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Iodine Solution as a Sporicidal Agent*,†
Iodine Solution as a Sporicidal Agent*J By LOUIS GERSHENFELD and BERNARD WITLIN Since the latter part of the 19th century, much has been written extolling the alleged virtues of iodine as an antiseptic. The sporicidal action of iodine has been reported but discrepancies appear in the results. Comparative studies have been made between water and water-alcoholic iodine solutions i n which the water solutions have consistently been shown t o be more efficient. This in vitro study reports o n the sporicidal efficiency of a 2 per cent water-iodine solution (iodine solution N. F. IX). Both “wet” and “dry” techniques were employed, using conditions with different pH values. Iodine solution N. F. IX was found to be effective against the spores of B. antbracis, B. subtilis, B. megatbwium, B. mesentericus, and CI. tetani. HE SPORICIDAI, ACTION of iodine has been Treported and known since 1873 (1). Data concerning t h e efficiency of iodine against t h e spores of B. anthrucis (2-4), Cl. tetani ( 3 , 6, 7), and Cl. welclzii (3, 4), both in ritro and in vivo findings, have been noted. However, there have been discrepancies (5) in the results reported. This may be due t o the variations in the techniques used in testing (5) and, in part, to t h e free iodine content of the preparations under test. Since the latter part of t h e 19th century, much has been written extolling the alleged virtues of iodine as an antiseptic. Preparations containing free iodine u ;ed as antiseptics are usually prepared today by dissolving iodine with soluble iodides in water or diluted alcohol. Comparative studies have been made between aqueous and hydroalcoholic iodine solutions in which t h e aqueous solutions have consistently been shown to be more efficient (7-10). This in d o study was undertaken t o determine the sporicidal efficiency of a 2 per cent aqueous iodine solution (iodine solution N. F. IX).
EXPERIMENTAL General Procedure.-Spores of B . anthracis, B . subtilis, B . megatherium. B . mesentericus, and GI. tetnni were employed. The procedure consisted of saturating sterile 1-cm. squares of \\;hatman No. 1 filter paper separately with saline suspensions of heat-treated (70’ for thirty minutes), one-year old cultures (spores). At room temperature (2227’1, the spores in each instance were washed off axar slants with isotonic saline and standardized so thzt the quantity of suspension (0.01 ml.) absorbed by each 1-cm. square of filtcr paper contained approxiiiately 300,000 spores of each species. The sterile i,lter-paper squares were dipped in the individual st\ore suspensions, the excess suspension was drained, nd the squares were placed in sterile
* Received February 14, 1952, from the Philadelphia College of Pharmacy and Science, Philadelphia. Pa. Presented t o the Pharmacy Subsection of the American Association for the Advancement of Science. PhiladelDhia. . December 28,1951. t This investigation was conducted a t the Philadelphia College of Pharmacy and Science, Department of Bacteriology, under a grant from the Chilean Iodine Educational Bureau Fellowship. Mellon Institute.
Petri dishes covered with Brewer “open type” covers. The squares were then placed in the incubator a t 37” and incubated to dryness (forty-eight hours). The dried spore-impregnated filter-paper squares were transferred to sterile Petri dishes and covered with 10 ml. of iodine solution, N. F. At thirtyminute intervals, for a period of six hours, the squares were removed with sterile forceps, rinsed in one 10-ml. tube of sterile broth’, traiisplanted to another tube of 10 ml. of broth, aricl incubated for seven days a t 37”. Anothcr series was treated as above, employing, however, a sterile isotonic saline rinse, followed by immersion in sterile, 1% sodium thiosulfate (until the disappearance of the iodine color), and then by another rinse in sterile isotonic saline (to remove the thiosulfate) prior to transplanting into broth. Finding.$.- In in nitro tests, iodine solution N. F. (2%) was capable of destroying the spores of R. subtilis, B . untltracis, and B. mesentericus within two and one-half hours and CZ. tetani within one and one-half hours. Under conditions of this test, spores of B . megntherium were not dcstroyed after an exposure of five and one-half hours. In vioo tests, using GI. tetani and other pathogenic spore-bearers, are being conducted. Increasing the Number of Spores.-To note the effect iodine solution N. F. would havc on a n increased number of spores, the first procedure was repeated, using 1,000,000 spores per ml. instead of 500,000 per ml. Findings.-The sporicidal eficiency of iodine solution N. F. IX was the same for 1,000,000 spores pcr ml. as for 500,000 per ml. pH Variation.-The general proccdure, using 1,000,000 spores per ml., was repeated with the test organisms against iodine solution iX.F. adjusted immediately before testing with hydrochloric acid to PH values of 1.0, 2.0, 3.0, and 4.0. Hydrogen-ion COIIcentration measurcments were made with a Beckman electric potentiometer (Model H). Controls of hydrochloric acid in distilled water, adjusted to the desired PI3 values, were run simultaneously with controls of iodine solution N. F. Findings.-Iodine solution N. F. I S possessing a pH value of 4.6 was capable of destroying the spores of B . subtilis, B unthracis, and H. mesentericus within two and one-half hours and CI. telani within one and one-half hours. The spores of B . mega1 With the exception of C1. tetani, I;. D. A. broth was employed for all experiments. For Cl. telani, thioglycollate medium (steamed and cooled prior to inoculation) was used throughout.
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t b r i u m were not destroyed after a n exposure of five and one-half hours. Controls of hydrochloric acid in water at fiH 4.6were ineffective against all of the test spores within five and one-half hours. Iodine solution N. F. I X adjusted t o pH 4.0 with hydrochloric acid destroyed the spores of B. subtilis, B anthracis, B. mesentericus, B. megathfrium, and Cl. tetani within ninety minutes. Controls of hydrochloric acid in water a t PH 4.0 were ineffective against all of the test spores within five and one-half hours. Iodine solution N. F. I X adjusted to fiH 3.0, 2.0, and 1.0 with hydrochloric acid was capable of destroying the spores of B . subtilis, B . anthracis, B . mfsentericus, B . megatherium, and CI. tetani within sixty minutes but not in thirty minutes. Hydrochloric acid controls were ineffective within five and one-half hours. Sporicidal Testing on Nonabsorbable Surface (Wet).-Injector-type razor blades (Schick*) were washed in acetone to remove any adhering oil or grease and then were sterilized in Pehi dishes in the hot-air oven at 170" for two hours. The blades were dipped, by means of a platinum hook, into the spore suspensions of 1,000,000 per ml., allowed to drain for one minute, and then placed individually in 15 tnl. of iodine solution N. F. IX contained in a seeding tube (85 x 25 mm.) At designated intervals of time, the blades were removed, drained of excess iodine, immersed in sterile 1% sodium thiosulfate solution, rinsed in sterile isotonic saline, and then transplanted into 10 ml. of broth. The transplants were incubated a t 37" for seventy-two hours and then were observed for the presence or absence of growth. Findings.-The times required for iodine solution N. F. IX to kill the spore suspensions adhering to the razor blades were as follows: B . anthracis, ninety minutes; B . subtilis, ninety minutes; B . mesentericus, ninety minutes; B. megatherium, four hours; and CI.tetani, ninety minutes. Sporicidal Testing on Nonabsorbable Surface (Dry).-The procedure using the razor blades was followed except that the blades, after being dipped in the spore suspensions, were allowed to dry for seventy-two hours a t 37" by placing them in sterile Petri dishes with Brewer open-type covers. The dry blades were then immersed individually in 15 nil. of iodine solution N. F. IX in seeding tubes. At designated intervals of time, the blades were removed, drained of excess iodine, immersed in sterile 1% sodium thiosulfate, rinsed in sterile isotonic saline, and transplanted into 10 ml. of broth. The transplants were incubated a t 37" for seventy-two hours and then were observed for the presence or absence of growth. Manufactured by Eversharp. Inc., New York. N. Y
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Findings.-The times required for iodine solution N. F. I X to kill the spore suspensions dried onto the razor blades were as follows: B. anthrucis, five hours; B. subtilis, three hours; B. mesentericuh, four hours; and Cl. tetani, two hours. B. megutherium spores were not destroyed within five and one-half hours of exposure. SUMMARY A N D CONCLUSIONS
An in rdro study was made t o determine the effect of iodine solution N. F. IX (pH 4.6) upon one-year-old spores of B. anthracis, R. subtilis, B. megatherium, B. mesentericus, and CL. tetani by both "wet" and "dry" techniques. T h e same procedures were used employing lower pH values (4.0,3.0, 2.0, and 1.0). T h e results observed in this series of experiments revealed t h a t : 1. Two per cent aqueous iodine (iodine solution N.F. IX) possesses sporicidal efficiency. 2 The efficiency of iodine solution N. F. IX against the spores of B. subtilis, B. anthrucis, B. mesenterkus, B. megatherium, and C1. felani was enhanced at lower pH values (4.0, 3.0, 2.0, and 1.0). Controls of hydrochloric acid in water at the same pH values were ineffective against all of the test spores under the conditions of the procedures used in this study. 3. Wet suspensions of spores on an absorbable surface (filter paper) required longer periods of exposure than those on nonabsorbable surfaces (razor blades) before being killed. 4. Spores dried on an absorbable surface (filter paper) required shorter periods of exposure than those on nonabsorbable surfaces (razor blades) before being killed. REFERENCES (1) Davaine, "Dictionaire Encyclopedie des Sciences hied.". P. Asselin and G. Masson, Paris, 1873, p. 335. (2) Claudius, Deul. Z. Chirurgic, 44. 489(1902). (3) Tinker, M. E., and Sutton, H. B.. 1. A m . M e d . Assoc., 87, 1347(192G). (4) Simmons, J . S., ibid., 91, 704(1828). ( 5 ) Gershenfeld, L., and W i t h , B., A m . N. Y. Acad s~i., 53, 17z(1950). (G) Gershenfeld, L.. and Miller, R. E., THSS JOURNAL, 21, 894 (1932). (7) Moll, T., Zenlr. Bakt. P a ~ o s i l e n k A . b f . I Ref., 6, 416 (1920). (8) Beal. G. D., Waters, K. I,., and Black, P., TAIF JOURNAL, 8, 354(1947). (9) Gershenfeld, L.. and W i t h , B., i b i d . , 39, 489(lbrFO). (10) Ficarra, B. J., 1. In f ern . C o f l . Surgeons. 16, 115 (1951).
EXPERIMENTAL General Procedure.-Spores of B . anthracis, B . subtilis, B . megatherium. B . mesentericus, and GI. tetnni were employed. The procedure consisted of saturating sterile 1-cm. squares of \\;hatman No. 1 filter paper separately with saline suspensions of heat-treated (70’ for thirty minutes), one-year old cultures (spores). At room temperature (2227’1, the spores in each instance were washed off axar slants with isotonic saline and standardized so thzt the quantity of suspension (0.01 ml.) absorbed by each 1-cm. square of filtcr paper contained approxiiiately 300,000 spores of each species. The sterile i,lter-paper squares were dipped in the individual st\ore suspensions, the excess suspension was drained, nd the squares were placed in sterile
* Received February 14, 1952, from the Philadelphia College of Pharmacy and Science, Philadelphia. Pa. Presented t o the Pharmacy Subsection of the American Association for the Advancement of Science. PhiladelDhia. . December 28,1951. t This investigation was conducted a t the Philadelphia College of Pharmacy and Science, Department of Bacteriology, under a grant from the Chilean Iodine Educational Bureau Fellowship. Mellon Institute.
Petri dishes covered with Brewer “open type” covers. The squares were then placed in the incubator a t 37” and incubated to dryness (forty-eight hours). The dried spore-impregnated filter-paper squares were transferred to sterile Petri dishes and covered with 10 ml. of iodine solution, N. F. At thirtyminute intervals, for a period of six hours, the squares were removed with sterile forceps, rinsed in one 10-ml. tube of sterile broth’, traiisplanted to another tube of 10 ml. of broth, aricl incubated for seven days a t 37”. Anothcr series was treated as above, employing, however, a sterile isotonic saline rinse, followed by immersion in sterile, 1% sodium thiosulfate (until the disappearance of the iodine color), and then by another rinse in sterile isotonic saline (to remove the thiosulfate) prior to transplanting into broth. Finding.$.- In in nitro tests, iodine solution N. F. (2%) was capable of destroying the spores of R. subtilis, B . untltracis, and B. mesentericus within two and one-half hours and CZ. tetani within one and one-half hours. Under conditions of this test, spores of B . megntherium were not dcstroyed after an exposure of five and one-half hours. In vioo tests, using GI. tetani and other pathogenic spore-bearers, are being conducted. Increasing the Number of Spores.-To note the effect iodine solution N. F. would havc on a n increased number of spores, the first procedure was repeated, using 1,000,000 spores per ml. instead of 500,000 per ml. Findings.-The sporicidal eficiency of iodine solution N. F. IX was the same for 1,000,000 spores pcr ml. as for 500,000 per ml. pH Variation.-The general proccdure, using 1,000,000 spores per ml., was repeated with the test organisms against iodine solution iX.F. adjusted immediately before testing with hydrochloric acid to PH values of 1.0, 2.0, 3.0, and 4.0. Hydrogen-ion COIIcentration measurcments were made with a Beckman electric potentiometer (Model H). Controls of hydrochloric acid in distilled water, adjusted to the desired PI3 values, were run simultaneously with controls of iodine solution N. F. Findings.-Iodine solution N. F. I S possessing a pH value of 4.6 was capable of destroying the spores of B . subtilis, B unthracis, and H. mesentericus within two and one-half hours and CI. telani within one and one-half hours. The spores of B . mega1 With the exception of C1. tetani, I;. D. A. broth was employed for all experiments. For Cl. telani, thioglycollate medium (steamed and cooled prior to inoculation) was used throughout.
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t b r i u m were not destroyed after a n exposure of five and one-half hours. Controls of hydrochloric acid in water at fiH 4.6were ineffective against all of the test spores within five and one-half hours. Iodine solution N. F. I X adjusted t o pH 4.0 with hydrochloric acid destroyed the spores of B. subtilis, B anthracis, B. mesentericus, B. megathfrium, and Cl. tetani within ninety minutes. Controls of hydrochloric acid in water a t PH 4.0 were ineffective against all of the test spores within five and one-half hours. Iodine solution N. F. I X adjusted to fiH 3.0, 2.0, and 1.0 with hydrochloric acid was capable of destroying the spores of B . subtilis, B . anthracis, B . mfsentericus, B . megatherium, and CI. tetani within sixty minutes but not in thirty minutes. Hydrochloric acid controls were ineffective within five and one-half hours. Sporicidal Testing on Nonabsorbable Surface (Wet).-Injector-type razor blades (Schick*) were washed in acetone to remove any adhering oil or grease and then were sterilized in Pehi dishes in the hot-air oven at 170" for two hours. The blades were dipped, by means of a platinum hook, into the spore suspensions of 1,000,000 per ml., allowed to drain for one minute, and then placed individually in 15 tnl. of iodine solution N. F. IX contained in a seeding tube (85 x 25 mm.) At designated intervals of time, the blades were removed, drained of excess iodine, immersed in sterile 1% sodium thiosulfate solution, rinsed in sterile isotonic saline, and then transplanted into 10 ml. of broth. The transplants were incubated a t 37" for seventy-two hours and then were observed for the presence or absence of growth. Findings.-The times required for iodine solution N. F. IX to kill the spore suspensions adhering to the razor blades were as follows: B . anthracis, ninety minutes; B . subtilis, ninety minutes; B . mesentericus, ninety minutes; B. megatherium, four hours; and CI.tetani, ninety minutes. Sporicidal Testing on Nonabsorbable Surface (Dry).-The procedure using the razor blades was followed except that the blades, after being dipped in the spore suspensions, were allowed to dry for seventy-two hours a t 37" by placing them in sterile Petri dishes with Brewer open-type covers. The dry blades were then immersed individually in 15 nil. of iodine solution N. F. IX in seeding tubes. At designated intervals of time, the blades were removed, drained of excess iodine, immersed in sterile 1% sodium thiosulfate, rinsed in sterile isotonic saline, and transplanted into 10 ml. of broth. The transplants were incubated a t 37" for seventy-two hours and then were observed for the presence or absence of growth. Manufactured by Eversharp. Inc., New York. N. Y
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Findings.-The times required for iodine solution N. F. I X to kill the spore suspensions dried onto the razor blades were as follows: B. anthrucis, five hours; B. subtilis, three hours; B. mesentericuh, four hours; and Cl. tetani, two hours. B. megutherium spores were not destroyed within five and one-half hours of exposure. SUMMARY A N D CONCLUSIONS
An in rdro study was made t o determine the effect of iodine solution N. F. IX (pH 4.6) upon one-year-old spores of B. anthracis, R. subtilis, B. megatherium, B. mesentericus, and CL. tetani by both "wet" and "dry" techniques. T h e same procedures were used employing lower pH values (4.0,3.0, 2.0, and 1.0). T h e results observed in this series of experiments revealed t h a t : 1. Two per cent aqueous iodine (iodine solution N.F. IX) possesses sporicidal efficiency. 2 The efficiency of iodine solution N. F. IX against the spores of B. subtilis, B. anthrucis, B. mesenterkus, B. megatherium, and C1. felani was enhanced at lower pH values (4.0, 3.0, 2.0, and 1.0). Controls of hydrochloric acid in water at the same pH values were ineffective against all of the test spores under the conditions of the procedures used in this study. 3. Wet suspensions of spores on an absorbable surface (filter paper) required longer periods of exposure than those on nonabsorbable surfaces (razor blades) before being killed. 4. Spores dried on an absorbable surface (filter paper) required shorter periods of exposure than those on nonabsorbable surfaces (razor blades) before being killed. REFERENCES (1) Davaine, "Dictionaire Encyclopedie des Sciences hied.". P. Asselin and G. Masson, Paris, 1873, p. 335. (2) Claudius, Deul. Z. Chirurgic, 44. 489(1902). (3) Tinker, M. E., and Sutton, H. B.. 1. A m . M e d . Assoc., 87, 1347(192G). (4) Simmons, J . S., ibid., 91, 704(1828). ( 5 ) Gershenfeld, L., and W i t h , B., A m . N. Y. Acad s~i., 53, 17z(1950). (G) Gershenfeld, L.. and Miller, R. E., THSS JOURNAL, 21, 894 (1932). (7) Moll, T., Zenlr. Bakt. P a ~ o s i l e n k A . b f . I Ref., 6, 416 (1920). (8) Beal. G. D., Waters, K. I,., and Black, P., TAIF JOURNAL, 8, 354(1947). (9) Gershenfeld, L.. and W i t h , B., i b i d . , 39, 489(lbrFO). (10) Ficarra, B. J., 1. In f ern . C o f l . Surgeons. 16, 115 (1951).