Effect of Increase in Acidity on ANTISEPTIC EFFICIENCY OTTO RAHN AND JEAN E. CONN New York State College of Agriculture, Cornell University, Ithaca, N. Y.
Benzoic acid, salicylic acid, and sulfurous acid are nearly a hundred times as efficient antiseptics in strongly acid solutions as they are in neutral solutions. With benzoic and salicylic acids, only the undissociated acid is antiseptic; the benzoate and salicylate ions appear to have practically no effect on yeast. Multiplication of the yeast was inhibited whenever the undissociated benzoic acid concentration was above 25 mg. per 100 ml. With
salicylic acid the limiting concentration was 4 mg. of undissociated acid per 100 ml. Sulfur dioxide in water dissociates to harmless SOs-ions, and to HSOI- ions which inhibit the multiplication of Bacterium coli but not of yeast. The yeast is inhibited only by undissociated HzS03. The rapid death of yeast is brought about by 7 to 8 mg. of undissociatedHzSOa per 100 ml.; B a c t e r i u m coli can tolerate nearly ten times as much.
T
almost completely dissociated in neutral or alkaline solution, but almost completely undissociated in a strongly acid medium. It seems possible that the undissociated acid is toxic while the ions are not. This is known t o be true for lactic acid. Rogers and Whittier (8) showed that Streptococcus lactis ceases to make lactic acid when the undissociated lactic acid is 0.017 molar, regardless of pH.
H E efficiency of most of the strong disinfectants is not materially affected by changes of acidity, chlorine being the most striking exception. Many weak disinfectants, however, which are primarily used as antiseptics, increase their power more than a hundred fold when the medium becomes strongly acid. The best known examples are benzoic and salicylic acid and sulfur dioxide, which are very efficient in preserving acid foods but are almost useless in neutral solution. Degering and associates (1) measured for many disinfectants the effect of p H on the concentration necessary to kill Staphylococcus aureus and Bacterium coli in 10 minutes. For some antiseptics they determined also the inhibiting concentrations a t different pH. They came to the conclusion that there is a “specific hydrogen-ion effect”. Hoffman, Schweitzer, and Dalby ( d ) studied the mold-preventing properties of some disinfectants and believe that “the fungistatic power of such a molecule (for example, benzoic acid) depends on the presence of both water- and oil-soluble groups ilythe molecule in some degree of balance. The benzene ring group is oil soluble (nonpolar), and the carboxyl is water soluble (polar) Apparently the hydrogen-ion concentration of the media affects the relation of polar and nonpolar groups and thus affects fungistatic power.” There is a much simpler explanation of the effect of p H on such compounds as benzoic or salicylic acid. They are weak acids,
BENZOIC’ AND SALICYLIC ACIDS
To prove this for benzoic acid, a glucose yeast extract broth was adjusted to different acidities with citric acid and potassium phosphate, and for each different p H the concentrations of sodium benzoate was determined which would barely permit and which would prevent the growth of a wine yeast. This yeast develops well a t p H 2.5, and even a t p H 2, though very poorly. From the concentrations of benzoate and the corresponding pH, it could be computed how much undissociated benzoic acid was present in this medium. The calculation is based on the formula for the dissociation constant,
. . ..
[HI [benzoate ions] =6.61 X 10-6 = [HID [undissociated acid] [.y 1 The fraction of undissociated acid is: undissociated fraction
TABLE I. INHIBITIOK OF GROWTH OF Saccharomyces ellipsoideus
pH
0.55 0 38 0.13 0 027 0,022 0 003
Sodium Benzoate 25 50 21 42 20 35 17 30 40 50 34 42 50 100 42 85 250 500 212 420 250 500 212 420 1000 1500 850 1270 Over 2500 Over 2120
2.93 4 10 5.14 5 70
0.527 0.070 0.0068 0.00188
10 80 600 1000
083 0 77
Sodium Salicylate 15 8.6 100 69 800 518 2000 864
11 86 691 1728
18 35 13 24 19 23 16 31 28 55 6.7 11.5 I9 28 Over 6 . 4 4,5 4.8 3.5 1.6
01
=
[HI
[H 1
+ 6.61 x 10-6
The fraction 01 multiplied by the concentration of total benzoic acid gives the concentration of undissociated acid a t any pH. Since present experiments were made with sodium benzoate, this value must be recalculated as benzoic acid by multiplication by 0.847. Table I gives the concentrations of benzoate which barely permitted growth of yeast and those which completely inhibited it. This concentration varies from 50 mg. a t pH 3.5 to over 2500 at p H 6.5 for 100 ml. of medium. The last column shows that the different concentrations at the differentacidities have one thing in common. They all represent nearly the same amount of undissociated acid-namely, about 25 mg. per 100 ml. It must be concluded that yeast, which can grow a t pH 2.5 as well as pH 7.0, ceases t o multiply when the concentration of undissociated benzoic acid reaches 25 mg. per 100 ml.
-Inhibitory Concn., Mg./100 MI.UndisSodium s a l t Total Acid Undis. Acid sociated No No No Fraction OL Growth growth Growth growth Growth growth
3 5 3.65 4.1 4.4 5.0 5 7 5 8 6 5
011
6,8 6.0
4.8 3.2
185
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
186
Vol. 36, No. 2
SULFUROUS ACID
Somewhat different is the case of sulfurous acid which is dibasic and has two dissociation constants:
3
s
4
7
6
PH
Figure 1. Growth Inhibition by Sodium Sulfite of Yeast (Double Lines) and Bacterium coli (Single Lines)
PH
TABLE
11.
Spa-ions
FractionsHSOaions
INHIBITION OF
HzSOs undis.
0.804 0.870 0.926 0.969 0,968-
0.196 0.130 0.074 0.031 0.032-
7.05-7.25
0,975-n am 0.996 0.997 0.998
0.019-n ni4 0.004 0.0032 0,002
0 0 0.001 0.005 0.047
0.165 0.630 0.836 0,932 0.948
0.855 0.370 0.163 0.063 0.0056-
0 332 0 858
0 142
0 667
0.0004 10-6
7.6 7 . 6 -7.8 8.0
~
020
NazSOa
B a c t e r i u m coli 10-5 60-80 0 Over 100 0 Near 200 0 Over 500 0 500-1000
5 . 9 -5.92 6.12 6.40 6.80 6.75-7.00
n ~ 7 n n
GROWTH BY
----Inhibiting
"--
SODIUM SULFITE
Conon., Mg./100 MI,-----HSOaH2S03 HzSOs ions undis. 39.1-52.1 Over 65.2 Near 130 Over 325 325-652
7.6-10.0 Over 8 . 5 Near 9 . 6 Over 1 0 . 0 10.4-12.8
0 0 0 0 0
0
1000-2000
652-1304
12.0-18.0
0
0
Over 2000 2000-5000 Over 5000
Over 1304 1304-3255 Over 3255
Over 5 . 2 5.2-10.5 Over 6 . 5
0 0 0
0 0
Saccharomyces ellipsoideus 1 .o
2.0 2.48 2.94 4.0 -4.1
n Y nn*n YU"
..... .....
.....
.....
.....
.....
Under5 5-10 110-120
Un'de'r'3.25 Under'2.7 3.25-6.5 3-6 71.4-77.9 68-74
Under0.53 0.21-0.41 0.40-0.40
1200-1400 Near 1600
779-909 S e a r 1040
0 32-0.37 Near 0 . 0 1
Y
4.97 6 08
518-605
Near 148
The case of salicylic acid (Table I) is a close parallel. The dissociation constant is 1.06 X By substituting this value for 6.61 X 10-6 in the above equation, the fraction of undissociated salicylic acid can be computed. Although the salt concentrations vary greatly with pH, the undissociated acid remains nearly the same, about 4.0 mg. per 100 ml.; it cannot be doubted that the antiseptic power of salicylic acid depends entirely upon the undissociated fraction, which is greatly affected by the p H of the medium. The strong antiseptic action of the undissociated acid as compared with its ion is probably due to the different rate with which they enter the cell. Jacobs (6) stated:
2 The equations as given by Michaelis contain a serious typographical error. I n all fractions containing the product of both dissociation constants RiKz,the corresponding [H +I value must be squared. This squareis omitted in the book.
TABLE 111. BACTERICIDAL CONCENTRATIONS OF SODIUM SULFITE
It is now generally believed that the permeability of cells to ions is a decidedly complex phenomenon, more or less limited in its extent and involving theoretical equilibria which may be approached very slowly and perhaps never be attained. The entrance of most undissociated weak acids and bases, particularly those of organic origin whose molecules contain considerable nonpolar hydrocarbon portions, seem to take place with great rapidity according to the simple laws of diffusion. This is similar to the theory of Hoffman and associates but is based on the definite conception of dissociation.
For each p H value there exists a corresponding concentration of HS03- ions, SO3-- ions, and undissociated HzS03. They were calculated by the Michaelis formulas2 (6) and are shown in Table 11. The first sets of experiments were made with Bacterium coli in broth buffered with phosphate mixtures. The source of H2S03 was Na2S03. At p H 8 one hundred times as much disinfectant is needed as a t pH 6. The corresponding concentrations of HSOs- ions, however, are quite constant, and i t seems evident that Bacterium coli ceases to multiply when the concentration of HSOa- ions reaches about 10 mg. per 100 ml. The concentration of these ions reaches a maximum a t p H 3.7. At still lower p H the HzS03 is largely undissociated. Therefore the toxicity of sulfurous acid should decrease below p H 3.7 if its efficiency depended exclusively on the HSOs- ions. As Bacterium coEi does not grow at such high acidities, the experiments were repeated with yeast which could grow feebly even a t p H 2.0. The result did not agree with this anticipation. The sulfite proved extremely toxic a t very low pH, and increasingly so as p H decreased. It was easy to show (Table 11) that inhibition of yeast was caused by undissociated H2S03 which is so toxic that 0.4 mg. per 100 ml. (4 p.p.m.) is sufficient to prevent growth. Near p H 6 some other factor enters; growth was stopped almost completely by 1.6% sodium sulfite which contained only 0.001 p.p.m. undissociated HzS03, and far less HS03- ions than were present a t p H 4.97. It may be that a concentration of more than 1% sodium sulfite brings about the change from alcoholic to glycerol fermentation which would interfere with the growth metabolism.
a
PH
NszSOa Causing Death i n 16 Min., , ME./ 100 M1.a
2.08
37
2.65 2.95 3.64
200 110 1530
1.84 2.53 3.00
245 830 5000
From Figure 2.
Undissociated HsSOFraction
IvIg./IOO ml.
Concn. exponent
Saccharomyces ellipsoideus 0.331 7.8 0.061 0.116 0.013
B a c t e r i u m coli 0.461 0.148 0.055
;:A)
3.20
13.0
g;
176
1
1.47
INDUSTRIAL AND ENGINEERING CHEMISTRY
February, 1944
5: .6
ELL1 PSOIDEUS
SACCHAROMYCES
,
4:
Log Scale Plot of Death Time at Various
Figure 2.
Acid Concentrations
Comparison shows that the two organisms are inhibited by different parts of the same molecule. HS0,- ions prevent the bacterium in concentrations of 10 mg. per 100 ml. while 500 mg. of this ion do not inhibit the yeast. Only the undissociated acid inhibits yeast. The different sensitivity of the two types of organisms to different ions of the same molecule induced us to test the concentrations of sodium sulfite required for killing. Solutions of different p H were prepared from citric acid and potassium phosphate and, after heat sterilization, were mixed with a solution of sodium sulfite in citric acid. To each 10 ml. of these standardized disinfecting solutions, 0.5 ml. of the culture was added, and the death time was determined by making transfers after 1, 2, 4, 8, 16, 30, 60, 90, and 120 minutes. The time intervals in which the organisms died are shown in Figure 1. Such results on a logarithmic scale must give straight lines (7’). Figure 2 shows that the lines are straight and that they are parallel for different acidities, which means that n is constant. However, the death-time curves for yeast have a slant different from that for the bacterium which indicates different concentration exponents. These exponents can be read directly from the logarithmic plots because n =
log t z log c1
- log tl - log c2
If we choose for yeast the line of p H 2.95 (any line is equally good since they are parallel), and select t2 = 120 and tl = 1, the corresponding logarithms of concentration are 0.04 and 0.69: n=
log 120 0.69 0.04
-
-
2*08
0.65 =
3.20
-
For Bacterium coli at pH 2.53, t2 - t l = 120 1, the corresponding logarithms of c are 0.74 and 9.32 10, and consequently
-
n =
log 120
0.74
2.08
+ 9.32 - 10 = 1.42
= 1*47
or nearly half the exponent for yeast. From these curves can be read the concentrations of disinfectant which will kill the organisms a t different p H in the same time.
187
The concentrations required to kill in 16 minutes are given in Table 111. From these values the amount of undissociated H2SOa was computed, and the values are sufficiently constant to indicate that the undissociated acid is the cause of death. When the concentration of the sodium sulfite exceeds 1000 mg. per 100 ml., death is slower, owing perhaps to “salt effects”, and the undissociated acid must be higher. The unexpected result is that ten times as much undissociated acid is needed to kill Bacterium coli, which was far more easily inhibited by sulfite than yeast, as to kill yeast. Among other antiseptics whose efficiency is controlled by acidity, the dyes are outstanding. Hoffmann (3) showed that with crystal violet the death rate is not greatly affected by acidity, while the inhibition of growth is greatly affected, not through electrolytic dissociation but because of the relation between pH and the oxidationreduction potential of the dye which is the real cause of growth inhibition. The effect of acidity on disinfection by chlorine was explained by Holwerda (4) as due t o dissociation of HOC1; however, this does not explain the entire rang6 of the pH effect and cannot be considered the final solution. SUMMARY
The toxic principle of sodium benzoate is the undissociated benzoic acid molecule which increases with the acidity of the medium. The test organism, a wine yeast, was completely suppressed whenever the concentration of undissociated benzoic acid reached 25 mg. per 100 ml. Salicylic acid acts in the same manner. Yeast multiplication ceased completely when 4 mg. of undissociated salicylic acid were present. Sulfur dioxide forms the dibasic sulfurous acid of which the SOI-- ion is without effect; the HSOa ion inhibits the growth of Bacterium coli when its concentration reaches 10 mg. per 100 ml., but yeast is not inhibited by it. The effect of SQZ on yeast is due t o the undissociated H2S03 molecule of which only 0.4 mg. per 100ml. prevents multiplication. At high acidities SO2 is a good disinfectant. The efficient principle is the undissociated HzSOa molecule; 7 mg. per 100 ml. kill the yeast. To kill Bacterium coli, 70 mg. are needed. Thus the bacterium, which is much more sensitive in regard to growth inhibition, can tolerate ten times as much disinfectant as yeast before it is killed. The different efficiency of undissociated molecules and ions of the same acid may be explained by the fact that it is difficult for ions to permeate living cell membranes. LITERATUR- CITED
(1) Degering and associates, J . Am. Pharm. Assoc., 27, 865 (1938), 28, 514 (1939); IND. ENQ. CHEM.,30, 646 (1938), 31, 742 (1939), 32,996 (1940). (2) Hoffman, Charles, Schweitzer, T. R., and Dalby, Gaston, IND. ENCI.CHEM.,33, 749 (1941). (3) Hoffmann, C. E., thesis, Cornel1 Univ., 1943. (4) Holwerda, K., Mededeel. Dienst Volksgezondheid Neder1and.India, 17, 251 (1928) (5) Jacobs, M. H., Cold Spring Harbor Symposia Quant. Biol., 8, 30
(1940). (6) Michaelis, L., “Hydrogen Ion Concentration”, p. 56, Baltimore, Williams and Wilkins, 1926. (7) Rahn, O., “Physiology of Bacteria”, p. 347, Philadelphia, J. Blakiston’s Son & Co., 1932. (8) Rogers, L. A., and Whittier, E. O., J . Bad., 16, 211 (1928).
