RICINOLEATE UPON BACTERIA

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1 A COMPARATIVE STUDY OF THE ACTION OF SODIUM RICINOLEATE UPON BACTERIA From the Division of Laboratories and Research, New York State Department of Health, Albany Received for publication, May 14, 1928 This study records the results of a comparison of the cellular susceptibility of different species of streptococci and certain. other bacteria exposed to the action of sodium ricinoleate under similar physiological conditions. The following microorganisms were investigated: Streptococcus vtiridans (no. 13), S. pyogenes (no. 12), S. equinus (no. 17), S. lacticus (no. ib), S. fecalis (no. 16), streptococci isolated from cases of measles (no. C205, no. C230, no. C231, no. C232), erysipelas (no. C174, no. C175, no. C176, no. C177), and scarlet fever (no. 14E Dochez N. Y. no. 5, no. 14F Dick 1, no. C203 United States Hygienic Laboratory), pneumococci (types I, II, III, and IV), Staphylococcus albus, B.diphtheriae, B. tuberculosis, B. paratyphosus A and B, and B. dysenteriae (Flexner and Shiga). Sodium ricinoleate was chosen as the most suitable soap because it dissolves readily in physiological salt solution. Preparation of ricinoleic acid. One hundred grams of commercial castor oil were mixed with 300 cc. of an 8 per cent solution of potassium hydroxide in alcohol, and allowed to stand at room temperature for four hours and at 4 C. for twenty hours. The sedimented soaps were separated by decantation and filtration, with the aid of suction through a Buchner funnel cooled with ice water. They were then dissolved in water and further treated separately; the alcoholic filtrate, together with the liquid decanted from the solidified soap, was mixed with an excess of water and extracted with ether to remove unsaponified substances. The clear soap solutions were acidified with an excess of 10 per cent hydrochloric acid; the liberated 203

2 204 fatty acids were extracted with ether, washed out several times with water, freed from ether under diminished pressure, and dried in a vacuum desiccator over sulphuric acid; the oily liquid was mixed with one-quarter volume of absolute alcohol and allowed to stand for several days at -7 C. The saturated fatty acids, which were found in a larger amount in the fraction derived from the soap insoluble in alcohol during the saponification of the oil, crystallized out. The crystals were removed at -7 C. by decantation and filtration through a folded filter paper. The filtrate was kept at the low temperature as long as the crystals were formed; finally, it was freed from a part of the alcohol by evaporation under diminished pressure, and further cooled until no sediment was deposited. The oily liquid was neutralized with a 4 per cent solution of sodium hydroxide in alcohol. The solidified soap was filtered off and further treated separately. The filtrate was made alkaline to phenolphthalein and was distilled off under diminished pressure. The soaps were dried in a vacuum desiccator over calcium chloride and extracted with ether. Those soluble and insoluble in ether were further treated separately. The sodium soaps were dissolved in water and precipitated with a 10 per cent solution of barium chloride. The barium salts were filtered off with the aid of suction, washed out with water, suspended in 95 per cent alcohol, and heated to 700C. until dissolved; the solution was cooled to 500C. and filtered through a filter paper placed in a double-walled metal funnel kept at a temperature of 400C. The barium salts which solidified in the filtrate at between 400 and 300C. were separated by decantation and further treated separately. The barium salt which remained in the solution below 300C. and was precipitated by cooling to 40C. was filtered off. All fractions of barium salt were further purified separately by the above method with 98 per cent alcohol, and finally with absolute alcohol. The barium salts were suspended in water, and decomposed with 10 per cent hydrochloric acid; the liberated fatty acid was extracted with ether, washed out with 1 per cent sulphuric acid and with water, and freed from ether by evaporation under diminished pressure. The iodine value of some fractions, determined by Wijs' method, was found to be somewhat lower, and that of others, a little higher than the theoretical value. The fraction of fatty acid with the iodine value of 85 was used for the preparation of sodium salt; for that purpose the ricinoleic acid was mixed with two volumes of absolute alcohol and neutralized with 4 per cent sodium hydroxide in alcohol. The soap was filtered off with the aid of suction and dried in a vacuum desiccator. It was a

3 ACTION OF SODIUM RICINOLEATE UPON BACTERIA 205 white powder; and its solution in an 0.85 per cent solution of pure sodium chloride was perfectly clear. A 2 or a 1 per cent solution was sterilized in the autoclave for ten minutes, and certain quantities of that solution were diluted with a sterile salt solution to make up to the different dilutions used in the experiment. A highly diluted solution, 0.01 per cent, of soap should not be heated as it turns opalescent and, therefore, is physiologically less effective. Sodium chloride used should be free from calcium and magnesium. Old samples of sodium ricinoleate were found to be less active than freshly prepared ones. All the bacteria studied were grown in streptococcus toxin broth, i.e., beef-infusion broth (ph 7.6 to 7.8) to which 0.02 per cent of glucose was added.' For B. tuberculosis, only a special broth was used to which 5 per cent of glycerol was added; a small disk of sterilized cork was placed in each test tube to support the bacterial pellicle. Investigation of the action of sodium ricinoleate was limited to establishing the highest dilutions of soap which inhibit growth and kill the bacteria. The method was the same as that described in a previous paper (Kozlowski, 1925). DISCUSSION OF RESULTS The presence of a certain amount of sodium ricinoleate in broth inhibited the growth of some strains of the inoculated bacteria completely, while that of some other micro6rganisms was only slightly retarded or was not even affected in concentrations of 1:1000. It this respect there is as great variation among the streptococci as among other kinds of bacteria (see table 1). The figures for the growth-inhibiting dilutions of soap represent only a relative value and show that the bacteria which were incubated in the same kind of broth and at the same temperature will react in the manner described. These figures will be much 1 The possible presence, in the broth, of substances which might weaken the action of the soap should be considered. Broths prepared by special methods used in this laboratory were compared; as, for instance, broth for pneumococcus, B. diphtheriae, and streptococcus. (New York, 1927.) Broth that gave a rich precipitate in twenty-four hours with sodium ricinoleate in a dilution of 1:1000 was found to be less suitable than broth that, under such conditions, formed only a slight turbidity.

4 206 lower if such substances as salts of magnesium, calcium, or serum, which bind soap, are mixed with the broth. One cubic centimeter of physiological salt solution added to the broth was enough to influence the reaction on account of an excess of-sodium in the medium. The inhibitory test, however, in which water was used as a soap solvent, showed that, in some cases only the highest inhibitory dilutions of soap were somewhat lower than TABLE 1 The action of sodium ricinoleate on bacteria in broth culture HOIGHIT BACTZRI- HIGHEST INEIBI- CIDAL DILUTION BACTERIA TORY DILUTION or SOAP IN BROTH OF 80AP IN BROTH (ACTION AT 35-C. IX 7 HOUS) Streptococcus viridans... 1: Streptococcus pyogenes... 1: Streptococcus equinus... 1:10,000 0 Streptococcus lacticus... 1: Streptococcus fecalis Streptococci (measles): C230,C232, C :10,000 1:5000 C :20,000 1:5000 Streptococci (scarlet fever): 14E, 14F... 1:20,000 1:5000 C :5000 1:5000 S3treptococci (erysipelas) :20,000 1:5000 Pneumococci... 1:20,000 1:10,000 Staphylococcus albus... WM 1:2000 B. diphtheriae... 1:20,000 1:2000 B. tuberculosis... 1: B. dysenteriae B. paratyphosw O B. coli communis 0 = no action in the dilution 1:1000. those obtained with the soap-salt solution which seemed, to a certain extent, to affect some strains of streptococci; namely, those isolated from cases of erysipelas, scarlet fever (no. 14F), and measles (no. C231), and S. pyogenes. The figures that express the bactericidal dilutions of soap correspond to the given time and temperature; they may increase considerably if the bacteria are in contact with the soap for a

5 ACTION OF SODIUM RICINOLEATE UPON BACTERIA 207 longer time. In many cases some bacteria, pneumococci and certain strains of streptococci, were killed in four hours instead of seven. Transplants made at different time intervals revealed that the bactericidal effect of sodium ricinoleate upon susceptible bacteria is conditioned by the concentration of active soap solution, the amount of bacteria, and the time and the temperature of exposure. The stronger the concentration of the active soap solution, and the higher the temperature (between 00 and 400C.) the sooner were the bacteria killed. The specific susceptibility of each kind, species, and strain, is the most important factor since, if the particular strain used is non-susceptible, the other factors in the reaction are nullified. Of the streptococci investigated, the strains isolated from cases of scarlet fever and measles were subjected to special study; a slight difference in the susceptibility of the different strains in both groups was observed in tests repeated six times. A comparison of a large number of strains might possibly lead to a more accurate knowledge of the streptococci which belong in these two groups and which have recently attracted so much interest. Of the bacteria investigated, pneumococci were found to be the most susceptible to the bactericidal action of sodium ricinoleate, types II and III being much more susceptible than type I; they were dissolved by that soap as readily as by bile or sodium oleate. However, they are not more susceptible to the inhibitory action than some of the streptococci. Sodium ricinoleate may be used in bacteriological research and in diagnostic work as well. The optimum dilution of sodium ricinoleate for a test of the solubility of pneumococci in a broth culture is about 1:5000; higher concentrations of the soap cause a turbidity in the broth which obscures lysis. The test can be carried out in the following manner: To a small test tube containing 0.8 cc. of broth culture are added 0.2 cc. of a 0.1 per cent solution of sodium ricinoleate in water. The turbidity of the broth culture disappears gradually and the medium becomes clear in from four to fifteen miainutes at room temperature. Lysis was accelerated markedly at a temperature of 37 C. Pneumococci, types II and IV, were dissolved much

6 208 sooner than pneumococci of types I and III. Pneumococci grown im the "streptococcus" broth were dissolved sooner than in the "pneumococcus" broth. No streptococci studied were dissolved by sodium ricinoleate. SUMMARY AND CONCLUSIONS Among the streptococci investigated those isolated from cases of erysipelas, measles, and scarlet fever were the most susceptible to the bactericidal action of sodium ricinoleate; they were killed by this soap in a dilution of 1:5000 in about seven hours or sooner at 35 C.; in a control culture, without soap, they survived for more than one week; their growth was inhibited by this soap in a dilution of 1:20,000 or less; in their cellular susceptibility they approach that of pneumococci which were killed by the same soap, under similar conditions, in a dilution of 1:10,000. Pneumococci are dissolved quite readily by sodium ricinoleate in a dilution of 1:5000; and in this regard they differ from other bacteria investigated. Of the bacilli, B. diphtheriae was found to be quite susceptible to the inhibitory, and much less to the bactericidal, action of sodium ricinoleate; B. tuberculosis showed a slight susceptibility, as it was inhibited in its growth by this soap in a dilution of 1:2000; B. paratyphosus, B. dysenteriae, B. coli communis, and S. fecalis are quite resistant to the inhibitory and bactericidal action of sodium ricinoleate. They are probably adapted to the action of soaps in their natural conditions of growth. An intermediate place between susceptible and non-susceptible bacteria is taken by some streptococci which are more or less saprophytic, i.e., S. lacticus, S. pyogeres, and S. viridan2; they were inhibited in their growth by sodium ricinoleate in a dilution of 1:5000 or lower, but were not killed in a dilution of 1: The bactericidal effect of sodium ricinoleate is conditioned by the specific cellular susceptibility of the bacteria, and further by the concentration of the soap solution, by the time and the temperature of exposure. The difference in the cellular susceptibility of various strains of the groups of streptococci isolated from cases of measles, scarlet

7 ACTION OF SODIUM RICINOLEATE UPON BACTERIA 209 fever, and other infections, might be helpful in bacteriological investigations. Sodium ricinoleate may be used instead of bile for dissolving pneumococci in diagnostic routine work. In conclusion, the author wishes to express his gratitude to Dr. Wadsworth, the director, and to thank Mr. J. Quigley for his kind assistance. REFERENCES KOZLOWSKI, A Comparative studies of the action on the pneumococcus of bile acids and unsaturated fatty acids, found in bile in the form of soaps. Jour. Exper. Med., 42, 453. New York, 1927 Standard Methods of the Division of Laboratories and Research of the New York State Department of Health, 86, 87, 88. Baltimore, Williams & Wilkins Co. Downloaded from on November 3, 2018 by guest