Gerrnicidal Power of Sodium Hypochlorite Effect of Addition of Alkali C. K. JOHNS,Central Experimental Farm, D e p a r t m e n t of Agriculture, Ottawa, Canada picked up by a hypochlorite soluH E influence of p H upon Employing Esch. coli and Staph. aureus as tion in passing over well-washed the germicidal speed of test organisms, the influence of added alkali salts metal surfaces, and little was to hypochlorites has been upon the germicidal power of sodium hypochlorite be gained by studying the influreported upon by a number of at 20" and at 50" C. is studied. The greater ence of such excessive quantities workers (1, 3, 4,5 , 7-15), and, destruction of Esch. coli by strongly alkaline hyof organic matter as Fabian and with two exceptions, there has his co-workers employed. been general agreement that inpochlorite solutions at 50", as reported by Fabian, The m e t h o d employed2 for creasing alkalinity slows down is shown to result f r o m the combined action of p H evaluating the efficiency of the germicidal speed to a m a r k e d and temperature and not to a stimulation of the chlorine solutions consisted in degree. Prucha ( I O ) , however, germicidal power of the hypochlorite by the added adding 1 cc. of bacterial suspenreported no difference in suscepalkali. At 20", however, added alkali greatly sion to 20 cc. of test solution, and tibility of Escherichia coli to a subculturing to duplicate tubes mildly and a strongly alkaline retards the destruction of this organism. W i t h of litmus milk (using two loops hypochlorite,' while Fabian and Staph. aureus the rate of destruction at both temfor simultaneous inoculation) a t co-workers (1) presented data peratures is markedly slowed down by the addition i n t e r v a l s of 3, 6, 9, and 15 indicating that "the addition of of alkali. The latter organism resists much minutes. So much irregularity alkalies to sodium hypochlorite higher concentrations of chlorine than Esch. was encountered in the results of increases its germicidal power." the m e t h o d that i t was later This latter statement, so comcoli and appears to be a more suitable test organmodified, and 1 cc. of the seeded pletely at variance with the findism for studies of this nature. solution was i n t r o d u c e d into ings of most workers, prompted Evidence is presented supporting the view that litmus milk tubes a t intervals the undertaking of the studies a n alkali-hypochlorite solution designed to comvarying from 0.25 to 30 minutes. reported here. bine the processes of washing and sterilizing is The tubes were incubated for 4 Fabian and co-workers in their days a t 37" C., since a number work studied the influence of the unlikely to be as effectice and economical as a addition of 0.5 per cent of sodium show growth after 4 days but not separate washing with alkaline detergents folafter 2 days. Even this method carbonate, trisodium phosphate, lowed by a sterilizing treatment with hypochlorite and sodium h y d r o x i d e to a was not free from irregularities, of lower alkalinity. sodium hypochlorite solution a t as the data presented in Table I i n d i c a t e . These data were 50" C. I n addition. the effect of amounts of ice cream mix up to 2 per cent was also determined. secured where each of the four solutions was replicated four Escherichia coli was selected as test organism. Subcultures times on the same day with each test organism. In order to were made into litmus milk a t intervals of 1 to 30 minutes and save space, data are given for a single representative test for incubated a t 37" C. for 48 hours. I n the studies reported in the each organism and temperature. Nevertheless, the results present paper, a basic solution prepared from 65 per cent recorded, which are in general agreement with the more calcium hypochlorite and sodium carbonate (ratio 4 to 3) was irregular findings of the earlier tests, make it possible to draw employed. The required amounts of C . P. sodium carbonate some conclusions. and trisodium phosphate and of commercial sodium metasiliRESULTSWITH Escherichia coli cate were added to portions of the same hypochlorite solution made up by suitably diluting the sodium hypochlorite stock It is evident that a t 20" C. Esch. coli behaves in the exsolution. Sodium metasilicate was substituted for the pected manner toward increased alkalinity of the hypochlorite sodium hydroxide of Fabian on account of its increasing use solution; i. e., increasing alkalinity means slower killing. At as a detergent. 50" C., however, the reverse holds true; i. e., increased alkalinity actually speeds up the killing process. This latter TESTS WITH ORGANISMS appears to confirm the results reported by Fabian and coSince extensive tests in this laboratory have confirmed the workers (1) while the contradiction between their results and claim of Myers and Johnson (9),that Staphylococcus aureus those reported from other laboratories results from the differis the most satisfactory nonspore-forming organism for ent behavior of this organism a t the higher temperature. germicidal testing of hypochlorites, parallel tests were con- That this greater destruction of Esch. co2i a t 50" C. by alkaline ducted with the U. s. Department of Agriculture No. 209 hypochlorites is due to the increased hydroxyl-ion concentrastrain of this organism, kindly furnished by R. P. Myers, tion rather than to an increase in the germicidal power of the along with those on Escherichia coli (strain No. 117). The hypochlorite is indicated by the data of Table 11. I n this 24-hour growth on nutrient agar a t 37" C. was washed off test, alkali salts were added to sterile distilled water instead of with sterile tap water and standardized to give a concentra- to the basic solution of sodium hypochlorite; in all other tion of ten million cells per cubic centimeter of seeded test respects the technic previously described was followed. The solution as employed by Fabian. The amount of organic results are essentially in agreement with those of Myers (8) matter added through the introduction of such a large number who reported that this organism was destroyed in 3 minutes of organisms was expected to be a t least as great as that a t 50" C. in a peptone solution made alkaline to p H 10.09,
T
1 In a more recent paper, Prucha (11) reports a decided slowing down of germicidal speed where alkali is added to hypochlorite.
2 Since then a more satisfactory method has been developed which will be described in another paper.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
588
TABLE I.
IXFLUENCE O F
Vol. 26, No. 7
ADDEDALKALI SALTS U P O S GERMICIDAL SPEED O F SODIUM HYPOCHLORITE
PLUS0.5% Na3POa.12HzO N o ADDEDALKALI( P H 8.4) ~ PLUS0.5% KazCOs (PH 10.8) (PH 11.7) Min. of exposure: 0 . 2 5 0 . 5 1 2 5 10 20 30 0 . 2 5 0 . 5 1 2 5 10 20 30 0.25 0 . 5 1 2 5 10 20 30 - - Esch.coli,O.5p.p.m.,5O0C. + b + + + + Esch. coli, 7.5 p,p.m.,20' C. - - - - S t a p L a u r e u s , 5p.p.m., 5 0 ' C . - + Staph.aureus, 50p.p.m.,2OoC. - - - a Determined on 10 p. p. m. available chlorine a t 20' C. with glass electrode. b groath; -, no growth.
+ - + + ++++ - - -
+- + + +- -
++ ++ ++ ++ +- + + + + + + + ++ ++ ++
+++ +
+++ +++ +++ ++- +++ -++ -++ +++++++
P ~ r 0.5% s Na?Si0~.5HzO (PH 12.0) 0 . 2 5 0 . 5 1 2 5 10 20 30
+++ +
++ ++
+++ +
+++ ++- -++ -++- ++ +++++
+,
TABLE 11. RESISTANCE O F Esch. coli CONTROL (DISTD.Hz0, p H a 5.7) 1
2
5
10
20
30
TO ALKALISOLUTION^ .4T 50" PLUS0.5% NazC03 (PH 10.8) PLUS0 5% P;ad'Od 12Hz0 (PH 11.7) Minutes of ezposure 1 2 5 10 20 30
+ ++ ++ ++ ++ ++ ++ + ? Determined by hydrogen electrode at 20' C
-
-
-
-
-
-
-
c. PLUS0.5% NazSi03 5HzO (PH 12.0)
-
In the present studies numerous attempts mere made to determine the p H of the hypochlorite solutions, using both quinhydrone and hydrogen electrodes. Both hydrogen peroxide and sodium thiosulfate were used to remove the active chlorine, but satisfactory readings could not be obtained. Recently, however, employing a glass electrode with a vacuum tube potentiometer of the Johnson (Wisconsin) type, the values shown in Table I were obtained. It is evident that the pH values of the solutions containing added alkali are sufficiently high that, in the light of the results reported by Myers, the germicidal action against Esch. coli a t 50 " may be attributed to the hydroxyl-ion Concentration, since such low concentrations of chlorine as here employed are rapidly dissipated a t this temperature.
TESTSWITH Staphylococcus aureus The reaction of Staph. aureus to hypochlorite of increased alkalinity is practically the same a t both 20" and 50" C. It lacks the susceptibility to hydroxyl-ion concentration a t 50 " displayed by Esch. coli; a t both temperatures the added alkali brings about a marked reduction in germicidal speed. The greater resistance of this organism to hypochlorite is also indicated in Table I. Over a number of tests, this organism has required approximately six times as great a concentration of available chlorine to achieve the same degree of destruction as was obtained with Esch. coli in the absence of added alkali.3 In consideration of this greater resistance and of the fact that its reaction to increased alkalinity is much the same at 50"as a t 20" C., Staph. aweus is a much more suitable test organism for studies of this nature than is Esch. coli. DI~CUSSION The work reported by Fabian and co-workers was undertaken largely to find out how strong a chlorine solution should be to obtain sterilization and also the possibilities of using alkaline detergents in combination with chlorine. In regard to the first point it is open to question whether the results obtained with a technic such as was employed here are applicable to the sterilization of surfaces upon which films of bacteria are growing. From the results of studies made in this laboratory, which will be published shortly, a much higher concentration of chlorine is required than where the bacteria are suspended in the hypochlorite solution. Furthermore, the comparative efficiency of different commercial alkaline hypochlorites at low concentrations as compared with 100 p. p. m. of available chlorine is greatly influenced by the buffer capacity of the solution, and the relative ranking of certain products a t 1 or 2 p. p.m. has been found to be totally different from that a t 50 or 100 p. p. m. With regard to the second point, if the use of alkaline detergents with chlorine implies an attempt to combine the processes of washing and sterilization, there would appear to 3 The chlorine concentration uas vaned for the t u o test organlsms and the two temperatures so that the differences in germicidal pouer uould stand out more clearly than where a uniform concentrailon uas employed
be a strong argument against this practice. The rate of loss of available chlorine in the presence of organic matter'(mi1k or ice cream) is decidedly greater where alkali has been added to the hypochlorite, as the results of Prucha and Fabian clearly show. Furthermore, the decline in germicidal value is even greater than the titration values indicate, for Mallmann (6) and Fay ( 2 )have reported that in hypochlorite rinse containing traces of milk the germicidal potency is lower than that of a fresh solution of equivalent concentration of available chlorine by titration. It is therefore difficult to see where combined washing and sterilization can be as effective and economical as the usual practice of washing with alkaline detergents followed by treatment with hypochlorite of lower alkalinity. Where a long period of contact between traces of hypochlorite and metal equipment is unavoidable, there may be a place for the more alkaline, less corrosive type of hypochlorite. However, the increasing employment of noncorroding surfaces of glass-enamel and stainless steel and the practice of sterilizing equipment immediately before use appear largely to remove the necessity for employing a less corrosive hypochlorite. ACKNOWLEDGMENT The writer wishes to acknowledge his indebtedness to A. G. Lochhead, Dominion Agricultural Bacteriologist, for advice and criticism; and to A. H. Jones of the Division of Bacteriology of the Central Experimental Farm for assistance in conducting the tests; to the Division of Chemistry for assistance with hydrogen electrode determinations; to I. L. Baldwin, University of Wisconsin, for details concerning the Johnson type vacuum tube potentiometer; and to George S. Field, Kational Research Council, Ottawa, for assistance in constructing one. LITERATURE CITED Fabian, F. W., Beavens, E. A . , Bryan, C. S., and Jensen, J. M . , IWD.ENG.CHEM.,23, 1169-73 (1931). Fay, A. C., Kans. h g r . Expt. Sta., Circ. 160 (1931). Johns, C. K., Sci. Agr., 10, 553-63 (1930). Ibid., 12, 3 8 4 2 (1931). Johns, C. K., 20th Ann. Rept. I n t e r n . Assoc. D a i r y and M i l k Inspectors, 1931, 197-209.
Mallmann, W.L., M i c h . Quart. B u l l . , 14, 244-5 (1932). Mallmann, W.L., and Schalm, O., Mich. Eng. Expt. Sta., B u l l . 44 (1932). Myers, R . P., J . A g r . Research, 38, 521-63 (1929). Mvers. R. P.. and Johnson, A. H.. Proc. I n t e r n . Assoc. M i l k b e a l e r s , Lab. Sect., 25, 21-55 (1932). Prucha, M. J., 16th Ann. Rept. Intern. Assoc. D a i r y and M i l k Inspectors, 1927, 319-28. I b i d . , 22nd Ann. Rept., 1933, 70-80. Ram Ayyar, C.S., A g r . J.I n d i a , 2 5 , 2 1 3 - 1 9 (1930). Rideal, E. K., and Evans, U. R., J . SOC.Chem. I n d . , 40, 64-6R (1921).
Tilley, F. W., and Chapin, R. M., J . Bact., 19, 295-302 (1930). Weissenbach, R . J., and Mestresat, W., Compt. rend. aoc. bid., 81, 93-6 (1918).
RECEIVED February 28, 1934.
