loopfull is removed from each dilution and transferred to capable of killing the test organism in 10 minutes but not GERMICIDAL SUBSTANCES

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1 A NEW METHOD FOR THE EVALUATION OF GERMICIDAL SUBSTANCES A. J. SALLE, W. A. McOMIE AND I. L. SHECHMEISTER Department of Bacteriology, University of California, Berkeley, California Received for publication March 27, 1937 The usual procedure in testing the germicidal strength of a compound is to note its effect on one or more species of bacteria and to compare the results with those obtained from phenol. A value known as the phenol coefficient may be calculated from the results of such a test. The phenol coefficient is defined as the killing power of a germicide, towards a specified organism, as compared to that of phenol. If the organism, Eberthella typhosa (B. typhosus) is used as the test organism the result will be an Eberthella-typhosa phenol coefficient, etc. The phenol coefficients will vary, naturally, with the test organism employed. Several methods are used to determine the phenol coefficient. That proposed by Reddish (1928), and recommended by the American Public Health Association, was followed by us in every case. Briefly, the phenol coefficient is determined by inoculating various dilutions of the germicide in sterile distilled water with the test organism. After an interval of five minutes a standardized loopfull is removed from each dilution and transferred to broth. This is repeated after ten minutes and again after fifteen minutes. The tubes are examined for turbidity after 48 hours. The same procedure is followed for phenol. The phenol coefficient is determined by dividing the greatest dilution of disinfectant capable of killing the test organism in 10 minutes but not in five minutes by the phenol dilution found to effect this result. 1 A 4 mm. loop, outside diameter. 267

2 268 A. J. SALLE, W. A. McOMIE AND I. L. SHECHMEISTER Various factors must be controlled in order that the results shall be of value. These include: species of organism, viability of the organism, temperature of incubation, proportion of culture to disinfectant, size of the inoculation loop, and composition of the culture medium. It is customary to rate disinfectants on the basis of their phenol coefficients. The method may be justifiable if the germicides are to be employed on external skin surfaces or in connection with the sterilization of non-living material. On the other hand, if disinfectants are internally administered or used on mucous surfaces, the above method presents serious objections. A germicide possessing a high phenol coefficient is usually preferred to one less potent. There would be no advantage in using a compound with a coefficient of 10 on mucous surfaces if it possessed ten times the toxicity of phenol. Phenol could be employed to equal advantage, with probably a considerable saving in the cost of the disinfectant. It is believed that a more accurate method of rating those germicides, recommended for internal administration or for use on mucous membranes, would be to test them for their effect on the growth of living embryonic tissue as well as for their ability to kill bacteria. A number, known as the toxicity index is determined by dividing the highest dilution required to prevent the growth of embryonic chick heart tissue during 48 hours by the highest dilution required to kill the test organism after an exposure of 10 minutes. For the preparation of tissue cultures it is necessary to have: (1) Plasma, (2) Tyrode solution, (3) tissue fragments and, (4) diluted embryonic fluid. 1. Plasma. Guinea-pig plasma is satisfactory and easily obtained. Ten cubic centimeters of blood are mixed with 1.0 cc. of a 1:1000 heparin solution. This prevents rapid coagulation of the plasma. The blood is centrifuged, the plasma removed and refrigerated until used. 2. Tyrode solution. This is a physiological salt solution containing 0.1 per cent glucose. It is used probably more than any other physiological salt solution for tissue culture work.

3 EVALUATION OF GERMICIDAL SUBSTANCES Tissue fragments. Chick hearts removed from 9-day-old embryos are used in pieces from 0.5 to 1.0 mm. in diameter. The size should be as uniform as possible, never larger than 1.0 mm. in diameter. 4. Dilute embryonic fluid. This is prepared by mincing chick embryos in a tissue grinder and diluting with 5 parts of Tyrode solution. The suspension is then centrifuged and the clear supernatant fluid removed. The embryonic fluid serves as food for the tissues. The various dilutions of the germicide under examination are made in the embryonic fluid. The tissue cultures are prepared as follows: The fragments are embedded in guinea-pig plasma diluted with three parts of Tyrode solution in Carrel flasks. The plasma coagulates shortly after the addition of the tissue. The fibrin matrix holds the fragments. The coagulated plasma is then covered with a layer of embryonic fluid containing a known concentration of the germicide. The flasks are capped to prevent evaporation and incubated at 380C. The plasma, Tyrode solution and embryonic extract are carefully measured so that the final concentration of germicide in each flask is known. The flasks are examined for tissue growth after an incubation period of 48 hours.2 The highest dilution of the germicide showing no growth of the tissue is taken for the calculation of the toxicity index. The following example will show the method for determining the index. The highest dilution of disinfectant preventing growth of tissue in 48 hours is 1: The highest dilution of the chemical showing no growth of the test organism, Staphylococcus aureus, after 10 minutes' exposure,' is 1:2500. The toxicity index is calculated by dividing 5000 by The result, 2.0, means that the germicide is twice as toxic for the tissues 2 A period of 24 to 48 hours is required in order to determine if the tissue fragments show proliferation. 3 The exposure time of the organisms to germicide was limited to 10 minutes because it is not believed that a chemical in contact with a mucous membrane or the cut surfaces of an incision will remain effective for a longer period of time.

4 270 A. J. SALLE, W. A. MoOMIE AND I. L. SHECHMEISTER as for the bacteria. If the toxicity index was 0.5 it would mean that the disinfectant was twice as toxic for the bacteria as for the tissue. Theoretically, the smaller the toxicity index the more nearly perfect the germicide. An index greater than one indicates that the germicide is more toxic to the tissue than to the bacteria. An index less than one indicates that the germicide is more toxic to the bacteria than to the tissue. In previous communications (Salle and Lazarus, 1935, a-h) comparisons were made of the resistance of Staphylococcus aureus and embryonic chick heart tissue to the following germicides: 1. Phenol (carbolic acid, C6H5OH) 2. Iodine (12) 3. Iodine trichloride (I Cls) 4. Merthiolate (sodium ethyl-mercuri thiosalicylate, C2H5. Hg.S. C6H4 *COONa, manufactured by Eli Lilly and Company) 5. Metaphen (the anhydride of 4,nitro-5-hydroxy-mercuri-ortho cresol, C2H2. CH3ONO2.Hg, manufactured by Dermatological Research Laboratories) 6. Hexylresorcinol (Normal hexylresorcinol, C6H3(OH)2C6H13, manufactured by Sharp and Dohme). 7. Mercurochrome (The disodium salt of 2:7 dibromo-4-hydroxymercuri-fluorescein, NaOOC * C6H4C: C6H2Br: OC6HBr(ONa) l I HgOH * 3H20, manufactured by Hynson, Westcott and Dunning) 8. Potassium mercuric iodide (K2HgI4) 9. Mercuric chloride (HgCl2) The Staphylococcus aureus phenol coefficients and their corresponding toxicity indices are recorded in table 1. When E. typhosa was substituted for S. aureus as the test organism a different set of figures was obtained. The germicides fall in a different order on the basis of their toxicity indices. The results are recorded in table 2. The results show that the simple inorganic compounds rate considerably better than some of the newer organic preparations. In both series iodine gave the lowest toxicity index while mercurochrome proved to be the most toxic.

EVALUATION OF GERMICIDAL SUBSTANCES 271 TABLE 1 Toxicity of germicides to chick heart tissue and Staphylococcus aureus GERMICID DILUTION SHOWING NO TOXICITY COCCUS ESHOWING GROWTH OF INDEX =/ ARU NO

5 EVALUATION OF GERMICIDAL SUBSTANCES 271 TABLE 1 Toxicity of germicides to chick heart tissue and Staphylococcus aureus GERMICID DILUTION SHOWING NO TOXICITY COCCUS ESHOWING GROWTH OF INDEX =/ ARU NO TIRSUE STAPHYLO- A/B PHENOL GROWTH = A COCCUS COEFFICIENT AUREUS = B Iodine... 1:1,800 1:20, Iodine trichloride... 1:2,400 1:6, Mercuric chloride... 1:45,000 1:16, Hexylresorcinol... 1:21,000 1:7, Metaphen... 1:76,000 1:6, Phenol... 1:840 1: Potassium mercuric iodide... 1:12,000 1: Merthiolate... 1:176,400 1:5, Mercurochrome... 1:10,500 1: TABLE 2 Toxicity of germicides to chick heart tissue and E. typhosa HIGHEST HGET DILUTION HIGHEST TOXICITY E. TYPHOSA GERMICIDE SHODIN SHOWING NO INDEX - PHENOL GETCIESH NONTIS EH. GROWTH TYPHOSA OF A/B COEFFICIENT GROWTH =A - B Iodine... 1:1,800 1:24, Potassium mercuric iodide... 1:12,000 1:110, ,100 Mercuric chloride... 1:45,000 1:180, ,800 Iodine trichloride... 1:2,400 1:8, Metaphen... 1:76,000 1:90, Hexylresorcinol... 1:21,000 1:7, Phenol... 1:840 1: Merthiolate... 1:176,000 1:5, Mercurochrome... 1:10,500 1: DISCUSSION HIGHEST HIGHEST DILUTION STAPHYLO- The determination of the phenol coefficient is not an accurate basis for evaluation of disinfectants employed under all conditions. The method is practical for rating disinfectants to be employed for the sterilization of non-living material. On the other hand the method fails to take into account the toxicity of germicides for living tissues when used internally or on mucous surfaces. A more valuable expression would be one based on a

6 272 A. J. SALLE, W. A. McOMIE AND I. L. SHECHMEISTER combination of the killing power of the disinfectant for bacteria with its toxic action toward living embryonic tissue. This method gives a figure which is known as the toxicity index. The toxicity index is defined as the ratio of the highest dilution of disinfectant required to prevent the growth of embryonic chick heart tissue during 48 hours to the dilution required to kill a given test organism in 10 minutes. The toxicity indices are not always proportional to the phenol coefficients. Iodine trichloride and metaphen have the same phenol coefficient yet metaphen is about 32 times more toxic to embryonic chick heart tissue than the iodine compound (table 1). As a germicide merthiolate is over 100 times more potent than mercurochrome but only about one-eighth as toxic to tissue. An important conclusion is that the older and simpler germicides rate considerably higher than the newer and more complex compounds. Iodine is one of the oldest germicides known. It is usually employed as tincture of iodine, an alcoholic solution, which is quite irritating, due to the presence of the alcohol. Because of this irritating property the alcoholic solution is gradually losing favor. In aqueous solution, however, the iodine is non-irritating and relatively non-toxic to tissue. It is the most ideal of any of the compounds studied when tested by the tissue culture technique. Of the newer organic preparations examined hexylresorcinol gave the lowest toxicity index on Staphylococcus aureus (grampositive), while metaphen gave the lowest figure when tested on Eberthella typhosa (gram-negative). The above germicides are being retested in an attempt to control some of the more obvious variables. Some of these are, (1) temperature, (2) time of action of chemical on bacteria and tissue, and (3) presence of organic matter. REFERENCES REDDISH, G. F Article on the Standardization of Disinfectants and Antiseptics, in Newer Knowledge of Bacteriology and Immunology, edited by E. 0. Jordan and I. S. Falk. University of Chicago Press. SALLE, A. J., AND LAZARUS, A. S. 1935a A comparison of the resistance of bacteria and embryonic tissue to germicidal substances. I. Merthiolate. Proc. Soc. Exper. Biol. and Med., 32,

EVALUATION OF GERMICIDAL SUBSTANCES 273 SALLE, A. J., AND LAZARUS, A. S. 1935b A comparison of the resistance of bacteria and embryonic tissue to germicidal substances. II. Metaphen. Proc. Soc. Exper.

7 EVALUATION OF GERMICIDAL SUBSTANCES 273 SALLE, A. J., AND LAZARUS, A. S. 1935b A comparison of the resistance of bacteria and embryonic tissue to germicidal substances. II. Metaphen. Proc. Soc. Exper. Biol. and Med., 32, SALLE, A. J., AND LAZARUS, A. S. 1935c A comparison of the resistance of bacteria and embryonic tissue to germicidal substances. III. Mercurochrome. Proc. Soc. Exper. Biol. and Med., 32, SALLE, A. J., AND LAZARUS, A. S. 1935d Comparison of resistance of bacteria and embryonic tissue to germicidal substances. IV. Hexylresorcinol. Proc. Soc. Exper. Biol. and Med., 32, SALLE, A. J., AND LAZARUS, A. S. 1935e Comparison of resistance of bacteria and embryonic tissue to germicidal substances. V. Iodine. Proc. Soc. Exper., Biol. and Med., 32, SALLE, A. J., AND LAZARUS, A. S. 1935f Resistance of bacteria and embryonic tissue to germicidal substances. VI. Iodine trichloride. Proc. Soc. Exper. Biol. and Med., 33, 8-9. SALLE, A. J., AND LAZARUS, A. S. 1935g A comparison of resistance of bacteria and embryonic tissue to germicidal substances. VII. Potassium mercuric iodide. Proc. Soc. Exper. Biol. and Med., 33, SALLE, A. J., AND LAZARUS, A. S. 1935h Comparison of resistance of bacteria and embryonic tissue to germicidal substances. VIII. Mercuric chloride. Proc. Soc. Exper. Biol. and Med., 37, Downloaded from on March 17, 2019 by guest

Gye and Cramer (1919) found that the ionizable salts of calcium injected together with the washed spores of Cl. tetani or of certain

Gye and Cramer (1919) found that the ionizable salts of calcium injected together with the washed spores of Cl. tetani or of certain STUDIES ON TETANUS TOXOID III. ANTITOXIC RESPONSE IN GUINEA PIGS IMMUNIZED WITH TETANUS ALUM-PRECIPITATED TOXOID FOLLOWED BY TET- ANUS SPORES F. G. JONES AND W. A. JAMIESON Lilly Research Laboratories,