-amyltricresol, o-hydroxy phenylmercuric chloride and a mixture, known as Mercresin, of the two chemicals. Information was also sought as to how solutions of these chemicals compared with some market mercurials and a phenol derivative in germicidal action.
Experimental Procedure Food and Drug Administration methods ( 2 ) were followed. Preliminary work with mercurials showed that the apparent phenol coefficients obtained were often higher than the true ones, as the quantity of chemical carried from the medication tube to the broth subculture tube in the 4-mm. loopful required by the F. D. A. method was large enough to cause bacteriostasis. For this reason a second tube of sterile broth was always inoculated with four to seven 4-mm. loopfuls of material from the tube which had been inoculated directly from the medication tube. In a large part of the work a standard quantity of seven loopfuls was secondarily transferred. The secondary transfers were completed at once after each test in order to secure greater accuracy. Although the F. D. A. method requires results to be based on the 48-hour observation, incubations over longer periods of time were frequently made to ascertain what part bacteriostatic action played.
sec-Amyltricresol, o-Hydroxyphenylmercuric Chloride, and a Mixture of the T w o Test Organisms Staphylococcus aureus Rosenbach ( I ) (Government strain
No. 209) and Eberthella typhosa (Zopf) Weldin (Hopkins strain) were secured from the Food and Drug Administration a t Washington. Cultures of the same two organisms were also obtained from the Boston Biochemical Laboratory. The Health Department of Massachusetts a t Boston furnished a culture of a hemolytic streptococcus. Corynebacterium diphtheriae (Flugge) Lehmann and Neumann was received from the Harvard Medical School, and Sarcina ventriculi Goodsir from Brown University. Staphylococcus aureus Rosenbach, Escherichia coli (Migula) Castellani and Chalmers, Pseudomonas aeruginosa (Shroeter) Migula, Serratia p yoseptica (Fortineau) Bergey et all Proteus vulgaris Hauser, Staphylococcus citreus (Migula) Bergey et al., and Streptococcus Zactis (Lister) Lohnis were taken from the stock collection of the Department of Biology and Public Health a t the Massachusetts Institute of Technology.
sec-Amyltricresol (Pentacresol) sec-Amyltricresol was dissolved to the extent of 1 per cent in a 2 per cent soap solution (a potassium soap prepared from two parts of coconut oil and 2 parts of castor oil); and the germicidal efficacy of the soap solution of the chemical was determined, using first Staph. aureus (Government 209) as the test organism at 20" C. A phenol coefficient of 1 was ob-
INDUSTRIAL AND ENGINEERIYG CHEMISTRY
610
tained. Its efficacy against E. coli was next determined. The test organism was destroyed by the undiluted 1 per cent stock solution of Pentacresol in 45 but not 30 minutes. Specific action towards Staph. aureus, a Gram-positive organism, was thus indicated. Since E. coli is a Gram-negative organism, other Gram-negative and Gram-positive bacteria were used in further tests. TABLE I. GERMICIDAL ACTIONOF PEINTACRESOL -Phenol Test Organism
1% solution
CoefficientFullstrength chemical
1% in 2% Soap Solution Staph. aureus (Gov. 209) 1 100 Staphylococcus aureus (Sherman) 1 100
Ternperature
c.
Gram Reaction
++ ++ +-
20 20 Staph. citreu8 2.6 250 20 Sarcina ventriculi 2 200 20 Streptococcus lactis 2 200 20 E. tvphosa (Hopkins) 7+ 37 O.07f 1% in 50% Alcohol-10% Acetone Solution 1+ A hemolytic Streptococcus" loo+ 37 C. diphtheriae" 0.4+b 40437 E. typhosa (Hopkins) 0.14 14 37 a Grown and subcultured in beef infusion broth (final pH 7.6). b Did not survive for 5 minutes in a 1: 80 dilution, the highe)st dilution run.
+ +
~~~
Staph. citreus, Sarcina ventriculi, and Streptococcus lactis, all Gram-positive bacteria, were easily destroyed by Pentacresol in high dilution; phenol coefficients of 2.5, 2, and 2, respectively, were obtained in tests conducted a t 20" C. On the other hand, P s . aeruginosa, Ser. pyoseptica, and Proteus vulgaris, Gram-negative bacteria, were much more resistant to this solution; the test organisms survived in the undiluted 1 per cent solution for more than 15 minutes. There were three possible causes for the specificity thus shown by the soap solution of the chemical. Either the Pentacresol, the soap solution, or the solution of the two, was specific in its action. To determine whether the Pentacresol was the cause of specificity and to eliminate any possible contributing action of the soap, a 1 per cent solution was prepared in 50 per cent alcohol and 10 per cent acetone. Dilutions of 1:2,1:3, 1:4, 1:5, l:lO, 1:20, and 1:60 (which correspond to 50, 33.3, 25, 20, 10, 5, and 1.67 per cent of the 1 per cent tincture, respectively) were prepared from this stock solution. At the same time dilutions were made u p from 5 per cent phenol for the purpose of showing, in the subsequent tests, that the resistance of each test organism employed was a t least equal to that demonstrated in the foregoing tests with the soap solution of Pentacresol (which proved to be the case). The following organisms were then used in germicidal tests a t 20" C.: Staph. aureus, Streptococcus lactis, E. coli, Ps. aeruginosa, and Proteus vulgaris. Results of the tests showed that the alcohol-acetone solution of Pentacresol was specific in its action towards Gram-positive bacteria, but that it was decidedly more effective towards Gram-negative bacteria than the soap solution of the chemical. The two Gram-positive bacteria, Staph. aureus and Streptococcus lactis, were destroyed in less than 5 minutes in the 1:20 dilution. E . coli and Proteus vulgaris were destroyed in less than 5 minutes, and P s . aeruginosa in less than 10 minutes, in the 1:2 dilution of the tincture, but all three of these Gram-negative bacteria survived for a t least 15 minutes in the 1 :4 dilution. I n order to ascertain what germicidal action could be attributed to the alcohol and acetone present in the stock solution of the 1 per cent Pentacresol, solutions containing varying percentages of 95 per cent alcohol and acetone were prepared, and germicidal tests were made. The solvents present in the stock solution were sufficiently germicidal to destroy each of the test organisms employed in the above tests in less
VOL. 28, NO. 5
than 5 minutes. But the solvents present in the various dilutions prepared from the stock solution of Pentacresol ( I :2, 1:3, 1:4, etc.) failed in all cases to destroy any of the test organisms in 15 minutes. Staph. aureus (Government 209), Streptococcus lactis, E. coli, P s . aeruginosa, Ser. pyoseptica, and Proteus vulgaris were subjected to the action of the 2 per cent soap (which contained no Pentacresol) a t 20" C. The bacteria survived for 45 minutes, the duration of the test, in all cases. The soap showed no germicidal properties and thus apparently contributed nothing to the specificity demonstrated by the solutions of Pentacresol. The results of germicidal tests with some of the common pathogens (Table I) showed that Pentacresol was specific not only for Gram-positive cocci but for Gram-positive rods as well. Table I shows that Pentacresol was highly efficacious as a germicide against Gram-positive bacteria, pathogenic and nonpathogenic. It also demonstrates the superiority of the tincture over the soap solution where E. typhosa was concerned.
Germicidal Action of Pentacresol in Presence of Organic Matter Germicidal tests were carried out according to F. D. A. methods (2) with the 1 per cent tincture of Pentacresol in the presence of 20 per cent sterile horse-blood serum with Staph. aureus (Government 209) and E. coli as test organisms a t 30" C. Comparative tests were conducted a t the same time with three of the leading market mercurials (tinctures), mercuric chloride excluded, a phenol derivative, solutions of ohydroxyphenylmercuric chloride (the Mercarbolides), and Mercresin. When Staph. aureus was used as the test organism in the presence of 20 per cent serum, the phenol coefficient of the 1 per cent tincture of Pentacresol was 0.16, or nearly three times that of the most effective mercurial (mercurial A) and over eight times that of the phenol derivative. Although there was a reduction in germicidal efficacy due to the presence of blood serum when Staph. aureus was employed, there was practically no reduction when E. coli was used as the test organism; a phenol coefficient of 0.05 was obtained. This value was just one-half that of the most effective mercurial (mercurial A), but was greater than that of market mercurial C and far superior to that of the phenol derivative.
The Mercarbolides "Mercarbolide" is the name given a preparation containing
A 0.1 per cent o-hydroxyphenylmercuric chloride,
()H&l. OH Three solutions of this mercurial were investigated-an aqueous solution, an aqueous glycerol-sodium chloride solution, and an aqueous alcohol-acetone-sodium chloride solution. Analyses of these solutions are given in Table 11. Table I1 and other data not presented here demonstrate several facts. The apparent phenol coefficients of the three solutions of Mercarbolide were four to ten times as great as the true coeficients when Staph. a w e u s was employed as the test organism; the difference was due to bacteriostasis. The aqueous glycerol-sodium chloride solution did not destroy Staph. aureus in the usual 5-minute period, but the solution possessed bacteriostatic properties in the lower dilutions which, over a period of time, were equivalent to bactericidal action. Phenol coefficients obtained with E. typhosa were eighteen to one hundred times greater than corresponding coefficients secured with Staph. uureus. Secondary subcul-
MAY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
The data in Tables I, 11, and I11 demonstrate clearly that Mercresin combines in many respects the advantages of both Mercarbolide and Pentacresol: the strong action of the former against such Gram-negative organisms as E. typhosa and E. coli, and of the latter against Gram-positive organisms such as the hemolytic streptococcus and Staph. aureus. When Staph. aureus was employed as the test organism, the germicidal efficacy of Mercresin varied directly with that of phenol as the temperature was changed.
TABLE 11. GERMICIDAL ACTIONOF MERCARBOLIDE Mercarbolide Solution" Aqueous
Test Organism Stc,z&kV. 2a9;eus Same E. typhosa (Hopkins)
E . typhosa
--Phenol Apparent 0.17f
.... ...
. . ..
Coefficientr-TruebA B 0.038
....
0 . 6 7 or greater
1.4*
38
. , ..
570 or greater
--TestOrganinm Not TemDilu- Dede- peration stroyedstroyed ture Min. Min. a C.
..
1:l
..
.. ..
..
..
15
37 20
..
37
611
1400
Effect of Exposure Period on Germicidal Action of Mercresin Tests to determine the effect of aqueous d i l u t i o n s of Mercresin on Staph. aureus (Government 209) were carried out according to F. D. A. methods a t 37" C., varying. the Deriods of exDosure from a few seconds to several hours. The time recorded for the short exposures was that elapsing between the instant when the first drop of culture struck the chemical solution in the medication tube and the instant when the 4mm. loopful of material from the medication tube was deposited into sterile Reddish broth. The average results of the tests involving the lower dilutions are shown in Figure 1. The test organism was destroyed in less than 15 seconds by
a Each stock solution contained 0.1% o-hydroxyphenylmercuric chloride besides the constituents listed. b The coefficients recorded i n column A are based on the 1:lOOO solution (0.1%) of o-hydroxyphenylmercurio chloride; those i n column B , on the original o-hydroxyphenylmercuric ohloride.
u
ture tubes, though used in the germicidal tests with E. typhosa, were found to be unnecessary because of the high dilutions in which Mercarbolide was lethal to this organism. 4 comparison of the results shown in Table I1 indicates that the tincture was superior to the aqueous glycerolsodium chloride solution and equal to the aqueous solution in respect to germicidal action against Staph. aureus a t 37" C. However, the aqueous glycerol-sodium chloride solution appeared to be best in germicidal efficacy towards E . coli and E . typhosa. The germicidal efficacy of the Mercarbolides varied directly with that of phenol as the medication temperature was changed and when Staph. aureus was employed. Bacteriostatic action in the subculture tubes, inoculated from the lower dilutions of o-hydroxyphenylmercuric chloride during'tests, corresponded to bactericidal action, for there was enough of the chemical present to prevent growth until the reproductive mechanism of the test organism was permanently disabled. This fact was proved by the absence of growth in secondary subcultures made after a few hours.
0
I
I
2
3
4 5 b 0 IO It I+ TIME I N M U T E S FOR OESTRUCTION
Germicidal Action of Mercarbolide in Presence of Organic Matter
I6
19
20
I
FIGURE 1
Germicidal tests were performed according to F. D. A. a 1:5 dilution, in less than 1 minute by all dilutions up to and methods with the exception that 20 per cent sterile horse-blood including 1:10, in 5 minutes or less by all dilutions up to and serum was present in the final chemical dilutions. A comincluding 1:16. A prolongation of the curve (indicated by the parison of the germicidal action in the presence of organic dotted line) demonstrated that Mercresin was practically matter was made with the chemicals listed in Table IV. instantaneous in its action against Staph. aureus. Other When Staph. aureus (Government 209) was employed as tests showed that Staph. aureus was destroyed in less than 15 the test organism a t 30" C., there was practically no reducseconds by a 1:5 dilution of Mercresin a t 30" C. and in less tion in the germicidal efficacy of Mercarbolide tincture due than 4 hours by a 1:60 dilution at 37" C. to the blood serum. Mercarbolide tincture was superior to Germicidal Action of Mercresin in Presence of mercurials B and C, aqueous solution of Mercarbolide, and Organic Matter the phenol derivative. Although the coefficient obtained with E. coli in the presence of organic matter was greater Regular germicidal tests were carried out a t 30" C., with than that with Staph. aureus, there was a reduction in efficacy the exception that normal horse serum, without preservative, due to the serum. Mercarbolide tincture showed greater germicidal action than mercurial C, aqueous Mercarbolide solution, and the phenol derivaTABLE111. GERMICIDAL ACTIONOF MERCRESIN tive, and nearly as much as the 1 per cent tincture Phenol Phenol Coefficient Coefficient a t Zoo C. of Pentacresol toward E . coli a t 30" C. Test Organism 37' C. 30' C. Test Organism True Apparent0
Mercresin Mercresin is a solution of 0.1 per cent 0-hydroxyphenylmercuric chloride and 0.1 per cent sec-amy~tr~cresol in per cent alcohol, per cent acetone, and water.
..
S t a p h . aureus (Gov. 209) 0.21 0.2 Staph. aureus 0.2 , E . t v p h o s a (Hopkins) 0.28 0.35 P s . aeruginoeo 0.5 A hemolytio atreptococcus~ 0.3 . Ser. pyoseptica 0.38 C . diphtheriaeb 0.18 B. hoffmanni 0 . IS+ E . coli 0 : ii+ E. C O Z ~ 0.23 a Values obtained by the F. D. A. method when no secondary subcultures were made. b Grown and subcultured in beef infusion broth (final PH, 7.6).
. ..
..
.. .. .. ... ...
INDUSTRIAL AND ENGINEERIXG CHEMISTRY
612
was substituted for some of the water in preparing dilutions of Mercresin and the other chemical solutions. Staph. aureus (Government 209) was destroyed in less than 5 minutes in the presence of 50 per cent horse serum (five times the quantity of serum advocated by F. D. A. methods, (2). Using the same test organism, the average phenol coefficient of Mercresin in the presence of 20 per cent serum was 0.055. A comparison of Mercresin with eight other chemical solutionsl demonstrated the following descending order of germicidal efficacy in the presence of 20 per cent serum, when Staph. aureus (Government 209) was used as the test organism a t 30" C.: iodine (7 per cent), Pentacresol (1 per cent), Mercresin and mercurial A (of nearly equal value), Mercarbolide, mercurial B, mercurial C, the phenol derivative, and aqueous Mercarbolide. When E.coli was used, the descending order was: iodine, mercurial A, mercurial B, Mercresin, Pentacresol, Mercarbolide, mercurial C, aqueous Mercarbolide (glycerol-sodium chloride solution), and the phenol derivative. Table I11 shows that E.coli was the most resistant to Mercresin of any of the eight test organisms employed. Yet the phenol coefficient of Mercresin, using E.coli as the test organism a t 30" C. in the presence of 20 per cent serum, was 0.054.
Germicidal Activity of Some Antiseptics and Germicides Germicidal tests were performed with the chemical solutions listed in Table IV, according to F. D. A. methods, using Staph. aureus (Government 209) and E.coli as the test organisms a t a medication temperature of 30" C. ACTIONOF ANTISEPTICS AKD TABLEIV. GERMICIDAL GERMICIDES Average Phenol Coefficient aureu-E. coliFullFullMarket strength LMarket strength solution chemicalb solution chemicalb 153 8.9+ 127 10.7 5 1.0 0.05 100 lll+ 0.11s 0.2+ 200 75 0.0740.075 70 17.8 0.047* 9.4 0.089 86 0.038 38 0.086 3.35 0.034 1.7 0.067 39 0.025 25 0.039 Survival for 0.16 160 10 min. ... ---Staph.
Solutiona Iodine (7%) Pentacresol (1%) Mercresin Phenol derivative Mercurial A Mercarbolide Mercurial B Mercurial C Aqueous Mercarbolide
-
++
a d l l the,solutions were tinctures except the phenol derivative and aqueous Mercarbolide. b Phenol coefficients calculated on the basis of the dry chemical.
Using phenol coefficients as the basis for comparing the germicidal activity of the market solutions of the chemicals listed in Table IV, the descending order of germicidal efficacy was as follows when Staph. aureus (Government 209) was employed as the test organism: iodine, Pentacresol, Mercresin, the phenol derivative, mercurial A, Mercarbolide, mercurial B, mercurial C, and aqueous Mercarbolide. The descending order with E.coli was: iodine, Mercresin, All t h e solutions compared were tinctures, with the exception of aqueous Mercarbolide and the phenol derivative. 1
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aqueous Mercarbolide, mercurial A, Mercarbolide, the phenol derivative, mercurial B, Pentacresol, and mercurial C. Mercresin was superior to all the other market chemical solutions tested, except iodine (7 per cent) and Pentacresol in respect to Staph. aureus, and except iodine and aqueous Mercarbolide in regard to E.coli. Mercarbolide tincture was superior as a germicide to mercurials B and C, and nearly as effective as mercurial A against both Staph. aureus and E . coli.
Summary and Conclusions Pentacresol (1 per cent in 2 per cent soap solution) at 20" C. demonstrated high germicidal action towards Gram-positive bacteria such as the hemolytic streptococcus, but was low in efficacy against Gram-negative bacteria such as E.coli. A 1 per cent solution of the chemical in 50 per cent alcohol and 10 per cent acetone served as a much more effective stock solution. Dilutions of the tincture, high enough to rule out the bactericidal action of the solvents, were germicidal towards Gram-negative bacteria and readily destroyed E. typhosa. In the presence of 20 per cent horse serum, Pentacresol (1 per cent) was more effective as a germicide against Staph. aureus than the six mercurials and the phenol derivative with which it was compared. 0-HYDROXYPHEKYLMERCURIC CHLORIDE. Mercarbolide tincture, in contrast to Pentacresol, was most effective in germicidal action towards Gram-negative bacteria, the phenol coefficient with E . typhosa being over 1100 on the basis of the dry chemical. The different solutions of Mercarbolides demonstrated marked bacteriostatic action towards Staph. aureus. In the presence of 20 per cent serum, Mercarbolide tincture was reduced in efficacy only slightly, if any, when Staph. aureus was employed as the test organism. There mas some reduction in efficacy when E.coli was used. Aqueous Mercarbolide solution showed higher germicidal action than the tincture towards E. typhosa and E. coli, but was not as effective as a bactericide against Staph. aureus, MERCRESIN.This chemical was fairly uniform in its germicidal activity towards all the Gram-positive and Gram-negative pathogens and nonpathogens examined a t 20", 30°, and 37 O C. Germicidal action was especially pronounced towards the hemolytic streptococcus and E . typhosa. Bacteriostasis was demonstrated to such a degree that secondary transfers were necessary in most cases to obtain true germicidal values. Short-time exposure tests showed Mercresin to be extremely rapid (almost instantaneous) in germicidal action against Staph. aureus. In the presence of 20 per cent horse-blood serum and using Staph. aureus as the test organism a t 30" C., Mercresin was superior in germicidal efficacy to all the market mercurials examined except one, to which it was equal in value. Mercresin has shown no signs of deteriomtion over long periods of time. SAC-AMYLTRICRESOL.
.
Literature Cited (1) Bergey, Manual of Determinative Bacteriology, 4th ed., Baltimore, Williams & Wilkins Co., 1934. ( 2 ) U. S. Food and Drug Administration, Methods of Testing Antiseptics and Disinfectants, Depl. Agr. Circ. 198 (1931). REC~IVE February D 4, 1936. Contribution No. 68 from the Department of Biology and Public Health, Massachusetts Institute of Technology. work was supported by a fellowship from the Upjohn Company.
The
