CoUege of ^fipsiciansi anli burgeons Digitized by tine Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/onmethodsusedresOOsedg EOGcWICK. Frederic S.Le Columbia Uuivtr K'«w York, [From Technology Quarterly, Vol. VI, No. 2, July, 1893.] On Methods Used and Results Obtained IN MAKING germicidal-efficiency tests DISINFECTANT FOR USE IN RAILWAY SANITATION. By WILLIAM T. SEDGWICK, Ph.D. BOSTON : Beacon Press; Thomas Todd, Printer, 7-A Beacon Street, _j 1899. — - 1_^ f From tho j Biological Laboratory, MASSACHUSETTS INSTITUTE rt l\f) io IS My/ jr>r ON METHODS USED AND RESULTS OBTAINED IN MAKING GERMICIDAL-EFFICIENCY TESTS OF A DISINFECTANT FOR USE IN RAIL WA V S ANITA TION. By WILLIAM T. SEDGWICK, Ph.D. Received August 18, 1893. The arrival of Asiatic cholera in the harbor of New York in the autumn of 1892 directed general attention to matters of public san- itation. The disease was brought upon steamships, and much of the alarm that was felt was due to the knowledge that cholera might be carried all over the land by the railroads. The importance of railway sanitation had long been known to experts, and the presence of cholera at our doors served to inform the public. Fortunately, in 1892, the disease was confined to New York. Recognizing the probability of a recurrence of the danger and the possibility of a more serious invasion in 1893, and anxious to be ready for any emergency, the officers of the Pennsylvania Railroad Company determined to have thoroughly tested, with respect to its efficiency or germicidal power, a disinfectant of their own manufacture, which, since 1885, they have been using in their cars, closets, urinals, pas- senger stations, freight-houses, shops, etc., or wherever a liquid disin- fectant is applicable upon a railway system. The extent and public importance of such disinfection may be realized from the fact that this company operates altogether nearly 8,000 miles of railway. It forms one of the principal avenues of communication between the principal seaports of the United States and the great West. It carries over its lines yearly many thousands of immigrants, and directly connects one with another the largest cities in the United States. It is therefore held to be one of the most important railway systems in America ; and it has a reputation for careful and scientific administration. I am informed by Dr. C. B. Dudley, Chief of its Chemical Depart- ment, that chemical studies on commercial disinfectants by this road began as early as 1880, and in 1884 these studies culminated in the thorough examination of some twenty-six commercial disinfectants. In view of the multiplicity of materials offered in the market as dis- infectants, and the very high price charged for many worthless com- pounds, it was decided to embody the results of all these studies in a specification for a disinfectant to be made for the use of the road. This specification was issued in February, 1885. A few years later, when the work of the Committee on Disinfectants of the American Public Health Association became available, this specifica- tion was revised accordingly, and the so-called P. R. R. Standard Dis- infectant was brought up to date. The disinfectant submitted to me for tests of its germicidal power was said to consist of a concentrated solution of chloride of zinc, chloride of copper, and bichloride of mercury, with a few drops of terebene. The terebene was added as a telltale, to inform the officers whether the disinfectant had been faithfully used, rather than as a disinfectant. I was also informed that the disinfectant contains one part in five hundred of bichloride of mercury and a little over 45 per cent, of chloride of zinc. The chloride of copper was added because a little copper salt was desired to fix sulphuretted hydrogen, and the chloride of zinc solution was made very strong on account of the well-known deodorizing properties of this material. I am also informed by Dr. Dudley that this disinfectant is made by the railroad company, for its own use, under the supervision of the officers of its laboratory. Old battery zincs which are no longer useful in the batteries, muriatic acid, sulphate of copper, common salt, and bichloride of mercury are used in its manufacture. The actual cost of the disinfecting material in an eight-ounce bottle is about two and one half cents. The material is distributed to the service in wooden boxes, holding twenty-four bottles each, and in barrels. If these boxes and bottles are returned, the real cost to the different parts of the service is the figure given above. If the bottles are broken and the boxes destroyed the cost is somewhat greater. In barrels which are not returned the cost is somewhat less than in bottles, no labor being required for bottling. There were made and used on the whole Pennsylvania railway system during the year 1892 366 gross of eight-ounce bottles and 69 fifty-gallon barrels. The specifications to which the disinfectant must conform are given as follows : " Disinfectant for the use of the Pennsylvania Railroad Company will consist of a neutral solution of the normal chlorides of copper and zinc and mercuric chloride put up in eight (8) ounce bottles and se- curely corked. Each bottle should contain 2,400 grains of zincic chlo- ride, 120 grains of cupric chloride, io.$ grains of mercuric chloride, and ten drops of a mixture of equal parts of terebene and turpentine." The bottles must have pasted upon them the following " Directions for Use," a perusal of which will convey a good idea of the scope and the importance to the community of efficient railway sanitation : DIRECTIONS FOR USE. For Use on Cars. — Empty the contents of this bottle into about one quart of water. With a sponge or waste, wash with the liquid all parts of the urinal, seat, and hopper, and the floor adjacent to them in the closet, allowing some of the liquid to run down the urinal pipes. The same solution should be used on the floors and woodwork of the whole car whenever washing with disinfectant is necessary. The carpets and plush of an infected car should not be treated with disinfectant, but should be fumigated instead. For Cleaning Privies, Closets, etc. — Empty the contents of this bottle into about one quart of water, and use as above. The disinfectant acts slowly on lead, tin, copper, brass, iron, etc., and it is not advisable to allow it to stand in the basins or traps. In the vault, use the disinfectant full strength, not less than half a dozen bottles at once, but it is better to disinfect privy vaults with crushed sulphate of copper, or chloride of lime, sprinkled freely over the mass. For Removing Bad Odors. — Empty the contents of this bottle into about one quart of water, and sprinkle the liquid in the place from which the odors arise; also saturate a towel, or other fibrous substance, with the liquid and hang it in the place from which it is desired to remove the odors. For use on towels, etc., a disinfectant without color can be furnished if desired. In Cases of Diphtheria, Scarlet, Typhoid, and Typhus Fevers, Cholera, Dys- entery, Consumption, etc. — Place a little of the disinfectant, full strength, in the vessel which receives the excrement, or vomit, or throat discharge, and pour more of the disin- fectant on the discharge after it has been received in the vessel. If the discharge soils the floor or furniture wash these soiled spots with the disinfectant mixed with water as above. Soiled bed and body linen, rags, or handkerchiefs used to receive discharges, and soiled clothing should be burned, or soaked in disinfectant diluted as above, for four hours and then rinsed with water. For the Dead, where Disinfectant is Necessary. — W^rap the body in a sheet thoroughly saturated with the disinfectant full strength. For Attendants on the Sick. — Empty the contents of this bottle into a pint of water, and wash the hands and other portions of the body, if sailed, with it, rinsing off with clean water. In sick rooms and hospital wards, wash all surfaces with tne same solution. 2i^="This disinfectant, when mixed with water, as described, will not injure the hands, but should not get into the mouth. As has been- stated above, in view of the probability of a fresh invasion of cholera in 1893, the officers of the road determined to be as fully prepared for it as possible, and accordingly, in December, 1892, Mr. Theodore N. Ely, then Superintendent, now Chief, of Motive ?ower, desired me to make a careful series of tests to determine, in the most rigid manner, the precise germicidal value of their standard disinfectant, and to suggest to them, if I could, how its efficiency might be improved. In view of the fact that the composition of the disinfectant is well known, that it is not for sale, except to its employees or where the interests of the road are directly involved, and is not in any sense a proprietary preparation and, above all, impressed with the im- portance of the inquiry to the public welfare, I consented to under- take the investigation, although with reluctance because of the peculiar difficulties involved in all efficiency tests upon germicides. I was for- tunate, however, in securing throughout the investigation the aid of Mr. Albert P. Mathews, S.B., Assistant in Biology at the Massachu- setts Institute of Technology, to whose skill and devotion the work owes much of whatever value it may possess. In the present state of opinion regarding the active cause of infectious disease, which holds that this is invariably due to specific microorganisms, it becomes simple in theory to determine the value of any germicide or antiseptic. It seems, at first sight, easy enough to test the efficiency of a disinfectant by simply observing under various conditions its germicidal power. But in arranging the details of the experiments difficulties arise which are hard to overcome. It might be supposed that if we exposed known numbers of spe- cific disease germs to the action of a fixed amount of the disinfectant for a definite period, and then made cultures from the mixture, we should readily settle the efficiency of the disinfectant at the given strength. But this is by no means the case ; for, unless great care be taken to- prevent it, we may carry over with the test sample of the mixture taken for planting enough of the disinfectant to inhibit later the de- velopment of the still-living germs. Errors of this kind are partic- ularly insidious and very difficult to avoid. Again, there is reason to believe that cultures of the germs submitted to examination vary in their endurance considerably, particularly if these are of kinds which produce spores. As in the higher plants some fields of corn or oats are strong and vigorous while others are weak and sickly, so here it is important to know that the cultures or crops of bacteria with which we are dealing are fairly representative of those which devastate the human body. 5 The problem in this case was strictly one of germicidal and not merely antiseptic properties. For the preservation of food the prob- lem is often one of antisepsis, but into disinfection this milder problem does not enter. The question is one of destruction, not in- hibition, and may be stated thus : How efficient is the disinfectant as a destroyer of the germs of infectious disease ? The methods used in the examinations of the germicidal power of ■disinfectants have been almost as numerous as the observers, and the results obtained have varied widely. Koch has employed a silk thread dipped into the culture and then exposed to the action of the disin- fectant. The thread was afterwards washed with sterile distilled water, and then planted on the surface of the plated medium or else sown in bouillon. Such a method is open to serious objection, as it is difficult to wash away all the disinfectant clinging to the thread and the slight amount left may continue to act antiseptically, and lead to erroneous conclusions. Sternberg, in this country, has proceeded in a different manner. He exposes the broken-down beef tea, or pure culture of the bacteria in bouillon, to a known strength of the germicide for a given length of time, after which he transfers by peculiar flasks a certain small amount to a large amount of fresh alkaline bouillon and allows incubation to proceed. The objection to this method is that he inevitably intro- duces into the second culture medium an uncertain small amount of the disinfectant. The methods of Koch and Sternberg are both, at least in theory, somewhat untrustworthy, for the reason that the action of the germicide cannot be checked at once and because each introduces into his control medium some of the germicide. It is self-evident that in testing any germicide it is necessary to stop its action quickly or to remove it wholly from the test medium. Only when we accomplish this can we hope to be sure of the results. In solving the present problem we were guided by the work of those experimenters who had also recognized the necessity of freeing the bacteria after their exposure for the given time from the possible antiseptic influences of small amounts of the germicide thus carried over. Two reagents have hitherto been used for this purpose for antiseptics containing corrosive sublimate, namely, ammonium sulphide and hydrogen sulphide. Our problem, however, was not so simple. In the first place, it was not only necessary to test the antiseptic effect of the precipitant upon every specific germ to assure ourselves that it was not itself more or less antiseptic ; but in the second place, we were here not merely working with a dilute solution of the subli- mate, but also with a strong solution of zincic and cupric chlorides^ and if either of the precipitants mentioned were used there would be formed a heavy, fiocculent precipitate likely to gather around the germs and drag them down, thus leading to serious error. Hence we were compelled to run control experiments with pure water in many cases,, and sometimes with other precipitants, while at the same time carefully testing the antiseptic action of the precipitant itself. As already stated, we endeavored to eliminate the various sources of error that had hitherto been recognized. The first question was that of the cultures themselves, and only the freshest cultures of pathogenic germs were used, since it is probable that such cultures are most nearly normal. We also satisfied ourselves that each culture was in a vigorous state, as indicated by the rapidity of its growth or the manner in which it formed spores. The cultures of the specific bacteria used were subjected to rigid differential tests in order to establish the fact that they were really the germs in question. The culture of typhoid fever bacilli had been isolated not long before from the spleen of a woman dead of typhoid fever. The culture of anthrax was comparatively fresh, having been obtained from a patient in the Boston City Hospital. The cul- ture of Staphylococcus pyogenes am-eus was derived from one isolated a few days previous by Dr. W. T. Councilman, Pathologist to the Bos- ton City Hospital. The culture of the Streptococcus of diphtheria was also derived from one isolated by Dr. Councilman a few days before it was used in these experiments. The culture of the cholera Spirilhivt of Koch was derived from one isolated by Dr. Dunham, of New York, from a case of Asiatic cholera there in September last. The culture of Bacillus subtilis had been in the laboratory for some time, but, as was proved by the resistant properties of its spores when subjected to heat, it was in a most vigorous condition. We may then safely con- clude that the cultures employed in the tests were in a fresh and vig- orous condition. It is generally believed that mercury forms a more or less stable compound with albumen, though whether this compound is or is not soluble, and whether it is or is not effective in making the germicide powerful, is a matter of dispute. Vaughan contends that it is very poisonous, while others hold that such an envelope forming about the bodies of the bacteria protects them from the further action of the germicide. How far these ideas are true, or how far they are im- portant if true, is uncertain. In the actual investigations we introduced a known amount of the fluid holding the germs in suspension into a known amount of the dis- infectant, and at the end of a given interval transferred a small known amount of this mixture to a large amount of dilute ammonium sulphide which was used as the inhibitor, sometimes making control experiments with stannous chloride and at other times with water in the place of the ammonium sulphide. We used ammonium sulphide in preference to hydrogen sulphide as inhibitor, for the reason that the solution of the germicide was found to be decidedly acid, and thus we were forced to neutralize the acid at the same time that the mercury was neu- tralized. The use of any sulphide we adopted with hesitation, owing to the formation of a heavy flocculent precipitate ; but by making control tests with stannous chloride, which simply neutralized the mercury and did not affect the other chlorides, and by using water in the place of the sulphide, we came to the conclusion that, in spite of this danger, the ammonium sulphide was the best inhibitor we could use, although it may be that further experiment would show the stannous chloride to be equally valuable. We thus succeeded in stopping the action of the disinfectant upon its arrival in the second flask, and by employing large dilutions further reduced the possibility of the contamination of the culture medium with an antiseptic salt. It will be noticed, also, in examining the detailed history of the experiments, that through planting a con- trol from the second flask directly into a large amount of bouillon we checked our final results and escaped the danger of losing the germs through too great dilutions. In all experiments controls were made into bouillon to determine the initial sterility of the dilution flask ; a sample was also planted from the precipitating flask to make sure that the bacteria were not lost in passing from the precipitating or inhibition flask to the third or dilution flask ; and finally, controls were made to discover how many bacteria would have appeared in a cubic centimeter of the third flask had the disinfectant been water. We have thus had a constant check on all results, and feel confident that error has as far as possible been eliminated. In order, however, to guard against all possible error, when the end 8 of the experiment was reached and the limit of the resistance of the bacterium had been determined by means of the precipitation methods, two other methods were used in order to check the results. One of these was that recommended by Sternberg, namely, exposing the cul- ture to the germicide for a certain length of time and then transferring a sm.all quantity of the mixture — as much as would cling to a platinum needle — into a flask of bouillon and allowing the latter to grow at optimum temperature. The other method was to transfer a platinum loopful of the mixed liquid culture and germicide to a weak solution of ammonium sulphide and then pour the whole of this into a large flask of bouillon. In the latter method we escaped both the diffi- culty of introducing the corrosive sublimate into the culture and the danger of great dilutions. The precise method of procedure ^ was this : Bouillon cultures of the species to be tested were grown for two days in the incubator at 37.5°C. Two cubic centimeters of such a culture, after prolonged shak- ing, and after microscopical and macroscopical examination had deter- mined that it was in good condition, were transferred to a 60 cc. sterile Erlenmeyer flask, and to them 1 3 cc. of sterile distilled water were added. An equal volume, or 15 cc, of the germicide was next added, and the time of exposure was determined by means of a stop watch. The flask here used may be called the action flask, and the time of exposure the action period. Towards the end of the action period one cubic centimeter was rapidly transferred to 500 cc. of dilute solu- tion of ammonium sulphide, or stannous chloride, and well shaken for thirty seconds, the precipitate not being allowed to settle. At the end of the thirty seconds one cubic centimeter from this flask, which may be called the inhibition flask, was transferred to a flask contain- ing 250 cc. of sterile distilled water; the last flask may be called the dilution flask. Immediately after this two flasks, containing each 25 cc. of bouillon, were planted, each with .3 cc, from the inhibition flask. This was done in order to have a dilution control. At the end of a couple of minutes two or three tubes of melted agar were sown with one cubic centimeter each from the mixture in the third or dilu- tion flask, and immediately plated. All plates and flasks were then placed in the incubator at 37.5°C. Immediately preceding the experiment one cubic centimeter of ' In formulating a definite mode of procedure we were guided, in part, by experiments made in this laboratory upon peroxide of hydrogen by the late George V. McLauthlin. water from the dilution flask was planted in bouillon in order to de- termine its sterility. This precaution was found to be necessary when working with the spores of B. sicbtilis and B. anthracis, both of which proved to be very resistant to ordinary sterilization methods. Immediately following each series of experiments a control experi- ment was made as follows : Two cubic centimeters of the bouillon culture used in testing were diluted with 28 cc. of water, and one cubic centimeter of this diluted culture carried into 500 cc. of am- monium sulphide in the inhibition flask. After thirty seconds one cubic centimeter from the mixture in the inhibition flask was carried to the dilution flask, containing 250 cc. of sterile water, and from the last flask agar plates were made by sowing in each tube of melted agar one cubic centimeter of the liquid and then plating the mass. Two other controls were obtained for each species by substituting for ammonium sulphide in the inhibition flask stannous chloride in the one case and distilled water in the other, and making the dilutions the sg.me as in the principal testing experiments. This routine was repeated for the different action periods until it was found that the germs were killed. When this knowledge had been obtained still another control was employed, namely, Sternberg's method and the modification of it already described. It will thus appear that all results obtained were carefully checked, and all tests finally made in duplicate or triplicate. After the actual theoretical value of the germicide had been thus established, its ac- tual value in practice was proved by its action on a typhoid fever stool. A DETAILED ACCOUNT OF THE EXPERIMENTS MADE TO DETERMINE THE GERMICIDAL EFFICIENCY OF THE DISINFECTANT. A. Experiments upon the Typhoid Fever Bacillus of E berth. Inasmuch as the tests with this bacillus were the first undertaken, it was necessary to develop the methods to be used in testing. The first few experiments were therefore intended to determine whether the stannous chloride or the ammonium sulphide were objectionable because of being germicides in themselves, and whether either method was superior to that of simple dilution in water. Experiment i, when compared with Experiment 2, indicated that the method of inhibition by precipitating with stannous chloride, was superior to that of diluting with water. Experiment i, in which the lO chloride was used, indicated that the germs were still alive after one minute's exposure, while Experiment 2 showed that all had been killed. If, then, we had relied upon the method of simple dilution we should have been led into error at the start. EXPERIMENT I. I volume of disinfectant diluted with 3 volumes of water. I cc. of 50-days-old bouillon culture of Eberth bacilli introduced into 250 cc. of this diluted disinfectant and exposed for one minute. I cc. of the mixture of disinfectant and culture then carried to 500 cc. of dilute, weakly acid stannous chloride, and left for 35 seconds. I cc. of liquid from the stannous chloride flask then mixed with 500 cc. of water and well shaken. From the last flask five agar plates were planted with one cc. each. Result : Average number of colonies per cc. of last flask, 3. EXPERIMENT 2. Same conditions as in preceding experiment. Disinfectant same strength and in same amount, and the same amount of the same culture used. Exposure to the disinfectant, one minute. 1 cc. of mixture of culture and disinfectant then carried to 500 cc. of sterile water and well shaken for 35 seconds. I cc. of this fluid then carried to 500 cc. of sterile water. Five agar plates were planted with i cc. of the last flask. Result : All the plates were sterile. These experiments having indicated the value of the stannous chloride as an inhibitor, we next proceeded to determine that of the ammonium sulphide. EXPERIMENT 3. 2^-days-old bouillon culture of Eberth bacilli. Grown at 37.5°C. I cc. of this culture carried to 250 cc. of disinfectant diluted as in i and 2. Exposed 20 seconds. I cc. of the disinfectant and culture then mixed wtth 500 cc. of ammonium sulphide. Exposed for 20 seconds. I cc. of this mixture from Flask 2 well washed in 500 cc. of sterile water. Five agar plates were then made from the last flask, each plate containing i cc. Result: Three plates were sterile ; two had one colony each upon them. In order to determine whether a longer exposure to the ammonium sulphide would in- jure the bacteria we allowed the inhibition flask to stand for three minutes, when we again diluted one cubic centimeter of it with 500 cc. of sterile water, and after shaking planted five cubic centimeters of the last dilution in five tubes of agar. Result: Two of the plates had one colony each; one of them contained three; and the third was sterile. This indicated that the sulphide possessed a very slight action upon the bacteria, if it exerted any at all. The slightly greater numbers of bacteria grown on these plates were wholly within the II range of variation in exactly similar series of experiments. Other- wise they would seem to indicate that a longer exposure of the bacteria to the sulphide was advantageous. The next experiment was made to test the value of washing the disinfectant from the bacteria by water. EXPERIMENT 4. Same conditions and culture as in Experiment 3, but the exposure was for 18 seconds. I cc. of mixture of disinfectant and culture was well shaken in 500 cc. of sterile water for 20 seconds, Flask 2. I cc. from Flask 2 then shaken in 500 cc. of sterile water, Flask 3. Six agar plates were planted with i cc. each from Flask 3. Result : three plates sterile ; two plates had one colony each upon them. The result of this experiment would seem to indicate that sim- ple dilution was as effective as ammonium sulphide precipitation. Further experiment proved that this conclusion was erroneous. Experiment 5 is the control for the two preceding, made in order to determine whether the bacteria had been lost by too great dilution. EXPERIMENT 5. I cc. of same culture as in Experiment 4 diluted with sterile water to the same degree that it would have been diluted had it passed through the action, inhibition, and dilution flasks. No disinfectant used. Six agar plates planted with i cc. each of Flask 3. Result : These plates had an average of eleven colonies each upon them. The result of Experiment 5 was sufficient to convince us that the dilution of the disinfectant was too great ; and thereafter, with the exception of the next control experiment, we reduced the last flask to 250 cc. ; reduced the disinfectant volume to 15 cc. ; and employed two cc. of the culture in the place of one. EXPERIMENT 6. This was made to test the germicidal power of the stannous chloride. I cc. of culture was carried to 250 cc. of sterile water. I cc. of this mixture then planted in 500 cc. of dilute stannous chloride and left for 20 seconds. I cc. of this then carried to 500 cc. of sterile water. Six agar plates were planted each with i cc. of last flask. Result : There was an average number of 14 colonies upon each plate. 12 This result proved that the stannous chloride in the length of time the bacteria was exposed to it, and in the strength in which it was used, exerted no appreciable deleterious effect upon the typhoid bacilli. Having now made a test of methods, in which it appeared that there was little advantage in using stannous chloride in preference to ammonium sulphide as inhibitor, we proceeded to determine the efficiency of the disinfectant. For this purpose we employed the disinfectant diluted to twice its normal bulk. This dilution was se- cured in the early experiments by means of the culture fluid itself. Later, however, we diluted with water. This strength of the dis- infectant was adopted as the standard both because it seemed likely that such a strength would be the one employed in practice and because we were thereby enabled to compare its efficiency with that of neutral solutions of mercuric chloride of the strength i : i,ooo as determined by many experimenters. EXPERIMENT 7. Fifteen cc. of a two-days culture in bouillon of Eberth bacilli were mixed with 15 cc. of disinfectant full strength. Thus the culture acted as the diluter of the disinfectant. The exposure was for 25 seconds. I cc. of the mixture was then carried to 500 cc. of stannous chloride and shaken for 20 seconds. I cc. from this flask was then transferred to Flask 3, containing 250 cc. of sterile water. Five agar plates were then planted from the last flask with one cc. each. Result: All the plates were sterile. EXPERIMENT 8. Identical with the preceding, except that the culture was exposed to the disinfectant for 45 seconds. Five agar plates planted from the last flask. Result: All plates sterile. EXPERIMENT 9. Control of 7 and 8. I cc. of culture transferred to 500 cc. of stannous chloride for 20 seconds. I cc. of this fluid then diluted with 250 cc. of sterile water. Two agar plates planted with i cc. each from the last flask. Result: 2,035 colonies upon each plate. It was obvious from the preceding experiments that twenty-five seconds' exposure sufficed to kill the bacteria. We now determined the minimum length of time of exposure necessary to accomplish this result. Here we adopted a slightly different method of pro- 13 cedure. It will be observed that the disinfectant was diluted in these cases with the culture medium itself. Considering that this medium was of an albuminous nature and therefore likely to intro- duce an element of uncertainty into the results, as the exact solu- bility of the albuminate of mercury is a matter of dispute, and its effect upon the germicidal value of mercuric salts has also been questioned, we freed the bacteria from this albuminous envelope as far as possible by thoroughly shaking two cubic centimeters of the culture in sterile water sufficient to dilute the disinfectant to twice its bulk. Hereafter, then, it will be understood that the 15 cc. of disinfectant were diluted with 13 cc. of water and 2 cc. of culture medium. In this way the bacteria themselves were exposed more directly to the action of the germicide. EXPERIMENT lO. Fifteen cc. of disinfectant mixed with 13 cc. of water and 2 cc. of culture of typhoid bacilli. Exposure of 10 seconds. I cc. of mixture carried to 500 cc. of stannous chloride for 20 seconds. I cc. from this flask, known as the inhibition flask, next transferred to a dilution flask containing 250 cc. of sterile water. Two bouillon flasks containing 25 cc. each of bouillon were then sown with 0.2 cc. each from the inhibition flask (No. 2). Two agar plates were made from Flask 3. Result: One bouillon flask sterile ; other bouillon flask growth. Agar plates, two sterile; the third contained two colonies. EXPERIMENT II. Identical with 10, except that the exposure to the disinfectant was for 15 seconds. Agar plates and bouillon flasks planted as in the preceding experiment. Result : both i^lates sterile. Both bouillon flasks sterile. EXPERIMENT 12. Identical with lo, except that the exposure to the disinfectant was for 20 seconds. Agar plates and bouillon flasks planted as in 10. Result : Agar plates were sterile. Bouillon flasks were sterile. From these experiments it follows that the Eberth bacillus of typhoid fever is killed by an exposure to the disinfectant — the latter being diluted to one half its original strength — of about twelve seconds. It is not killed in ten seconds, but it is in fifteen. We now proceeded to control this result by inhibition with ammonium sulphide in the place of the stannous chloride. EXPERIMENT I3. Identical with 11, except that ammonium sulphide was placed in the middle flask in place of the stannous chloride. The culture was the same and the dilutions were to the same amount and of the same kind. Exposure of culture to the disinfectant for 15 seconds. Carried to the sulphide and ex- posed for 30 seconds. Agar plates made from Flask 3, and bouillon flasks from Flask 2. Result: Agar plates sterile. Bouillon flasks remained sterile. EXPERIMENT I4. This experiment the same in all respects as 13, except that the exijosure to the disin- fectant was for TO seconds only. Result: Bouillon flasks sterile; agar plates sterile. It will be seen that the inhibition with ammonium sulphide con- firmed the results of inhibition with stannous chloride. The only difference was that the latter method showed some of the bacteria to be still alive after ten seconds' exposure to the disinfectant, while the former indicated that all had been killed. It may have been that in the former case the exposure was for a second or two longer than in the latter, as it was impossible to work with an accuracy within a second. The control experiments for the above, to guard that the bac- teria should not be lost from too great dilution, showed the last flask to contain 150 colonies to the cubic centimeter, and proved the dilutions to be safe. We also made another control, to deter- mine that the stannous choride was harmless. The plates showed an average of 104 colonies to the cubic centimeter — a result within the range of unavoidable differences in the number of bacteria trans- ferred from flask to flask. The result was then tested by Sternberg's method as follows : I cc. of culture was introduced into i cc. of disinfectant. At the end of lo seconds a little of the mixture was carried on a platinum needle and mixed with 25 cc. of bouillon. The flask remained sterile after being in the incubator for three days. At the end of 15 seconds another flask was sown in the same manner. This flask also remained sterile. A further confirmation was secured in the following way : I cc. of culture was introduced into i cc. of disinfectant. At the end of 10 seconds a plati- num loop of the mixture was mixed with i cc. of dilute ammonium sulphide, and after 30 sec- onds here the mixture was poured into a flask of bouillon containing 25 cc. The bouillon flask at the end of 24 hours contained a fine crop of typhoid bacilli. At the end of 15 seconds' exposure to the disinfectant a second platinum loop of the mixture was precipitated in the same manner and sown in bouillon. This flask remained sterile. Conclusion : The typhoid fever bacillus of Eberth is killed by an exposure of 15 seconds to the disinfectant when the latter is diluted to twice its bulk. 15 B. Experiments tipon Spores of Bacillus Siibtilis. In all experiments upon Bacilhis subtilis we employed a bouillon culture grown for three days at 37.5° C. and microscopically determined to be in the spore state. EXPERIMENT I 5. 2 cc. of culture of B. siibtilis mixed well with 13 cc. of sterile water. 15 cc. of disinfectant then poured into this. Shaken well for 5 minutes. 1 cc. carried to 500 cc. of ammonium sulphide. Left here for 30 seconds, i cc. from Flask 2 then carried to Flask 3, containing 250 cc. of sterile water. Bouillon flasks were sown from Flask 2, and agar plates from Flask 3. Result: Growth in bouillon flasks. Agar plates contained an average of 5 colonies. EXPERIMENT 1 6. Identical with Experiment 15, except that stannous chloride was employed as inhibitor. Exposure to disinfectant of 5 minutes. Bouillon flasks and agar plates as usual. Result: Growth in bouillon flasks. Agar plates had an average of 7 colonies each. EXPERIMENT I7. Identical with 15. Ammonium sulphide, inhibitor. Exposure of 7 minutes to disinfectant. Agar plates from Flask 3, bouillon flasks from Flask 2. Result : Bouillon showed a copious growth. Agar plates an average of 8 colonies. EXPERIMENT l8. Same culture as preceding. Stannous chloride, inhibitor. Exposure to disinfectant for 7 minutes. Result : Growth in bouillon flasks. Agar plates an average of 6 colonies. EXPERIMENT I9. Identical with 17, except that exposure was for 10 minutes. Ammonium sulphide, inhibitor. Result : Growth in bouillon flasks. Agar plates an average of 3 colonies. EXPERIMENT 20. Identical with i8, except exposure of lo minutes. Stannous chloride, inhibitor. Result : Growth in bouillon flasks. Agar plates an average of 3 colonies. EXPERIMENT 21. Control for experiments from 15-20. 2 cc. of culture. 28 cc. of water. I cc. of this then diluted with 500 cc. Ammonium sulphide. I cc. of mixture then carried to 250 cc. water. Result: Agar plates showed an average of 12 colonies. EXPERIMENT 22. Identical with 17. Exposure for \2.\ minutes. Inhibitor, ammonium sulphide. Result: Growth in bouillon flasks. Agar plates sterile. i6 EXPERIMENT 23. Same as preceding. Exposure of 15 minutes. Inhibitor, ammonium sulphide. Result : Bouillon flasks sterile. Agar plates sterile. EXPERIMENT 24. Control for 22 and 23. 2 cc. culture. 28 cc. water. I cc. of mixture carried to 500 cc. Ammonium sulphide. I cc. of this to 250 cc. water. Result : Agar plates an average of 44 colonies. It having appeared from the above that B. subtilis spores are destroyed in fifteen minutes the result was confirmed by Sternberg's, method. The results were as follows : Flask I. Exposed 15 minutes showed a growth. Flask 2. Exposed 17 minutes remained sterile. We also employed the modification of this method already no- ticed. The results of this were as follows : Flask I. Exposed 15^ minutes and inhibited with ammonium sulphide. Growth. Flask 2. Exposed 17^ minutes and inhibited with ammonium sulphide. Growth. Flask 3. Exposed 20 minutes and inhibited with ammonium sulphide. Sterile. Conclusion: Bacillus subtilis ^'^ox^^ are, under certain conditions, killed by the germicide diluted to twice its bulk in 20 minutes. Under other conditions they appear to be killed ia 17 minutes. C. Experitnejits tipon the Streptococcus of pseudo-Diphtheria. This Streptococcus has been repeatedly isolated from the mem- brane in the throats of patients sick with scarlet fever, and others- erroneously supposed to have true diphtheria. It apparently bears an aetiological significance in the cases of such membranous in- flammations of the throat. It has already been established at this- laboratory that this Streptococcus, believed by some to be identical with Streptococcus pyogenes, is even more resistant to germicides, than the bacillus of true diphtheria, i.e., the bacillus of Klebs-Loffler. EXPERIMENT 25. 2 CC. culture, 13 cc. water, and 15 cc. of disinfectant, mixed for 10 seconds. I cc. of the mixture then mixed with 500 cc. of ammonium sulphide for 30 seconds. I cc. of this mixture transferred to Flask 3, containing 250 cc. water. Bouillon flasks, planted from Flask 2. Agar plates with i cc. each from Flask 3. Result : Agar plates sterile. Bouillon showed a growth. 17 EXPERIMENT 26. Same as 25, except the exposure of 12 seconds. Ammonium sulphide Result : Agar plates sterile ; bouillon flasks sterile. EXPERIMENT 27. Control of 25 and 26. 2 cc. of culture mixed with 28 cc. of water. I cc. of mixture to 500 cc. of ammonium sulphide for 30 seconds. I cc. of sulphide flask then transferred to 250 cc. of water. Agar plates from Flask 3. Result : Plates showed an average of 39 colonies. From the above it appeared certain that an exposure for twelve seconds to the disinfectant when the latter is diluted to twice its volume is sufficient to kill the Streptococacs diphtherice. This result was confirmed by Sternberg's method and the modi- fication thereof employed in the case of B. siibtilis and th& Eberth bacillus. D. Experiments upon Staphylococcus pyogenes mireus. The culture for these experiments came originally from the labora- atory of Dr. Councilman. Three-days-old bouillon cultures were used. EXPERIMENT 28. 2 cc. bouillon culture, 13 cc. water, and 15 cc. disinfectant. Exposed 10 seconds. I cc. of mixture then carried to Flask 2, containing 500 cc. Ammonium sulphide. Ex- posed here for 30 seconds. I cc. than transferred from Flask 2 to Flask 3, containing 250 cc. sterile water. Bouillon flasks containing 25 cc. bouillon were planted with .2 cc. of mixture from Flask 2. Agar plates with i cc. each from Flask 3. Result: Agar plates sterile. Bouillon flasks sterile. EXPERIMENT 29. Same as above, but exposed to disinfectant for 20 seconds. Ammonium sulphide as inhibitor. Result . Agar plates sterile. Bouillon flasks sterile. EXPERIMENT 30. Control for 28 and 29. 2 cc. culture mixed with 28 cc. water. I cc. then to 500 cc. ammonium sulphide. I cc. of this then diluted 250 times with water. Result : Agar plates from last flask showed 530 colonies each. i8 The conclusion from these experiments is that Staphylococcus pyog- enes atcretis is killed by a ten seconds' exposure to the germicide when the latter is diluted to twice its bulk. This result was confirmed by Sternberg's method and the modifi- cation of it already described. In each case the flasks of ten seconds remained sterile, as did also those exposed for twelve and a half seconds. E. Experi'ments upon the Sph'illmn of Asiatic Cholera. For these experiments we used a bouillon culture grown for four days at 37.5° C. The culture came originally from Dr. Dunham's laboratory. EXPERIMENT 3I. 2 cc. of culture, 15 cc. of disinfectant, and 13 cc. of sterile water were mixed for 10 seconds. I cc. of mixture precipitated in 500 cc. Ammonium sulphide. 30 seconds. 1 cc. of this flask then carried to 250 cc. of sterile water. Bouillon flasks were planted from Flask 2. Agar plates from Flask 3. Results : Agar plates sterile. Flasks sterile. EXPERIMENT 32. Same as 31, except the exposure to the disinfectant was for 15 seconds. Result : Agar plates sterile. Bouillon flasks sterile. EXPERIMENT 33. Control for 31 and 32. 2 cc. of culture diluted with 28 cc. water, then i cc. passed through same amounts of ammonium sulphide and water as usual. Agar plates were planted from Flask 3. Result : Agar plates showed an average of 5 colonies to a cubic centimeter of liquid from last flask. The dilutions here were evidently a little too great, but when it is remembered that on this basis the middle flask should contain 1,250 cholera bacilli to the cubic centimeter, if they had not been killed, we considered the fact of the sterility of the flasks of bouillon planted from this flask as evidence enough that the bacteria were killed by the exposure given them. The experiments upon the Spirillum of Asiatic cholera would indi- cate that this bacterium is killed by an exposure to the germicide diluted to twice its volume for the space of ten seconds. This result was confirmed by Sternberg's method and the modifica- tion of it described. Bouillon flasks at 10 seconds remained sterile. 19 F. Experiments 7ipon Spores of Bacillus anthracis. The culture of Bacilhis anthracis used in these tests was from a luxuriant culture isolated by Prof. G. R. Tucker from a patient in the Boston City Hospital about one year before. A three-days' bouillon culture was used. EXPERIMENT 34. 2 cc. of culture well shaken in 13 cc. of water; 15 cc. of disinfectant mixed with it tor 15 minutes. I cc. of the mixture was then inhibited by 500 cc. of ammonium sulphide. 1 cc. from the last flask was, at the end of 30 seconds, carried to Flask 3, containing 250 cc. of distilled water. Agar plates made from the last flask. Bouillon flasks planted from the inhibition flask. Result : Agar plates had an average of 3 colonies each. Bouillon flasks showed copious growth. EXPERIMENT 35. Control for 34. 2 cc. of culture mixed with 28 cc. of water. I cc. of mixture then transferred to 500 cc. of ammonium sulphide for 30 seconds. I cc. of mixture then diluted in 250 cc. of sterile water. Agar plates from last flask. Result : Plates contained an average of 52 colonies each. We next passed on to determine the vitality of the spores and found them exceedingly resistant to heat. EXPERIMENT 36. I cc. of the culture used in the preceding two experiments was placed in a sterile test tube and immersed in boiling water. Every two minutes a sterile platinum needle was plunged into the hot culture and then mixed with 6 cc. of sterile bouillon. In order to deter- mine the exact degree of heat to which the spores were exposed we placed next to the tube containing the culture a second tube containing i cc. of sterile water, and within this tube suspended a thermometer so that it was at every point separated from the glass of the tube. This thermometer registered as follows : 5 seconds, 60 degrees C. ; 10 seconds, 70 degrees; 15 seconds, 80 degrees; 30 seconds, 92 degrees; 45 seconds, 95 degrees ; 60 seconds, 97 degrees ; 70 seconds, 99 degrees. It remained at this temperature during the course of the tests. The result proved that some of the spores were still alive at eleven minutes after immersion. We made no test later than that time. Experiment 34 indicated that nearly all the spores were killed after fifteen minutes' exposure to the disinfectant. EXPERIMENT 37. Identical with 34, except that the exposure was for 20 minutes. Result : Agar plates sterile. Bouillon flasks sterile. 20 Having now ascertained that anthrax spores of a very resistant type are killed by the disinfectant in twenty minutes, we confirmed the result as usual, by Sternberg's method, and also by the modifica- tion of that method already described. It will be remembered that in the modification of the method we precipitated a platinum loopful of the mixture of culture and disin- fectant in ammonium sulphide before pouring the whole mixture into bouillon. Result : li minutes . . . Direct. Growth. 14 minutes . . . Direct. Growth. 3 minutes . . . Direct. Growth. 17 minutes . . . Precipitated. Growth 5 minutes . . Direct. Growth. 19 minutes . . . Direct. Sterile. 8 minutes . . Precipitated. Growth. 17^ minutes . . . Precipitated. Growth lo minutes . . Direct. Growth. 19 minutes . . . Precipitated. Sterile. 12 minutes . . . Precipitated. Growth. From this result we may conclude that anthrax spores of this high degree of resistance are killed by an exposure to the disinfectant of nineteen minutes, the disinfectant being diluted to twice its volume. G. A Practical Test of the Disinfectant. In order to test the working of the disinfectant in actual use we mixed a stool of a typhoid patient with an equal amount of disinfect- ant. The stool was diarrhoeic, but contained many hard lumps of fasces. The mixture was shaken three or four times during the test. In order to determine at what time the stool became sterile we trans- ferred a small portion upon a platinum loop at the end of thirty sec- onds, and for every minute's interval thereafter up to twenty minutes, to a tube of melted agar containing perhaps 6 cc. of agar. At the end of twenty-four hours the plates planted at thirty seconds' exposure proved to be well spotted with colonies of bacteria, but far less than the control made at the beginning. From this time on they rapidly diminished until at the end of three minutes and a half there were none apparent, nor did any appear upon the subsequent plates or in the bouillon flasks. The efficiency of the disinfectant with faecal bacteria was thus established. 21 SUMMARY OF EXPERIMENTS ON B. TYPHI ABDOMINALTS. No. of the Experiment. Age of Culturft. Strength ot Dis- infectant. Time of Ex- posure to Disinfectant Precipitant used as Inhibitor. Colonies on Agar Plates. Bouillon Flasks. Number of Colonies on Control. 1. 50 days I 1 minute SnCl, 3 • • . 2. 50 " \ 1 " HoO 3. 1\ " i 20 seconds (NH4)oS and 1 11 4. 1\ " i 18 <' H2O and 1 11 5. 2\ " not exposed SnCl2 11 7. 2 " i 25 seconds SnCIa 2035 8. 2 " i 45 " SnCl2 2035 10. 2 " \ 10 " SnClo 0and2 growth. 150 11. 2 " \ 15 " SnCU sterile. 150 12. 2 " \ 20 " SnCl. " 150 13. 2 « \ 15 " (NH4)2S a 150 14. 2 " \ 10 " (NH4)2S " 150 Control: Sternberg's Method, Flask at 10 seconds, sterile. Control: Modification of Sternberg's Method, Flask at 10 seconds, growth; 15 seconds, sterile. SUMMARY OF EXPERIMENTS ON SPORES OF BACILLUS SUB TILLS. No. of the Experiment. Age of Culture. Strength of Dis- infectant. Time of Ex- posure to Disinfectant. Precipitant used as Inhibitor. Colonies on Agar Plates. Bouillon Flasks. Number of Colonies on Control. 15. 3 days i 5 minutes {NH4)2S 5 growth 12 16. 3 " 1 5 " SnCl2 7 12 17. 3 " i 7 " (NH4)2S 8 12 18. 3 " I 7 " SnCla 6 12 19. 3 " 1 2 10 " (NH4)2S 3 12 20. 3 " \ 10 " SnCl2 3 12 22. 3 " k 12^ " (NH4)2S 44 23. 3 " \ 15 " (NH4)2S sterile 44 Control : Sternberg's Method, Flask at 15 minutes, growth ; 17 minutes, growth. Control : Sternberg's Method, modified. Flask at 15 minutes, growth; 17 minutes, growth ; 20 minutes, sterile. 22 SUMMARY OF EXPERIMENTS ON STREPTOCOCCUS DIPHTHERIA. No. of the Experiment. Age of Culture. Strength of Dis- infectant. Time of Ex- posure to Disinfectant. Precipitant used as Inhibitor. Colonies on Agar Plates. Bouillon Flasks. Number of Colonies on Control. 25. 26. 3 days 3 " 10 seconds 12 " (NH4)2S (NH4)2S growth sterile 39 39 Control : Sternberg's Method, Flask at 10 seconds, growth ; 12 seconds, sterile. Control : Sternberg's Method, modified, Flask at 10 seconds, growth ; 12 seconds, sterile. SUMMARY OF EXPERIMENTS ON S. PYOGENES AUREUS. No. of the Experiment. Age of Culture. Strens;th of Dis- infectant. Time of Ex- posure to Disinfectant. Precipitant used as Inhibitor. Colonies on Agar Plates. Bouillon Flasks. Number of Colonies on Control. 28. 29. 3 days 3 " 10 seconds 20 " (NH4)2S (NH4)2S Sterile sterile 530 530 Control : Sternberg's Method, Flask at 10 seconds, sterile ; at 12 seconds, sterile. Control : Sternberg's Method, modified. Flask at 10 seconds, sterile ; at 12 seconds, sterile. SUMMARY OF EXPERIMENTS ON SPIRILLUM OF ASIATIC CHOLERA. No. of the Experiment. Age of Culture. Strength of Dis- infectant. Time of Ex- posure to Disinfectant. Precipitant used as Inhibitor. Colonies on Agar Plates. Bouillon Flasks. Number of Colonies on Control. 3L 32. 4 days 4 " 1 2 10 seconds 15 " (NH4)2S (NH4)2S sterile sterile 5 5 Control: Sternberg's Method, 10 seconds flask, sterile. Control : Sternberg's Method, modified, 10 seconds flask, sterile. SUMMARY OF EXPERIMENTS ON SPORES OF BACILLUS ANTHRACIS. No. of the Experiment. Age of Culture. Strength of Dis- infectant. Time of Ex- posure to Disinfectant. Precipitant used as Inhibitor. Colonies on Agar Plates. Bouillon Flasks. Number of Colonies on Control. 34. 37. 3 days 3 " 15 minutes 20 " (NH4)2S (NH4)2S 3 growth sterile 52 52 Control : Sternberg's Method, Flask at 19 minutes, sterile. Control : Sternberg's Method, modified. Flask at 17 minutes, growth ; 19 minutes, sterile. 23 CONCLUSIONS. The foregoing experiments appear to establish the fact that the Standard Disinfectant used by the Pennsylvania Railroad Company will, under favorable circumstances, destroy the germs of typhoid fever, diphtheria, Asiatic cholera, and Staphylococcus pyogenes aureus in a few seconds, and the spores of Bacillus subtilis and Bacillus anthracis in a few minutes. It must, therefore, be regarded as satisfactory and of excellent efficiency. There is no evidence that the zinc chloride materially contributes to its value as a disinfectant, and however great the value of zinc chloride as a deodorant may be, it is probable that if a portion, or all of this, could be replaced by a still more concentrated solution of corrosive sublimate the disinfectant might be improved. The recent paper of Green {Zeit. fiir Hygiene, etc., 13, 511) suggests that the copper chloride also has some value. Finally, the decidedly acid re- action of the disinfectant as received is, from the germicidal point of view, an advantage. COLUMBIA UNIVERSITY LIBRARIES This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the rules of the Library or by special ar- rangement with the Librarian in charge. DATE BORROWED DATE BORROWED 2a(1 140)M100 GAYLAMOUNT PAMPHLET BINDER Manu/acluted by 1 GAYLORD BROS. Inc. Syracuse, N. Y. Slocklon, Calif. RA615 Sedgwick Se2 Se.Z