The term “phenol coefficient” is the figure denoting the germicidal efficiency of phenol-like compounds against Bacillus typhosus as compared to the germicidal power of pure phenol against this test organism under the specified conditions of a special bacteriological test. The special procedure employed is the Rideal-Walker test (16) or one of its modifications, such as the Hygienic Laboratory method (5) or the Food and Drug Administration method (18). The figure obtained by means of this test is used (a)as a specification figure for selecting disinfectants by the purchaser and (b) as a factor to be used in calculating the dilutions to be made for use of such disinfectants in practice. Since 5 per cent carbolic acid is generally recognized as a satisfactory germicide for use on inanimate objects, dilutions of phenollike disinfectants for practical use are made so that they will equal this standard germicide in bactericidal efficiency. The dilution is easily calculated by multiplying the phenol coefficient figure by twenty. Such solutions of phenol-like compounds will kill disease-producing microorganisms of epidemiologic importance when employed under practical conditions (19). The phenol coefficient test serves a useful purpose in supplying this necessary information and is used for this purpose throughout the world.
Limitations
of the Phenol Coefficient GEORGE F. REDDISH St. Louis College of Pharmacy, St.Louis, Mo.
Although the phenol coefficient test is designed especially for determining the germicidal efficiency of phenol-like disinfectants as compared to phenol, the test has often been used for other purposes for which it is inapplicable. Compounds which are totally unlike phenol in chemical and germicidal properties have been compared to this standard germicide by means of the test. I n other instances pure chemicals which are insoluble in water have been tested by this method after dilution in special solvents although the test specifies only water for this purpose. Although special methods are available for determining the germicidal efficiency of antiseptics, the phenol coefficient test has often been employed for this purpose even though the figures obtained are meaningless, since the phenol coefficients obtained do not evaluate the relative germicidal efficiency of such preparations. Such misuses of the phenol coefficient test have caused much confusion and misunderstanding.
T
HE phenol coefficient test was developed in England 33
years ago and is now employed in practically all the civilized countries for testing the germicidal activity of disinfectants. Its use for this purpose constitutes its principal function today. If this were the only use made of this test, there would be no reason for discussing the subject a t this time. Although designed especially for testing disinfectants, the phenol coefficient test is being employed for three purposes: ( a ) for testing the germicidal efficiency of disinfectants, ( b ) for determining the germicidal values of pure chemical compounds, and (c) for estimating the value of antiseptics. Although the test should be employed only for testing phenol-like compounds, its use for other purposes has gradually developed in recent years and much confusion has resulted.
Disinfectants Disinfectants are chemical agents used to destroy pathogenic microorganisms on inanimate objects. The standard method for testing the germicidal efficiency of disinfectants in the United States is the Food and Drug Administration phenol coefficient method (7, 8, 18). This method is especially applicable to the testing of phenol-like disinfectants, such as coal-tar and cresylic acid compounds and other phenolic preparations, and gives results quite comparable to that obtained with the Hygienic Laboratory method which is limited only to phenol-like compounds (1). The method is not well adapted for the testing of chlorine compounds or for oxidizing agents in general since the presence of organic matter adversely affects the germicidal effectiveness of such preparations under practical conditions. A phenol coefficient figure for chlorine compounds would be misleading to the consumer since such a figure would not indicate the actual germicidal efficiency of such disinfectants under practical conditions in the presence of organic matter. The phenol coefficient test as simplified in the Food and Drug Administration method is a test of the germicidal efficiency of phenol-like compounds against Bacillus typhosus a t 20’ C. as compared to the ability of phenol to kill the same test organism under the same test conditions. A specified quantity of broth culture of Bacillus typhosus (0.5 cc.) is added to 5 cc. of the dilutions (in water) of disinfectant and phenol control a t 20’ C., and transfers are made into 10 CC. of sterile broth after 5 , 10, and 15 minutes. The greatest dilution of disinfectant which kills the test organism under these conditions in 10 minutes and not in 5 minutes is divided by the greatest dilution of phenol giving the same result; the figure obtained is the phenol coefficient. For example, if the results obtained by this test are as follows, the phenol coefficient is 5.0: Diluti VI Disinfectant. 1:300 1:350
1:400 1:450 1:600
1:600 Phenol: 1:90 1:lOO
5 Min.a
10 Min.
0
++0 ++ ++
450/90 = 5.0, the phenol coefficient. a
1044
+
I
gioath; 0 = no growth.
16 Min.
0
0
++ +0
+0 +0
0 0 0
0 0
0
SEPTEMBER, 1935
INDUSTRIAL AND ENGINEERING CHEMISTRY
If another test organism is used in this test, the figure obtained is designated accordingly-for example, F. D. A. (special) Staphylococcus aureus, 20" c. The use of special test organisms other than Bacillus typhosus is not necessary for testing disinfectants of the phenolic type since the dilutions as calculated by multiplying the Bacillus typhosus phenol coefficient by 20 are sufficiently germicidal under conditions of use to kill the various pathogenic organisms which cause epidemiological diseases. For example, a coal-tar disinfectant with a (Bacillus typhosus) phenol coefficient of 5.0 when diluted 1 to 100 (20 X 5.0) for use in practical disinfection is capable of killing all epidemiologic pathogenic microorganisms present with a considerable margin of safety ( I S ) . The use of test organisms other than Bacillus typhosus is therefore usually limited to special tests by individual manufacturers to demonstrate for their own satisfaction the effectiveness of their products against the various disease-producing bacteria.
Pure Chemical Compounds The phenol coefficient test has also been widely used for comparing the germicidal efficiency of pure chemical compounds. During recent years, however, there have been a number of instances of departures from the standard phenol coefficient test which have caused considerable confusion. Germicides have been tested by this method which are not chemically related to phenol and whose chemical and germicidal activity differ so greatly from that of this standard germicide that they should not be compared in this wayfor example, chlorine compounds, mercury compounds, formalin, hydrogen peroxide, iodine, picric acid, certain essential oils, etc., and a large number of organic compounds. Another departure has been the liberties which certain investigators have taken with the test itself, changing it to suit the compounds tested. Many germicides which are soluble in alcohol but not in water, or only slightly so, have been tested by this method in dilutions of alcohol instead of water, although water is specified as the diluting agent in all the standard phenol coefficient tests (6, 16, 18). Certain other germicides which are soluble in alkali but not in water, or only slightly so, have been tested in dilutions made in alkali solutions instead of water, and the figures obtained have been designated as phenol coefficients. As a result, so-called phenol coefficients have been reported in the chemical literature which do not represent a true comparison of the germicidal activity of such compounds with phenol, which is watersoluble. The test itself is often blamed when discordant results are reported, whereas the unwarranted modifications made by certain investigators are entirely responsible. The term "phenol coefficient" has had a definite meaning for over 30 years. Information relative to the germicidal properties of new compounds which are insoluble in water could better be reported in terms of the dilutions which kill the test organisms when tested in the presence of specified solvents. This would cause no confusion, and the bacteriological phenol coefficient test would not then be subject to the present abuse. A better solution of this problem would be the use of a "theoretical phenol coefficient" as suggested by Carswell and Doubly (2). Pure compounds which are insoluble in water, or only slightly so, may be tested in special solvents such as sulfonated oil, and then the phenol coefficients may be plotted against concentrations of sulfonated oil or whatever solvent is used. I n this way a theoretical phenol coefficient of aqueous solution of these germicides can be estimated. This method has not yet been standardized but deserves further study.
Antiseptics Antiseptics are chemical agents used to kill or inhibit infectious bacteria on the human and animal body. The
1045
phenol coefficient test is not suitable for testing antiseptics and should never be used for this purpose. Antiseptics are not used for killing Bacillus typhosus, and there is no logical reason for testing antiseptics against this organism. A figure showing germicidal activity against Bacillus typhosus does not indicate the effectiveness of antiseptics against pusproducing and other infectious bacteria which are present in and on the body. Antiseptics must be tested against the kinds of bacteria which they are expected to kill under practical conditions. Since Staphylococcus aureus is the most common cause of suppuration and the most resistant of those microorganisms causing infections in cuts, scratches, wounds, abrasions, etc., it is the logical test organism for testing antiseptics. Special tests for determining the germicidal efficiency of antiseptics have been developed in the laboratories of the United States Food and Drug Administration and adopted as the official methods. Although methods for determining bacteriostatic as well as germicidal activity of antiseptics have been developed, the procedures for testing the germicidal efficiency of soluble liquid preparations are of most interest in the present discussion. In 1925 the author (6) published the results of a study of the resistance to phenol of twenty-five strains of Staphylococcus aureus and from this investigation established a standard of resistance for this organism for use in testing antiseptics and disinfectants. The special test employed for testing soluble liquid antiseptics ( 1 l , I 2 )makes use of Staphylococcus aureus of known normal resistance to phenol. I n this test 0.5 cc. of a 24-hour F. D. A. broth culture of Staphylococcus aureus is added to 5.0 cc. of antiseptic a t 37" C., and transfers are made from this mixture to 10 cc. of sterile broth after 5 minutes (officially designated as the F. D. A. method Staphylococcus aureus 37" C., 18). If all of the microorganisms (approximately 350,000,000) in the 0.5 cc. of culture are killed by 5.0 cc. of antiseptic in 5 minutes, the product is an effective antiseptic. The culture of Staphylococcus aureus employed in this test must not be killed by 1:80 phenol in 5 minutes nor by 1:90 phenol in 10 minutes at 37" C. under the conditions of the test as just outlined. (No comparison of any kind is made to the results obtained with phenol, t h e phenol test being only a control on the resistance of the culture.) Such antiseptics are approximately equal in germkilling power to the 1:40 solution of carbolic acid used in antiseptic surgery. Antiseptics which pass this test a r e effective germicides when used in practice, as has been proved in the case of antiseptics for oral use (9). The classes of preparations to which this test is applicable are the various germicidal solutions ordinarily used for first aid, for oral use, and for general applications about the body for the prevention of infections. Tincture of iodine, Liquor Antisepticus, aqueous solutions of iodine, solutions of phenol, mercury compounds, essential oils, halogen compounds, etc., should be tested by this method. Solutions of antiseptic dyes, however, should be tested by the agar cup plate method (IO). The test is not especially applicable for the testing of antiseptics for skin disinfections, as has been shown by Tinker and Sutton (17), Reddishl and Drake (14)) and Simmons (16). The F. D. A. method Staphylococcus aureus 37" C. is applicable to those soluble liquid antiseptics which are employed for short periods of contact, and for this purpose it has proved both practical and satisfactory. It is apparent that the phenol coefficient method has no place in the testing of antiseptics. However, some bacteriologists have used and are using the phenol coefficient method for testing antiseptics and have actually criticized certain antiseptic preparations because of their low phenol coefficients. The official F. D. A. method Staphylococcus aureus 37" C.
INDUSTRIAL AKD ENGINEERING CHEiMISTRY
1046
for testing antiseptics is not a phenol coefficient test although essentially the same technic is employed (18): The limitations of the phenol coefficient make it necessary in some cases to judge the germicidal preparation by other tests or by additional tests. This is particularly true of preparations that are not completely soluble or miscible in water. It is also $rue of certain preparations designated as antiseptics. Soluble antiseptics or antiseptics completely miscible with water can be tested, of course, by the procedure already described as the F. D. A. Staphylococcus aweus phenol coefficient method. In the testing of these substances, however, the phenol coefficient is not obtained necessarily, the phenol figure being used merely as a check of the resistance of the test organism. The information desired is the concentration which will kill in 5 minutes.
In spite of this warning that a phenol coefficient test should not be made on antiseptics and that a phenol coefficient figure should not be applied to antiseptics, a number of instances are on record in which phenol coefficients have been applied to well-known germicides in this classification. Table I gives the phenol coefficients of some of these antiseptics as taken from the Handbook of Chemistry (4),along with a number of disinfectants; both the Bacillus typhosus and Staphylococcus aureus phenol coefficient figures are given in most cases.
TABLE I. PHENOL COEFFICIENTS
Compound Chlorine group: Chloramine Dakin’s solution IC18 (0.5% aqueous soln.) Phenolic group: Lysol Lysol Lysol Creolin Kreso-Dip Tricresol Ethvlohenol n-P;opylphenol n-Butylphenol n-Amylphenol n-Hexylphenol Naphthol Hexylresorcinol Mercury compounds: Mercury chloride Mercurochrome (220 soluble)
Method Disinfectants Rideal-Walker
A. P. H. A. A. P. H . A.
T:mz.,
37 20 20
--PhenolCoefficient Staph. B. a w e u s typhosus 133
.... ....
100 0.78 2.40 5.0
.... ..
Rideal-Walker Rideal- Walker Rideal-Walker Rideal-Walker Rideal-Walker Hygienic Lab.
25 25 25 25 25 20
6.0 16.5 50 139 375
....
9-10 6 2.6 7.4 21 6 68 197 500 11.4
Antiseptics Reddish (modification)
20
150
72
Reddish
20
143
100
Reddish (modification) Merthiolate (Cz’HsHgSCsH4Reddish C02Na) Metaphen (CiiHi107NHga) Reddish (modification) Miscellaneous : Rideal-Walker Formalin Rideal- W a1ker Hydrogen peroxide Tincture of iodine U.S.P. Reddish (modification) Tincture of iodine U. S.P. (3% alcoholic Iz U. S. P. tincture dilute CzHaOH) Reddish A. P. H. A. Lugol’s iodine Hygienic Lab. Menthol Menthol isomers: Rideal-Walker 2-Menthol Rideal-Walker &Menthol Rideal-Walker &Menthol (racemic) Rideal-Walker dl-Isomenthol Hygienic Lab. Methyl salicylate Picric acid (2-3% in Rideal-Walker CzHaOH)
20 20
1.7 40-50
20
1500
37 20
....
20
38
20 20 20 12-16 12-16 12-16 12-16 20 37
0.3
6.3 5.1 5-a 7-12 7-12 7-12
...
.... 40-50
0.7 0.01
.... 5.8 5.0 5.1
....
....
.... 1.76 6.0
I
What do these phenol coefficient figures mean and what use can be made of them? What, for example, can be done with chloramine having a Staphylococcus aureus phenol coefficient of 135 and a Bacillus typhosus phenol coefficient of 100? Chloramine is a chlorine compound whose chemical and germicidal properties are quite different from that of
VOL. 29, NO. 9
carbolic acid. Chlorine compounds are unstable, especially in the presence of organic matter. Phenol is stable and is not counteracted by organic matter. A germicidal substance which is counteracted by organic matter as are the chlorine compounds should not be compared with a germicide which is not affected by organic matter. It is untrue to state that a sodium hypochlorite solution with a phenol coefficient of 5.0, for example, has the same germicidal strength as a cresylic acid disinfectant with the same phenol coefficient; when these disinfectants are diluted to twenty times their phenol coefficients (1 to 100 in this case), they are not equally effective under practical conditions of use in which organic matter is almost always present. It is unfair to the purchaser of such preparations to make comparisons between chlorine compounds and phenol-like disinfectants through the medium of the phenol coefficient since he is led to believe that these two classes of disinfectants are equally effective germicides. The purchaser has a right to assume that two disinfectants with the same phenol coefficient should have the same germicidal efficiency when used in the same dilution. This is not true in the case of chlorine and phenol-like compounds, and the phenol coefficient of chlorine compounds is actually misleading to the consumer. The Bacillus typhosus phenol coefficient figures of the phenolic group of disinfectants in Table I do mean something and can be interpreted in terms of technical and practical values. These figures indicate the relative values of these disinfectants for practical use. When dilutions are made on the basis of these figures (twenty times the phenol coefficient), the consumer has the assurance that such solutions will be as effective as 5 per cent carbolic acid under all practical conditions, even in the presence of large amounts of organic matter. The user has additional protection in the fact that such phenol-like disinfectants are not specific in their germicidal activity and, in the dilutions used, will kill all kinds of epidemiologic disease-producing microorganisms under practical conditions. Klarmann (3) does not agree, since he has shown by laboratory tests that there are exceptions to this rule, particularly as regards Streptococcus hemolyticus. However, the author (15)showed that there are such margins of safety in the practical use of these dilutions of phenol-like disinfectants as to assure the killing of all such pathogenic microorganisms under practical conditions. Varley and Reddish (19) also proved this to be true with a large number of disinfectants of the coal-tar group, the cresol compound group, and with 5 per cent carbolic acid. Phenol-like disinfectants in dilutions twenty times their phenol coefficients are as germicidal under practical conditions as 5 per cent carbolic acid, a standard germicide which has proved effective under practical conditions for over 70 years. The phenol coefficients of the antiseptics, however, as given in Table I are meaningless.1 It cannot be assumed, for example, that tincture of iodine, which has a phenol coefficient of 38 against Staphylococcus aureus, is seven times better as an antiseptic than is Lugol’s iodine solution with a phenol coefficient of 5.1. Tincture of iodine is more germicidal than Lugol’s iodine solution but not seven times more effective as an antiseptic under general practical conditions than is the aqueous Lugol’s solution. The 5 and 2.5 per cent solutions of carbolic acid have phenol coefficients of‘ 0.05 and approximately 0.02, respectively. These solutions are effective antiseptics as used in clinical practice, and yet tincture of iodine has a phenol coefficient 760 times that of 5 per cent carbolic acid and approximately 1900 times that of 2.5 per cent carbolic acid. Although tincture of iodine is 1 This is not intended as a criticism of the Handbook of Chemistry for publishing these figures, but only of those originally responsible for determining phenol coefficients of antiseptics.
SEPTEMBER, 1937
INDUSTKIAL AND ENGINEERING CHEMISTRY
apparently 760 times more germicidal than 5 per cent phenol, as judged on the basis of phenol coefficient figures of the two germicides, it is not actually 760 times better as an antiseptic under practical conditions. The phenol coefficient of antiseptics, therefore, cannot be interpreted in terms of practical value. Other examples could be given of the impracticability of the phenol coefficient as applied to antiseptics. There is no justification on scientific, technical, or practical grounds for the use of this figure as a means of indicating the germicidal efficiency and practical value of this class of preparations. The phenol coefficient figure cannot possibly be used as an index of the value of such preparations for general use or even for special applications. Also, since antiseptics are marketed ready for use or the dilutions specified on the label, the phenol coefficient need not and really cannot be used for determining the proper dilution for use in practice. Many antiseptics act in a manner so different from phenol that any comparison to the germ-killing power of carbolic acid is out of the question. Antiseptics should therefore be tested directly on the bacteria which will be met in practice by the special F. D. A. tests for antiseptics designed specially for the purpose.
1047
Literature Cited Brewer, C. M., and Reddish, G. F., J. Bad., 17,44 (1929). Carswell, T. A., and Doubly, J. A., ISD.ENG.CHEM.,28, 1276 (1936). Klarmann, E., and Shternov, V. A.,Ibid., 28,369 (1936). Lange, N. A,, Handbook of Chemistry, p. 1185, Sandusky, Ohio, Handbook Publishers, Inc., 1934. P u b . Health Repts. (Reprint 675), 36, 1559 (1921). Reddish, G. F., Am. J . P u b . Health, 15, 534 (1925). Ibid., 16, 283 (1926). Ibid., 17, 320 (1927). Reddish, G. F., S.Am. P h a r m . Assoc., 25, 1117 (1936). Ibid., 18, 237 (1929). Reddish, G. F., J . L a b . Clin. l e d . , 14, 649 (1929). Reddish, G. F., in Jordan and Falk’s “Newer Knowledge of Bacteriology and Immunology,” Univ. Chicago Press, 1928 Reddish, G. F., Soap, 11, 95 (1935). Reddish, G. F., and Drake, W. E., S.Am. Med. Assoc., 91,712 (1928). Rideal, S., and Walker, J. T. A,, “Approved Technique of the Rideal-Walker Test,” London, H. K. Lewis & Co., 1921. Simmons, J. S.,J. Am. Med. Assoc., 91,704 (1928). Tinker, M. B., and Sutton, H. B., Ibid., 87,1347 (1926). U. S.Dept. Agr., Circ. 198 (1931). Varley, J. C., and Reddish, G. F., J . Bact., 32,215 (1936). RBCEIVED September 10, 1937. Presented before Section X, Subsection on Pharmacy a t t h e meeting of the American Association for the Advancement of Science, December 30, 1935.
PAPAIN Effect of Storage upon Activity ROBERT R. THOMPSON University of Hawaii, Honolulu, Hawaii
I
N VIEW of the fact that papain is inactivated by oxidation, it is of practical importance to ascertain t o what extent air affects the stability of the dried papaya latex. A number of samples of papain were prepared from fresh latex several years ago and furnish an opportunity to observe the changes that occur upon storage. The proteolytic activity of papain preparations is dependent upon the presence of a sulfhydryl group. Most samples of papain require reduction before they reach maximum activity. By the use of a reducing agent such as hydrogen sulfide, hydrogen cyanide, cysteine, etc., it is possible to reduce the oxidized sulfhydryl group and restore the preparation to a point near its original activity. Using the methods of Balls and Hoover (1) and of Balls, Swenson, and Stuart ($), the activity of the various preparations was determined on the inactive as well as the activated samples. Some of the samples were collected by the author, others were from local known sources; all were prepared and kept under identical conditions. Two samples of foreign origin were also used for comparison. Each sample was dissolved in water and activated with twice its weight of cysteine hydrochloride (in solution a t pH 5.0) for 60 minutes a t 40” C. The results were obtained as shown in Table I. It is unfortunate that the methods used in the assay of preparations 1-7 when first made, were descriptive only of the activity and not of the total enzyme content after suitable activation. No figures are now available for these early samples, but from past experience it appears that they would have compared a t the time of preparation with the present sample 8. The table shows that the first change in a papain preparation with age is inactivation, accompanied by a loss of the sulfhydryl substance that reacts with nitroprusside, but without the destruction of the enzyme. This, however, is followed
by a gradual loss of proteolytic power even in the presence of cysteine. It must therefore be concluded that the enzyme is slowly inactivated and even more slowly destroyed on exposure to air in the dry state. ThBLE
1
Sample Date Milk Clottinga Alcoholic Titrationb Origin No. Obtained Inactive Bctivated Inactive Activated Hawaii 1 0.43 Nov., 1931 0 00 0.18 0.40 0.44 2 June, 1932 0 00 0.19 0.50 0.46 3 Aug., 1932 0 00 0.22 0.65 0.37 4 April, 1934 0.07 0.18 0.55 0.43 5 Aug., 1934 0 00 0.24 0.50 0.48 April, 1936 0 15 0.30 0.50 6 0,49 7 June, 1936 0 22 0.32 0.40 2.85 2 50 8 April 1937 1.80 1.80 0.43 Ceylon 9 Oot.,’1936 0 00 0.35 0.40 0.45 S. E. Africa 10 Feb., 1937 0 36 0.35 0.35 a The unit is t h e amount of enzyme required t o clot 10 cc. of a standard milk meoaration in 1 minute a t 40” C.: l/(et) units uer mg., where e = enzymes;mg t = time, min. b The prot:in digestion observed on casein after 20 minutes a t 40’ C b y alcoholic titration. The’cc. of 0.1 N KOH divided b y the mg. of sa&e acted upon.
Frank T. Dillingham, Unithe investigation and supplys. Acknowledgment is also due
erature Cited (2) Balls, A. K., Swenson, T. L., and Stuart, L. S., J. Assoc. Oficial A g r . Chem., 18, 140 (1935). RECEIVDD June 7, 1937.
