applied repeatedly to the conjunctiva of albino rabbits (fig. 1). RIOPHAGE, VACCINES AND VENOMI SOLUTIONS) CHLORIDE*

Transcription

1 PRESERVATION OF BIOLOGICAL FLUIDS (BACTE- RIOPHAGE, VACCINES AND VENOMI SOLUTIONS) WITH ALKYL-DIMIETHYL-BENZYL-AMM\IONIUM- CHLORIDE* EUGENE AJAIER Florida Medical Center, Venice, Florida Received for publication January 12, 1939 The general properties of this compound have been evaluated rather extensively in other reports. The reported phenol coefficients are as follows: 150 at 20'C. (1\Iaier and 1\Iiller, 1936), 279 at 20'C. (Dunn, 1937), and 275 at 370C. (Heineman, 1937), respectively for Staphylococcus aureus no. 209 Dept. of Agriculture and 200 at 20'C. (MIaier and Aiuller, 1936), 250 at 20'C. (Dunn, 1937), and 176 at 20'C. (Heineman, 1937), for Eberthella typhosa (Rosenbach) Dept. of Agriculture at the same temperature tested by the F. D. A. method. Domagk (1935) shows that an aqueous solution of this compound in a concentration of 1: 1,000 can be given by mouth to guinea pigs as their only source of fluid for months without apparent deleterious effect upon their health. These toxicity studies indicate that rabbits tolerate 1.2 ml. per kilogram of body weight of a 10 per cent solution of the compound, when given intraperitoneally or subcutaneously. A solution ranging in concentration from 1:2,500-4,000 appears to be safe for use in the eye as it was shown to produce little or no reaction, when applied repeatedly to the conjunctiva of albino rabbits (fig. 1). To demonstrate this, the procedure was as follows: Of the solution 1:4,000, one half ml. was instilled into the conjunctiva of the rabbit and kept there for one minute. Immediately after, the eye was washed out with saline. For comparison we used a 3' per cent concentration of tincture of iodine * Trade name Zephiran. 33

2 34 EUGENE MAIER which was administered under the same conditions to a second animal. Only one application was necessary to produce the described effect with the tincture. Severe reactions resulted. Within three weeks the animal treated with the tincture of iodine showed a completely opaque cornea and severe inflammation with discharge of pus existed during most of the period. The animals which were treated with the ammonium chloride com- _in. _.~~~V FIG. 1. CROSS SECTION THROUGH CORNEA OF RABBIT Alkyl-dimethyl-benzyl-ammoniumchloride 1:4,000 was applied daily for ten days to this eye. There is little if any deviation from the aspect of the normal untreated cornea. pound showed hardly any reaction and the solution was administered daily for 10 days without any ill effects, except slight defensive movements of the animal during and after the application which stopped soon after the saline was instilled. After three weeks the animals were killed and the bulbi were enucleated. After fixation and hardening, the cornea was taken off the bulbs and sectioned and stained with Mayer's Hemalaun. The

3 PRESERVATION OF BIOLOGICAL FLUIDS histological examination confirmed the gross anatomical findings, namely, no injury to or thickening of the anterior epithelium in the case of the ammonium chloride, enormous thickening, fibrotic changes and scar tissue formation extending through the cornea into the substantia propria in the animal treated with tincture of iodine (fig. 2). 4k ~ ~ 4. 4I& FIG. 2. CROSS SECTION THROUGH CORNEA OF RABBIT TREATED WITH TINCTURE OF IODINE 3 2 PER CENT One application of tincture of iodine 3- per cent was sufficient to produce these profound changes in the cornea. There is enormous thickening, fibrotic changes and scar tissue formation extending through the cornea into the substantia propria. From such studies as these it appeared probable that the absence of mercury and phenol radicals and the low toxicity of this compound might make it very useful for the preservation of biological fluids. In the following, results obtained in treating bacteriophage, vaccines and venom solutions with the compound are recorded. 35

4 36 EUGENE MAIER STAPHYLOCOCCAL BACTERIOPHAGE We began with the determination of the germicidal range of alkyl-dimethyl-benzyl-ammoniumchloride. The chemical structure of the compound is: CH3 CH3 X / ~CH2N: R1 Cl The alkyl residue (R1) is composed of the radicals C811l7, C1oH21, C12H25, C14H29, C16H33, and C18H37, their source being the mixture of fatty acids of cocoanut oil in original proportion. The product is soluble in water, acetone and alcohol. Staphylococcus aureus (Dept. of Agriculture no. 209) was killed within and below 24 hours by concentration of the ammonium chloride of 1:80,000 to 1:100,000. Tests show that at 30'C. these organisms are killed within 10 minutes by concentrations of the compound between 1: 30O000-35,000. The range of inhibition we found to be very large, varying from 1:100,000 to 1:800,000. At a concentration of 1: 800,000 in the original tube after a contact of 24 hours at room temperature, the upper limit of bacteriostatic activity has therefore been reached, both the original and the subculture tubes showing growth. Test tubes containing 10 ml. of bouillon (Difco nutrient) were inoculated with 0.5 ml. of a 24 hour broth suspension of Staphylococcus aureus (Dept. of Agriculture no. 209). The broth contained the disinfectant in solution in concentrations ranging from 1:40,000 to 1: 800,000. Within 24 hours the tubes were opened again and subcultures were made. Table 1 shows the results obtained. From table 1 it will be noticed that the germicidal range reaches up to 1:100,000, the bacteriostatic range from there up to 1:800,000, and the inactive range from there on. Marshall and Hrenoff (1937) propose the term "disinfectant spectrum" for the relative evaluation of disinfectants whose bactericidal, bacteriostatic and inactive concentrations have thus been determined. On the basis of these results, it was decided to choose a dilution

5 PRESERVATION OF BIOLOGICAL FLUIDS of 1:50,000 of the disinfectant in staphylococcal bacteriophage. The following scheme was used in these tests: ml. bouillon plus 0.1 ml. of a 24-hour culture. Growth control tube ml. bouillon plus 0.5 ml. phage disinfectant mixture. 3. Same as 2. (Tube three is used in addition to no. 2 tube, as there might be in tube 2 or 3 an initial lysis with a secondary growth following a short time later. In case there is initial TABLE 1 Test organisms: Staphylococcus aureus. Incubation temperature 37'C. Time of contact 24 hours ORIGINAL SUBCULTURE RANGE CONCENTRATION TUBES TUBES 1:40,000 - _ Germicidal _ 1:60, :100,000 _ + 1:200, :300, :400, Inhibition.1:500, :600, :700, :800, Inactive... - no growth. + growth. lysis in tube 2 or 3, one of the tubes 2 or 3 can be filtered and the resulting phage propagated separately, regardless of the outcome in the other tube.) 4. Sterility Control of Phage. After phage lysis had taken place, filtration by Berkefeld N followed. The phage obtained was kept in the refrigerator and tested at intervals and after preservation with the compound in proportion of 1: 50,000. After intervals of two and three weeks, checks were made, to insure the viability of the phage. The longest period so far checked was three months, in which interval 37

6 38 EUGENE MAIER the phage had not lost any of its lytic power in the presence of the alkyl-dimethyl-benzyl-ammoniumchloride at the concentration under test. VACCINES In the preparation of autogenous vaccines, alkyl-dimethylbenzyl-ammoniumchloride was added to the vaccine to make a final concentration of 1:50,000. This concentration was agreed upon as previous tests had shown that the germicidal range was as high as 1:100,000. VENOM SOLUTIONS It has been stated (Macht, 1936) that rattle-snake venom solutions (Crotalus adamanteus) are very unstable, as they are affected by heat and rapidly decompose even at room temperature. To unaltered venom solutions prepared from dried venom (the venom was obtained freshly and frozen and dried rapidly in vacuo and over sulphuric acid), was added the ammonium chloride compound to make a final concentration of 1:50,000. In the same way, we treated venom solutions of the cotton-mouth moccasin (Ancistrodon mocasen). Over a period of 6 months we have not been able to demonstrate loss of toxicity, when tested in mice. Fresh venoms tested by intraperitoneal injection of the venom gave a minimum lethal dosage of gamma for the rattle snake and 150 gamma for the moccasin venom in mice averaging 20 grams. However, such venoms when sterilized by filtration through Berkefeld N filters did show an appreciable loss in toxicity, as determined in the mouse test, a loss which probably is due to absorption by the filter. To venom solutions the compound was added in the same concentration, with no apparent change or precipitation of protein material. DISCUSSION As to the significance of such studies, recent investigations, (Bronfenbrenner, 1928), have shown that in many respects the behavior of the phage toward chemicals is not essentially different from that of enzymes on one hand and from that of viruses on the other. Prausnitz and Firle (1924) mention that the

7 PRESERVATION OF BIOLOGICAL FLUIDS adaptation of phage to phenol and mercuric chloride was necessary. In these studies no perceptible degree of adaptation was necessary, to produce bacteriophage in the presence of alkyldimethyl-benzyl-ammoniumchloride. CONCLUSIONS A new compound, alkyl-dimethyl-benzyl-ammoniumchloride has been studied in reference to its germicidal, bacteriostatic and inactive zones in accordance with the term "disinfectant spectrum" as proposed by Marshall and Hrenoff (1937). The effect of this compound in dilutions of 1: 4,000 has been studied on the cornea of the rabbit by histological methods. Comparisons were made with 3' per cent tincture of iodine. It was shown that one single application of the iodine tincture was sufficient to destroy the cornea completely and produce permanent opaqueness in the same. The compound under discussion has been applied to the cornea daily for ten days with no consequences. A dilution of 1: 50,000 in staphylococcal bacteriophage was found not to interfere with the reproduction of the phage. Furthermore, the phage-disinfectant mixture was found to be capable of reproduction of new phage after 3 months contact in the refrigerator. The same concentration of the compound was employed in the preservation of vaccines and venom solutions. REFERENCES BRONFENBRENNER, J In: Filtrable Viruses, ed. by Th. M. Rivers. William & Wilkins, Baltimore. DOMAGK, G Eine neue Klasse von Desinfektionsmitteln. Deut. med. Wochschr., 61, DUNN, C. G Antiseptic and germicidal properties of a mixture of high molecular alkyl-dimethyl-benzyl-ammonium chlorides. Am. J. Hyg., 26, HEINEMAN, P. G Antiseptic properties of alkyl-dimethyl-benzyl-ammoniumchloride. J. Am. Pharm. Assoc., 26, MACHT, D. I Therapeutic uses of snake venom. Med. Record, 144, MAIER, E., AND MtLLER, E Die Wirksamkeit der gebriuchlichsten Desinfektionsmittel. Fortschr. Therap., 12, MARSHALL, M. S., AND HRENOFF, A. K Bacteriostatic action of disinfectants. J. Infectious Diseases, 61, PRAU5NITZ, C., AND FIRLE, E Neuere Untersuchungen tiber das Wesen des Bakteriophagen. Zentr. Bakt. Parasitenk, Orig., 93, Beih.,