Bactericidal Properties of Effect of pH
Phenylmercuric nitrate, Merthiolate, Metaphen, Mercurochrome, tincture of iodine, phenol, Hexylresorcinol, and acriflavine were studied under controlled conditions of pH with respect to their bactericidal properties against Staphylococcus aureus and Escherichia coli. Metaphen and Merthiolate could not be adjusted, and acriflavine gave negative results at the concentrations tested. -411 the others showed increased potency with an increase in the hydrogen-ion concentration of the menstruum. Phenylmercuric nitrate, for example, is effective in a dilution of 1 part in 10,000 at a pH of 7; at a pH of 3 its potency against Staphylococcus aureus is increased tenfold. At a pH of 4 phenol showed a 25 per cent increase in effectiveness. The results indicate that, except for interfering
W. A. BITTENBENDER WITH ED. F. DEGERING AND P. A. TETRAULT Purdue University, Lafayette, Ind.
7 INCE the first studies in bactericidal action, it has been
b
recognized that acidity is an important factor. The classical work of Pasteur (1879) contains suggestions as to the effects of acidity on bacterial growth. For this reason the influence of acid media on microorganisms has been studied in much detail and from many points of view. For any given species of bacterium there is an optimum and relatively narrow pH range which allows vigorous growth. A wider range exists on each side of the optimum over which growth occurs less readily. For most bacteria this optimum pH lie8 a little to the alkaline side of neutrality (about 7.27.6). The wider range of p H over which growth is possible has not been accurately determined for a great many bacterial species. For most pathogenic bacteria, however, it appears to extend between pH values 5.0 and 8.0 ( 2 1 ) . On the other hand, it has been shown that chemical compounds are more highly bactericidal in acidic media. The study of the bactericidal power of hydrogen ions began with the work of Paul and Kronig (18)in 1896. They studied the action of mercuric chloride in the presence of salts of the noble metals, acids, and bases, and were the first to indicate that bactericidal action in acid media is proportional to the hydrogen-ion concentration or the degree of the electrolytic dissociation. Subsequently, Winslow and Lochridge (66)in 1906 substantiated the results obtained by Paul and Kronig (18). They calculated the pH of the solutions they studied by means of conductivity data. All of these early workers, however, were handicapped by the lack of a method for the direct determination of hydrogen-ion concentration. As the methods of adjusting and determining pH were improved and made more readily available, additional work confirmed the previous results. Chick (4) in 1910 studied the coagulation of bacteria with hot water and found increasing effectiveness when acidity was produced by an amount of acetic acid too small to be bactericidal in itself. Other investigations made by Xorton and Hsu (17) in 1916 and Friedenthal (6) in 1919 gave similar results. I n 1920 Bigelow and Estey ( I ) , studying the sterilization of food juices, found that those of low p H were sterilized much more rapidly. These investigators employed electrometsic methods for evaluating the pH of the test solutions. I n general, there are two methods of adjustment and determination-the first, by use of buffers and indicators in a comparator, and the second, by direct adjustment with acid or base and electrometric determination (6).
There are many examples of each and many types of compounds that have been studied so that only a few instances may be mentioned. Using glycine and citrate buffers, Joachimoglu (12) found that mercuric chloride is most effective over a p H range of 5.0 to 6.6 and practically ineffective from 7.8 to 10.1. The same type of buffer solution was also used by Northrop and De Kruif (16) in 1922. Using buffered solutions, salicylic, benzoic, and related aromatic acids, and phenols were studied by Vermast (ZS), Waterman and Kuiper (94),and Kuroda (14). Each found that the free acids are much better than the sodium salts a t pH values from 3.4 to 8.4. I n addition, the use of various compounds as urinary antiseptics has provided a further field for investigation of the relationship between pH and antiseptic action. Herrold and Ewert (10) studied the antiseptic action of mallophene (0phenylazo-a,a-diaminopyridine hydrochloride) in urine at various pH values attained by the use of phosphate buffers. Mallophene was also studied by Tetrault (20) in an aqueous solution of nutrient agar and glucose. He found minimum effectiveness a t a p H of 6.6 and increasing effectiveness at lower pH values. Mandelic acid, which is rapidly becoming a popular urinary antiseptic, was also found to give better results in acidic media. Rosenheim (19) and Holling and Platt (11) first used the sodium salt of the acid with ammonium chloride and later the ammonium salt t o obtain an acidic urine of p H 5.2 which produced high bacteriostatic action. More recently, Carroll, Lewis, and Kappel (3) adjusted the pH of their solutions in urine with a Coleman glass electrode and found that highly acidic media gave good results. In 1926 Keysser and Ornstein (13) postulated that optimum p H is the most important factor in disinfection, stating that “antiseptics are most effective a t some definite p H which is different for each group of antiseptics.” Thus they found silver nitrate most effective a t p H 7.6 and acriflavine most effective a t pH 8.0-8.4, but quite ineffective a t p H 7.0. Herrold and Ewert (10) stated similarly: “It is evident that if the hydrogen-ion concentration influences the optimum range of action of urinary antiseptics, more importance should be given to such observations in urinary infections.” 742
Commercial Antiseptics trodes, and stirrer were rinsed well with water of the same pH as that of the solution, and the rinsings were added to the volumetric flask. The flask was then filled to the mark with water of the proper pH. (The pH of the water was also adjusted with dilute sodium hydroxide and acetic acid.) After the contents of the flask had been thoroughly mixed, the p H was rechecked with the glass electrode, a drop or two of alkali or acid being added if necessary. The effect of the small concentrations of extraneous ions introduced in adjusting the pH of these solutions was shown to be negligible by the work of Goshorn, Degering, and Tetrault (9).
factors such as increased insolubility or instability, these diversified antiseptics show a definite increase in effectiveness with an increase in the hydrogen-ion concentration. This is in agreement with the “hydrogen-ion effect” postulated by Goshorn, Degering, and Tetrault (9), in their studies of the phenylalkanoic acids. The results do not entirely confirm the statement of Keysser and Ornstein (13) that “antiseptics are most effective at some definite pH which is different for each group of antiseptics.” They indicate, instead, that the “hydrogen-ion effect” increases progressively up to a maximum for benzoic acid, the phenyl cilkanoic acids, phenyl mercuric nitrate, Merthiolate, tincture of iodine, Hexylresorcinol, phenol, closely related compounds, and some inorganic salts.
Bactericidal Tests Escherichia coli and Staphylococcus aureus were used as test organisms. The sample solution was made up to a given dilution with water of the same pH. The technique was essentially that described by the United States Department of Agriculture (22). The culture was adjusted to the pH of each test before use. The dilution indicated in the table killed in 10 but not in 5 minutes a t 37” C. I n testing the mercurials, the “retransfer culture” modification was used as described in the Department of Agriculture circular (22). Controls of inoculated nutrient broth were run for each pH value tested. The results of these tests on standard pharmaceutical preparations are shown in Table I.
Recent. results of Goshorn, Degering, and Tetrault (9) on the bacteriostatic and bactericidal action of benzoic acid corroborated these findings and led them to postulate a “hydrogen-ion effect” which holds for the bacteriostatic and bactericidal action of benzoic acid, related compounds, and salt solutions. It was with the hope of adding further verification to this hypothesis that the present work of studying commercially available antiseptics a t controlled hydrogen-ion concentrations was undertaken.
OF BACTERICIDAL TESTS TABLE I. RESULTS
PH
Solutions of Definite pH The pII of the solutions was measured by the use of a membrane type glass electrode ( 2 ) with a saturated calomel electrode. They were connected to a Universal potentiometer assembly constructed according to the design of Goodhue ( 7 ) ; it contained modifications by H. W. Swank and R. H. Goshorn of Purdue University. The glass electrode was checked against buffer solutions of known hydrogen-ion concentration, before and after each period of use. The following commercial products were tested as dispensed and a t p l l values of 3.0, 4.0, 5.0, 6.0, and 7.0: Phenyl mercuric nitrate,a 0.067% Merthiolate,b 1-1000 Metaphen,* 1-500 Mercurochrome,b 2% Tincture of iodineb (7% iodine, 5% potassium iodide, 82.57, alcohol) Phenol, 5% Hexylresorcinol,* 1-1000 Acriflavineb (neutral), 1-1000 Elixir of mandelic acidb (27.27, ammonium mandelate)
Highest Killing Dilution Staph. aureus Esch. coli
Lowest Dilution, Positive Growth Staph. a u r m s E w h . cold
Phenyl Mercuric Nitrate 400,000 600,000 40,000 20,000 20,000 30,000 10,000 30,000 30,000 20,000
3.0 4.0 5.0 6.0 7.0
100,000 10,000 10,000 20,000 10,000
3.0 4.0 5.0
6.0 7.0 10.0
75,000 75,000 10,000 5,000 10,000 10,000
Merthiolate 100,000 25,000 10,000 5,000 5,000 5,000
100,000 100,000 25,000 10,000 25,000 25,000
50,000 25,000 10,000 10,000 10,000
10.0
1,000
Metaphen 10,000
10,000
25,000
300
600
50,000 40,000 20,000 40,000 a
Mercurochrome 10.0
3.0
4,000 3,000 2,000 1,500 1,000
3.0
90 75 70 65
4.0 5.0 6.0 7.0
Supplied by The Hamilton Laboratories, Hamilton, Ohio. Prepared by t h e School of Pharmacy, Purdue University. e I n adjusting solutions of the lower pH values, concentrated acetic acid was used.
100
a
4.0
5.0 6.0 7.0
An 80 ml. portion of each of the tesfsolutions was placed in a Berzelius beaker into which the electrodes were inserted. The pH of the solution was adjusted by adding 0.1 N sodium hydroxide or 0.1 N acetic acid dropwise”; adequate mixing was ensured by the use of a mechanical stirrer. When the final adjustment of the pH had been made, the solution was transferred to a 100-ml. volumetric flask; the beaker, elec-
3.0 4.0 5.0 6.0 7.0 a
743
60
100,000 12,500 5,000 3,000 3,000
300 Tincture of Iodine 4,000 4,000 3,000 2,000 1,500
a
Phenol ( 1 6 ) 100 100 100 90 75 Hexylresorcinol 100,000 12,500 5,000 5,000 5,000
Contiols in water a t pH 3 did not grow.
a
4,000 3,000 2,000 1,500
5,000 4,000 3,000 2,000
a
90 75 75 65
~
125 125 100 90
a
a
25,000 12,500 5,000 5.000
25,000 12,500 12,QOO 12.a00
744
INDUSTRIAL AND ENGINEERING CHEMISTRY
Consideration of the data reveals a marked increase in bactericidal effectiveness with an increase in the hydrogen-ion concentration of the menstruum. These results are in agreement with the data obtained from a similar study of the phenyl alkanoic acids (8). The marked difference in the structure of these compounds seems to indicate that the “hydrogen-ion effect” (9) may be somewhat independent of structure, provided instability or insolubility in acid media are not interfering factors. Metaphen and Mercurochrome could not be adjusted to the desired pH, hence their solutions could not be checked with respect to the “hydrogen-ion” effect (9). Acriflavine gave negative results in the concentrations tested.
Acknowledgment The authors wish to thank H. Hunt for conditioning of the p H apparatus, and R. N. Shreve of the Chemical Engineering Department of Purdue and V. H. Wallingford of the Mallinckrodt Chemical Works for their interest and support of this project.
Literature Cited (1) Bigelow, W. D., and Estey, J. R., J . Infectious Diseases, 27, 602 (1920). (2) Britton, H.T. S., “Hydrogen Ions, Their Determination and Importance in Pure and Industrial Chemistry”, New York, D. Van Nostrand Co., 1932. (3) Carroll, G., Lewis, B., and Kappel, L.,J. Urol., 39, 710 (1938).
VQL. 31, NO. 6
Chick, H., J Hyg., 10, 237 (1910). Clark, W.M., and Lubs, H. A.,J. Bact., 2, 1, 109,191 (1917). Friedenthal, H., Biochem. Z.,94,47 (1919). Goodhue, L. D., Iowa State CoZZ. J . Sci., 10,7 (1935). Goshorn, R. H.,and Degering, E. F., paper presented before Div. of Medicinal Chemistry a t 94th Meeting of A. C. S., Rochester, N. Y., Sept. 6-10, 1937. Goshorn, R. H.,Degering, E. F., and Tetrault, P. A., IND. ENG.C H ~ M30, . , 646 (1938). Herrold, R. D., and Ewert, E. E., J. Lab. CZin. Med., 17, 49 (1931). Holling, H. E.,and Platt, R., Lancet, 1936,769. Joachimoglu, G., Biochem. Z . , 134,489 (1923). Keysser and Omstein, Klin. W O C ~ S C5, ~ 404 T . , (1926). Kuroda, T., Biochem. Z . , 169, 281 (1926). Lundy, H.W., J . Bact., 35, 633 (1938). Northrop: J. H.,and De Kruif, P. H., J. Gen. PhysioZ., 5 , 127 (1922). Norton, J. F., and Hsu, P. H., J . Infectious Diseases, 18, 180 (1916). Paul, T., and Kmnig, B., 2. physik. Chem., 21,414 (1896). Rosenheim, M. L.,Lancet, 1935, 1032. Tetrault, P. A.,Med. J . Record, Sept. 6,1933. Topley, W. W. C., and Wilson, G. S., “Principles of Bacteriology and Immunity”, Baltimore, Wm. Wood and Go., 1936. U. S. Food and Drug Administration, Dept. Agr. C ~ T C198 . (1931). Vermast, P.G.F., Biochem. 2..125, 107 (1921). Waterman. H.I.. and Kuioer. P.. Rec. trav. chim..43.232 (1924). Winslow, C. E. A., and Lochridge, E. E., J. Injectidus D&asei, 3, 547 (1906). PRE~SQNTED a t the 96th Meeting of the American Chemical Society, Milwaukee, Wis.
SUGAR MANUFACTURE
By Giovanni Stradano (1 s 3 6-1 60s 1
T h e second chemical plate in the “Nova Reperta” shown here, represents the manufacture of sugar as carried out in Sicily about the year 1570. Like the first plate shown in February, it is an engraving by the Brothers Galle of Antwerp from an original painting by Stradano. This is No. 102 in the Berolzheimer Series of Alchemical and Historical Reproductions. Again we are indebted to Professor E. C. Watson of the California Institute of Technology, who is the proud possessor of this very rare set of plates known as the “Nova Reperta” (New Discoveries), for his courtesy in supplying a photograph of this engraving. It is interesting to note the proximity of the factory to the field in which the sugar cane is growing. 50 East 41st Street New York, N. Y.
A complete list of the hrac 96 reproductions appeared in our January, 1939, issue. page 124. An addiriond rcproducrion appears each month.
D*D‘ BERoLZHEIMBR
