Class '"'■ '■ _ Book GopyrigM°_ COPYRIGHT DEPOSffi A HANDBOOK OX ANTISEPTICS THE MACMILLAN COMPANY NEW YORK • BOSTON ■ CHICAGO • DALLAS ATLANTA • SAN FRANCISCO MACMILLAN & CO., Limited LONDON • BOMBAY • CALCUTTA MELBOURNE THE MACMILLAN CO. OF CANADA, Ltd. TORONTO A HANDBOOK ON ANTISEPTICS BY HENRY DRYSDALE DAKJN, D.Sc, F.I.C., F.R.S. AND EDWARD KELLOGG DUNHAM, M.D. EMERITUS PROFESSOR OF PATHOLOGY, UNIVERSITY AND EELLEVUE HOSPITAL MEDICAL COLLEGE MAJOR, MEDICAL OFFICERS RESERVE CORPS, U. S. ARMY THE MACMILLAN COMPANY 1917 All rights reserved lb* Copyright, 1917, By THE MACMILLAN COMPANY. Set up and electrotyped. Published October, 1917. OCT 18 1317 J. S. dishing- Co. — Berwick & Smith Co. Norwood, Mass., U.S.A. ©CI. A 47665*5 LOUIS PASTEUR JOSEPH LISTER 1822-1895 1827-1913 An Extract from Pasteur's Address at the Celebration of his Seventieth Birthday You lastly, delegates of foreign nations, who have come from so far to give proof of jour sympathy with France — you bring me the deepest happiness which a man can experience who believes implicitly that science and peace will triumph over ignorance and war, that people will learn to agree to- gether, not for purposes of destruction but for improvement, and that the future will belong to those who shall have done the most for suffering humanity. I address myself to you, my dear Lister, and to all of you illustrious representatives of Science, of Medicine, and of Surgery. Young people, young people, confine yourselves to those methods, sure and powerful, of which we as yet know only the first secrets. And all, however noble your career, never permit yourselves to be overcome by scepticism*, both un- worthy and barren ; neither permit the hours of sadness which pass over a nation to discourage you. Live in the serene peace of your laboratories and your libraries. First ask your- selves, What have I done for my education ? then, as you advance in life, What have I done for my country ? so that some day that supreme happiness may come to you, the con- sciousness of having contributed in some manner to the prog- ress and welfare of humanity. PREFACE The main object of this handbook is to give a concise account of the chief chemical antiseptics which have been found useful for surgical purposes during the present war. Some of the publications on this subject are not readily accessible to many who wish to inform themselves as to cur- rent European practice, and the requisite information has not yet, so far as we know, been collected into a form handy for reference. It appeared, therefore, that the present work might prove of use to surgeons and others in this country who are now taking up military duties connected with the care of the wounded. The unparalleled severity and frequency of wound in- fections in the present war has led to considerable advances in our knowledge of antiseptics and of methods for their successful employment. These advances have already proved to be of great value in the treatment of septic condi- tions in civil and industrial practice. No endeavor has been made to make a complete com- pendium of the innumerable antiseptics and disinfectants that have been proposed from time to time, for text-books already exist in which most of these substances are ade- quately described, and no good purpose would be served by duplication. Our principal aim has been to collect in con- venient form the methods of preparation and use of various new antiseptics and modifications of old ones which have received some measure of endorsement by military surgeons during the past three years. viii PREFACE The utility of antiseptics, properly used as adjuncts to efficient surgery, is becoming more firmly established as the war proceeds. Surgeon General Sir George Makins in the Hunterian Oration for 191 7 says: "The most useful practi- cal test of the efficiency of any method of wound treatment is furnished by the observation of the dates at which micro- organisms disappear from the surface of the exposed tissues and at which the wound may be safely and permanently closed by suture or other means. When subjected to this test the antiseptic method has proved itself more rapid and more trustworthy than the phylacagogic or saline method of treatment." A large part of the investigations on antiseptics carried on during the last three years has been done at the instance of the British Medical Research Committee. We are in- debted to Dr. W. Morley Fletcher of that Committee and to the editors of the British Medical Journal and the Journal of the Royal Army Medical Corps for permission to use parts of the reports published in the journals referred to. Purely surgical details concerning the use of antiseptics do not lie within the scope of the present book although some of the principles essential to the successful use of antiseptics are briefly referred to. A short statement of the use of antiseptics in the treatment of carriers of infectious organ- isms is, however, included. Owing to similarity in the substances used we have deemed it advisable to include a brief statement of the use of certain disinfectants of the chlorine group for the sterilization of drinking water and the disinfection of hospital ships. September 1, 191 7. The Herter Laboratory New York. CONTENTS PAGE Preface . . . . ' vii Chapter I. General Introduction : Classification — laws of disinfection — influence of media — choice of antiseptics — modes of application . . i Chapter II. Antiseptics of the Chlorine Group : Hypochlorous acid and hypochlorites, eupad, eusol, chlora- mine-T, dichloramine-T, chemical determina- tion of the strength of chlorine antiseptics . 17 Chapter III. The Phenolic Group of Antiseptics : Phenol, cre- sols, ]ysol, thymol, salicylic acid, /3-naphthol, picric acid, etc 43 Chapter IV. Salts of the Heavy Metals as Antiseptics : Mer- cury, silver, bismuth and zinc salts . . 50 Chapter V. Dyes as Antiseptics : [Malachite green, brilliant green, acriflavine, proflavine . . . .61 Chapter VI. Miscellaneous Antiseptics : Peroxides, ozone, iodine, boric acid and salts, persulphates, acids, alcohol, ether, formaldehyde, hexamethylene- tetramine, iodoform, permanganates, quinine, chinosol, acetanilide 68 Chapter VII. Methods of Testing Antiseptics : Lethal concen- trations — influence of media — time relations — velocity — results 77 Chapter VIII. Certain Special Applications of Antiseptics : Dis- infection of carriers — disinfection of water — disinfection of hospital ships, etc., with electrolytic hypochlorite .... 99 A HANDBOOK OF ANTISEPTICS CHAPTER I GENERAL INTRODUCTION The terms " antiseptic," " disinfectant/' and " germicide" are frequently used irrespective of their precise significance. Strictly speaking, an antiseptic is a substance which inhibits the reproduction of microorganisms, but it need not of neces- sity manifest great killing or "germicidal" action on such organisms. Substances such as boric acid or sodium benzoate are examples of compounds which are fairly effective in re- straining the multiplication of bacteria though possessing feeble germicidal properties. Most of the substances used in the prevention of wound sepsis possess both antiseptic and germicidal properties though perhaps they are most commonly termed antiseptics. The term " disinfectant " should clearly connote a substance which destroys infecting agents and hence is identical with a germicide, but the word has come to be used in a popular sense, irrespective of complete sterilization, to indicate some of the phenomena commonly associated with efficient disinfection, such as a deodorant effect. 1 The use of the words " disinfectant " and " disinfection " in any other sense than that first indicated is to be deprecated. 1 An account of many important hygienic applications of disinfectants which are beyond the scope of the present work will be found in Colonel Lelean's book on "Sanitation in War." Churchill. London. 2 A HANDBOOK OF ANTISEPTICS Classification of Antiseptics. — For various reasons it is quite impossible to formulate a perfectly logical classifica- tion of antiseptics. In the first place, almost every soluble substance, provided it can be obtained in sufficient concen- tration, is capable of exerting some antiseptic action, so that theoretically it would be necessary to classify most known organic and inorganic substances, and such an un- dertaking is obviously useless and unnecessary. For prac- tical considerations it is sufficient to limit ourselves to those substances which have found fairly extended use as antiseptics. In classifying these we may endeavor to di- vide them according to the nature of the substances them- selves or according to their mode of action. The latter method has much to commend it, but unfortunately we know but little of the intimate details of the act of disinfec- tion as carried out with even the simplest substances, such as phenol or hydrogen peroxide. Chemical antiseptics naturally fall into two main classes of compounds — inorganic substances as exemplified by bodies such as hydrogen peroxide, chlorine, and metallic salts, such as those of mercury and silver, on the one hand, and on the other organic, or carbon, compounds such as phenols, aromatic acids, dye-stuffs, such as malachite green, etc., etc. But in these two main groups of inorganic and organic antiseptics are found substances of widely different properties and above all showing enormous variations in sta- bility. This instability of some of the most widely used anti- septics is a point of fundamental importance, for upon a clear recognition of this fact will depend, to a considerable extent, success or failure in their practical use. This chemical insta- bility is of various kinds, as a few examples will serve to show. Hydrogen peroxide is a good example of an unstable in- organic disinfectant. The exact way in which it destroys microorganisms is not clear but is undoubtedly connected with its oxidizing action. Now when hydrogen peroxide is GENERAL INTRODUCTION 3 placed on the surface of an infected wound, it is rapidly de- composed, part of the oxygen of the peroxide being used up in oxidizing organic compounds and much of it being liber- ated as oxygen gas owing to the decomposition of the peroxide by a widely distributed enzyme, "catalase." The net result is that after a period of time which may be almost incredibly short no undecomposed hydrogen peroxide re- mains and disinfection proceeds no further towards comple- tion. For, as will be shown later, the rate at which disinfec- tion proceeds is directly related to the concentration of the antiseptic. Obviously, this instability indicates that hydrogen peroxide should be either frequently renewed or restricted in its use to conditions where temporary action only is required. The chlorine group of antiseptics includes a number of important substances such as chlorine itself, hypochlorous acid and its sodium and calcium salts, and organic "chlor- amines" — i.e. substances containing chlorine attached to nitrogen in the form of XC1 groups. They are all char- acterized by marked instability, since in disinfection they react not only with the cell constituents of microorganisms but also with most other substances which are apt to ac- company bacteria. In so reacting, the active chlorine of the antiseptic is eventually converted either into inert chlo- rides or into inert organic substances in which the chlorine has become united to carbon. Thus in using the chlorine anti- septics, as with hydrogen peroxide, the process of disinfec- tion will only go on so long as some of the active substance remains undecomposed, and in practical use means must be taken for the renewal of these antiseptics at suitable inter- vals, in cases where sterilization is not promptly completed. In the examples of unstable antiseptics just cited, the active substances are decomposed during disinfection and cannot be regenerated. In another large class of antiseptics the active agent is not totally destroyed during disinfection but is rendered relatively inactive. Good examples of this 4 A HANDBOOK OF ANTISEPTICS are found among many metallic salts; mercuric chloride and silver nitrate, for example, are much less effective when acting on bacteria suspended in hard water than when acting on washed bacteria suspended in distilled water. Similarly, phosphates and other salts frequently found in wound exu- dates inhibit the action of metallic antiseptics. In these cases disinfection by the metallic salts is impeded not by the complete destruction of the compound, as with hydrogen peroxide or chlorine antiseptics, but through the conversion of the metallic salt into an inactive form. This conversion may be effected either by precipitation of the metallic com- ponent in an insoluble form, or by changing its state — e.g. changing its condition of ionization in aqueous solution. In such cases the antiseptic substance is rendered inert but not destroyed and, theoretically at least, could be regenerated by suitable chemical means. Lastly, there are other substances used as antiseptics which apparently are stable during disinfection or at least com- paratively so. Familiar examples of compounds of this type are found in the phenols and in aromatic substances of the dye class. These compounds apparently are not readily decomposed by the cell constituents of bacteria nor by most substances which are apt to accompany the latter. It has been suggested that some of these antiseptics act by virtue of changes they produce in the surface layer of the bacterium so that the latter is rendered permeable by the antiseptic. But even with these relatively stable substances there is a definite limit to the amount of disinfection which any given quantity of the compound can accomplish and this amount will vary according to the conditions under which the antiseptic acts. This fact will be noted again in the following section in which some reference is made to the laws governing disinfection. The following table shows the relative stability or insta- bility of some of the common groups of antiseptics : GENERAL INTRODUCTION Inorganic Antiseptics Hydrogen Peroxide and some of its derivatives Chlorine Hypochlorous Acid and its Salts Bromine and Iodine Boric Acid and its Salts Mercury Salts Silver Salts Bismuth Salts Zinc Salts Unstable, easily decomposed during disinfection Unstable, easily decomposed during disinfection Unstable, easily decomposed during disinfection Less unstable than chlorine Stable Often inactivated by precipitation or otherwise Often inactivated by precipitation or otherwise Often inactivated by precipitation or otherwise Often inactivated by precipitation or otherwise Alcohol, Ether, etc Iodoform Formaldehyde Hexamethylenetetramine and its de- rivatives Phenols, Naphthols, and derivatives Aromatic Chloramines Dyes such as malachite green, acri- flavine, etc. Organic Antiseptics Stable Fairly stable Unstable Mostly stable Mostly stable Unstable, easily decomposed during disinfection Mostly stable, though sometimes re- duced to leuco-forms, and often adsorbed by tissues It is a surprising fact that this varying stability of anti- septics is so little realized by many workers. Current litera- ture is full of reports of experiments in which bacteriologists have added reactive unstable substances, such as hypo- chlorous acid, to media which cause their prompt disap- pearance in dilute solution ; the mixture containing little or no remaining antiseptic is subsequently sown with organ- isms and astonishment is expressed at the inefficiency of the substance employed as a disinfectant. This important point will be referred to repeatedly in later sections. The Laws Governing Disinfection. — These have been chiefly worked at by Miss Chick. 1 Earlier experiments were 1 Journal of Hygiene, 8, p. 92, 1908, and 10, p. 238, 1910. 6 A HANDBOOK OF ANTISEPTICS carried out by Kronig and Paul 1 and by Madsen and Nyman. 2 It would be out of place to go into the details of the elaborate experiments and calculations necessary to elucidate the principles of disinfection but a brief summary of the essential results may be given. Miss Chick observed the number of bacteria that survived, at varying intervals of time, the action of a constant quantity of a stable antiseptic, such as phenol, at a constant temperature, on a known number of organisms. By utilizing these results mathematically she was able to calculate a velocity coefficient for the disinfecting action of the substance. The main result of these experi- ments was to show that in all essential particulars the act of disinfection could be regarded as obeying the laws govern- ing a simple chemical reaction, the disinfectant representing one reagent and the bacteria the other. This conception is of the greatest importance since the cardinal points of efficient disinfection, namely adequate active mass or con- centration of antiseptic, time of action, and perfect contact, are thereby experimentally established. The influence of temperature upon the rate of disinfection is interesting since here again a close analogy exists be- tween ordinary chemical reactions and disinfection. The velocity of disinfection increases with rise in temperature in a manner similar to that of an ordinary chemical reaction. Some idea of the magnitude of the effect of temperature may be gathered from the fact that with metallic salts the mean velocity of disinfection increased 2-4 fold for a rise in temperature of io° C, while with phenol it was as high as eightfold, using B. paratyphosus as test organism in each case. It must be remembered, however, that the similarity in the mathematical relations governing the velocity of simple chemical reactions and disinfection does not of necessity prove that the act of disinfection is a chemical one, although with some antiseptics this is almost certainly the case. 1 Zeitschr. f. Hygiene, 25, p. 1, 1897. 2 Ibid., 57, P- 388, 1907 GENERAL INTRODUCTION 7 Influence of Media. — The capacity of a disinfectant to kill microorganisms is dependent to an extraordinary de- gree upon the conditions under which it acts. Almost in- variably the greatest germicidal activity is shown when the substance acts upon bacteria freed from contaminating cul- ture media and suspended in distilled water or salt solution. The presence of proteins and similar substances, e.g. peptones, usually causes a huge reduction in the germicidal activity of most substances. In the presence of pus, in which many of the organisms are partly ingested in the bodies of the dead leucocytes and hence difficult to attack, the results are still less favorable. The following table gives some idea of the reduction in germicidal activity of some of the common antiseptics acting for two hours at room temperature on staphylococcus aureus (i drop of 24 hours broth culture) in a total volume of 5 cc. of either water or 50 per cent horse serum. The antiseptics were in all cases added last of all to the mixtures : Antiseptic Phenol .... Salicylic Acid . . Hydrogen Peroxide Iodine .... Mercuric Chloride 1 Silver Nitrate x . . Sodium Hypochlorite Chloramine-T . . Staphylococci in Water ' 1 : 250 - , 1 : 500 + 1 : 2500 — k 1 : 5000 + ' 1 : 3500 - k 1 : 8000 + 1 : 100,000 — L 1 : 1,000,000 + 1 : 5,000,000 — > 1 : 10,000,000 + 1 : 1,000,000 — k 1 : 10,000,000 + 1 : 500,000 — . 1 : 1,000,000 + ' 1 : 500,000 — 1 : 1,000,000 + Staphylococci in 50% Horse Serum 1:50 - 1 : 100 + 1 : 100 — 1 : 250 + 1 : 1700 — 1 : 2000 + 1 : 1000 — 1 : 2500 + 1 : 25,000 — 1 : 50,000 + 1 : 10,000 — 1 : 25,000 + 1 : 1500 - 1 : 2000 + 1 : 2000 — 1 : 3000 + negative subcultures. The — sign indicates sterilization as evidenced by while the + sign indicates incomplete disinfection. 1 No antidote was used in these experiments to prevent the action of traces of the salt in the subcultures. (Cp. p. 52.) 8 A HANDBOOK OF ANTISEPTICS In order to illustrate the difficulty of sterilizing pus, some selected experiments made by W. Parry Morgan x may be cited. In these experiments one part of pus was mixed with nine parts of antiseptic and after ten minutes 10 cmm. were planted in liquid agar which was then shaken and incubated. It was found that when the antiseptic was strong the num- ber of colonies could be counted readily, but when it was weak the agar became opaque with innumerable colonies. The results are, of course, comparative and do not lend themselves to strict quantitative expression, for the specimens of pus vary much among themselves. Table showing the Effects of Antiseptics upon the Growth of Bacteria in Pus Iodine i : 400 1 : 800 1 : 1600 1 : 3200 16 hours -f ++ + + ++ + + Mercury Biniodide 1 : 400 1 : 800 1 : 1600 1 : 3200 16 hours + ++ ++ + + + Phenol 1 : 40 1 : 80 1 : 160 16 hours . ; + ++ + + + Sodium Hypochlorite 1 : 200 1 : 400 1 : 800 1 : 1600 (Dakin's Solution) 18 hours — — — + 3 days — - - + Eusol (Bleaching Powder and Boric Acid) available chlorine 1 : 200 1 : 400 1 : 800 1 : 1600 18 hours — — — + 3 days ........ — - - + + When the proportion of pus to antiseptic was higher than in the above experiments, the disinfecting action was still fur- ther diminished. A few substances, such as some derivatives of hexameth- ylenetetramine, are known whose germicidal action is said not to be materially affected by the presence of serum, and one striking example exists of a substance which is apparently actually more active in serum than in water. This compound prepared by Benda and termed trypaflavine but now known 1 Brit. Med. Journ., May 13, 1916. GENERAL INTRODUCTION 9 as acriflavine (sometimes simply flavine) has been shown by Browning l and his colleagues to kill staphylococci in serum at one tenth the concentration necessary when the same or- ganisms are suspended in peptone solution. Experiments with pus, however, showed much higher concentrations were necessary to achieve sterilization than with peptone or serum media. The causes of these huge variations in the germicidal ac- tion of antiseptics under various conditions are but slightly understood. The reduction in the case of members of the chlorine group can be accounted for to a considerable extent by the rapidity of the chemical interaction of the disinfectant with the protein medium, with the production of inert substances if the proteins be in sufficient excess. But other causes must be at work where the more stable antiseptics such as phenol and the dye-stuffs are concerned. It is frequently said that the antiseptic is " quenched " or " fixed " by the protein medium, but these terms do not carry much enlightenment, nor does the suggestion that the molecular condition of the antiseptic is influenced by the presence of colloids in the medium. The low germicidal action shown by most antiseptics against pus is due in part no doubt to the mechanical difficulties of penetrating the mucoid particles in the pus. Parry Morgan has shown, moreover, that when the organisms in pus, or added to it, have undergone phago- cytosis they are less readily destroyed by antiseptics. The reduction in antiseptic activity of a substance in the presence of any particulate matter has been often observed 2 and is probably connected with the surface adsorption of the anti- septic by the particles with consequent reduction in its ef- fective concentration. The Choice oe Antiseptics. — The selection of anti- septics for various purposes requires the consideration of a 1 Brit. Med. Journ., Jan. 20, 1917. 2 Chick and Martin, Journ. of Hygiene, 8, p. 654, 1908, 10 A HANDBOOK OF ANTISEPTICS number of factors. The possession of high germicidal ac- tivity, as ordinarily tested in the presence of media comparable with those in which the disinfectant is to act, is of course desirable. But it is equally important to bear in mind the concentration at which the substance may be used, for a relatively innocuous substance used in fairly high concentra- tion will often give much more satisfactory results than lower concentrations of more active substances. The speed of disinfection is also an important question for it varies enor- mously with different types of antiseptics. Antiseptics of the chlorine group and iodine are among the most rapid, while the dyes and some metallic salts are relatively slow. An inspection of the tables in Chapter VII, in which a heavily infected mixture of blood serum and muscle extract was treated with various antiseptics at about the concentration recommended for wound treatment will give a good idea of their relative speed of action and potency under the conditions selected. The ideal surgical antiseptic should effect complete steri- lization within its sphere of action without causing any damage to animal cells. At the moment such a substance does not appear likely to be found, but on the other hand it is surprising to see how little damage may be done to animal tissues by some active antiseptics. An important method of judging of the injurious action of antiseptics is to investigate the condition of the leucocytes in wounds re- cently treated with the substance under consideration. In general it appears from experiments in vitro that, with the strength of antiseptics commonly used in surgery, mercury salts and hypochlorites have relatively little effect on phago- cytosis as compared with phenol (Parry Morgan). It is a regular phenomenon to observe activity of the leucocytes obtained from wounds which have been recently treated with hypochlorites. Ingenious methods for determining the influence in vivo GENERAL INTRODUCTION 11 of antiseptics on the activities of leucocytes have been worked out by Col. C. J. Bond. 1 Indigo impregnated threads are laid in aseptic or septic wounds with or without antiseptics. After varying periods of time the threads will be found to have become decolorized owing to the ingestion of pig- ment particles by the leucocytes. It was found that the application of a strong antiseptic, e.g. i : iooo mercury biniodide or i : 20 phenol, to a wound such as that made for the radical cure of hernia or for the removal of varicose veins, does delay to some slight extent the decolorization of an indigo thread placed in it as a drain. But in general it ap- peared that antiseptic solutions in moderate concentration exercise less influence over emigration and phagocytosis than many surgeons had supposed. There are, however, rea- sons for concluding that many antiseptics do exert a con- siderable inhibitive effect on the return immigration of liv- ing phagocytes. But if the use of antiseptics does bring about a reduction of the numbers, or inhibit the activities, of pathogenic organisms, not merely in vitro but in the actual wound, then although these reagents undoubtedly do cause the death of a certain number of body cells and prevent others from again reaching the tissues, this is relatively of slight importance if the invading organisms are at the same time materially diminished in numbers or offensive capacity. Other criteria which need consideration in judging of the suitability of any particular substance as an antiseptic con- cern the absence of marked irritation of the skin or other tis- sues to which the substance may be applied, and also its effect on the rate of dissolution of necrotic tissue and the rate of formation of healthy granulations. In general, too, it will be found that antiseptics which coagulate the proteins in wound exudates, e.g. salicylic acid, or strong phenol, are less desirable than those which do not act in this way, 1 Brit. Med. Journ., June 3, 1916, Feb. 3, 191 7. 12 A HANDBOOK OF ANTISEPTICS since anaerobes are apt to multiply in necrotic tissue more or less surrounded with impermeable coagulum. In contrast with this effect, the solvent action of hypochlorites and the related dichloramine-T on dead tissue is marked and con- stitutes a valuable property. But the problem of selecting the most desirable antiseptic is by no means limited to the preceding considerations. It is of paramount importance that judgment should be exercised in choosing a substance which is likely to be effec- tive under the conditions of its employment. Thus any of the hypochlorite solutions which give excellent results when used for the intermittent flushing of infected wounds, partly on account of the rapidity of their action and of their gen- erally inoffensive character, are almost useless when ap- plied in small amounts as a simple wet dressing which is infrequently renewed. In the latter case no active anti- septic persists for any significant length of time and not much more is accomplished than the prevention of secondary rein- fection. When prolonged action is required and frequent application is impossible, recourse must be had to more stable mixtures which yield up their store of antiseptic slowly. Examples of such substances are the bismuth iodoform par- affin mixture (B.I. P.) and the oi]y solution of dichloramine- T. These substances will be referred to later. Finally, it must be once more emphasized that antiseptics at best are only accessories to, and not substitutes for, efficient surgical treatment of infected wounds. The disinfecting action of antiseptics is practically restricted to the wound surfaces and the cavity inclosed by them and none of them appears to have any considerable power of penetration with- out simultaneous loss of activity. The prompt excision of damaged tissue has now become a routine method in war surgery on the Western Front, and this procedure, renders the effective employment of antiseptics much more certain than it was earlier in the war. GENERAL INTRODUCTION 13 Modes of Application. — Antiseptics are applied in various ways, according to the character of the wound, the nature of the substance, and the kind of action desired. Aqueous solutions are used most extensively. When un- stable antiseptics, such as hypochlorites and chloramine-T, are used and prolonged antiseptic action is required — as in the treatment of freshly infected or septic wounds — it is necessary to employ some means of frequently renewing the antiseptic. This end is- commonly accomplished either by irrigation or by intermittent instillation of fresh antiseptic solution into the wound by means of short rubber tubes so arranged that the solution may reach every recess of the wound. This method has been used extensively in the pres- ent war and has given excellent results. The technique of the method will be found fully described in a book by Carrel and Dehelly. 1 To obtain the best results with this mode of treatment, a careful observation of details is essential, and as only an outline can be given here reference should be made to the book just mentioned or to numerous original papers in recent publications. The first step is the mechanical cleansing of the wound by excision, this being omitted as dangerous if the phenomena of inflammation have already set in. The wound is then subjected to active antiseptic treatment. For this purpose sodium hypochlorite — so-called Dakin's solution — is em- ployed at a concentration of 0.5 per cent (see page 23). Great care is taken to insure uniform distribution of the antiseptic by means of the rubber tubes w T hich are usually of fine bore, closed at one end and provided near the closed end with a number of fine lateral per- forations. A number of these tubes, depending on the size of the cavity, are disposed within the wound, care being taken that no gauze intervenes between the tubes and the 1 Le traitement des plaies infectes, Collection Horizon, Masson et Cie., Paris, 191 7, 14 A HANDBOOK OF ANTISEPTICS tissues and also that the tubes are not so closely applied as to block the perforations. The tubes are led through the outer dressing and kept in position with safety pins. When, as is always the case with large wounds, more antiseptic solution is needed than can be conveniently supplied by using a glass syringe, the ends of the tubes are connected by T pieces to a single tube which in turn connects with a glass reservoir holding a liter of solution which is suspended sev- eral feet above the level of the bed. The supply of solution to the wound is arranged by opening for a few seconds every two hours the clip attached to the main tube. An important point is the absence of a drainage opening at the most dependent part of the wound; in fact, the ideal ar- rangement is attained when the wound is cup-shaped; when it is on the inferior surface of a limb the aperture is partly plugged with gauze to hinder free escape of the fluid. It is important to note that vessels in the base of the wound should not be ligatured with silk since this material is at- tacked by hypochlorites. The routine bacteriological ex- amination of the wound is important. It is found that when practical sterility has been maintained for two or three days it is safe to close the wound. In the case of wounds that have suppurated, it is advisable to wait a little longer. In place of the hypochlorite, the more stable chloramine- T has been used to a considerable extent and with particular success in fresh industrial accidents. It is usually used in 0.5-2 per cent solution and its action is similar to that of hypochlorite save that necrotic tissue is less rapidly removed. The methods of application of the ordinary solutions of less unstable antiseptics such as phenol, iodine, and the metallic salts, call for no special comment. When the frequent renewal of the antiseptic is impracti- cable or not desired, recourse may be had to pastes or oils of various kinds, which embody a store of antiseptic that may be gradually utilized. The mild antiseptic action GENERAL INTRODUCTION 15 of a paste containing about one per cent of chloramine-T and five per cent of sodium stearate in water is often useful to maintain sterility and prevent reinfection of wounds which have previously received more vigorous treatment. A much more intensive action can be obtained from the use of dichloramine-T dissolved in eucalyptol and paraffin as described on page 39. The amount of active antiseptic which may thus be applied to the wound surface is extremely large and its action is correspondingly prolonged. The method of using this oil is particularly simple and the results appear to be exceptionally good, both as regards the prevention and treatment of sepsis. A paste possessing moderate potency, introduced by Rutherford Morison, composed of bismuth subnitrate, iodoform, and paraffin, is being widely used with good results both for wounds of the soft tissues and for fractures. The fact that the wounds do not need dressing for several days gives it a great advantage when the number of cases re- quiring treatment is very large, and because of this it also secures a maximum of rest for the wound. It must not be forgotten that the employment of pastes as a primary dressing is not free from serious risks and should only be practiced with caution. The use of salicylic acid pastes for the immediate treatment of wounds other than superficial ones is said to have given particularly poor results as anaerobes are apt to flourish in walled off recesses of the wound. The coagulating properties of salicylic acid would especially facilitate such a result. The administration of antiseptics by intravenous injection is the only method other than that of local application that is at all practiced for military purposes. Eusol has been used to some extent for this purpose l though it is abundantly clear that any action it may have is unconnected with any 1 Lorrain Smith, Ritchie, and Rettie, Brit. Med. Journ., Nov. 13, 191 5. Frazer and Bates, Brit. Med. Journ., Aug. 5, 1916. 16 A HANDBOOK OF ANTISEPTICS germicidal effect since the actively haemolytic hypochlorite, in therapeutic doses, is immediately decomposed by the blood. 1 A few striking cases of favorable results of its use have been recorded but as the procedure has not found wide acceptance and is not free from danger, it is well to suspend judgment as to its value. A number of years ago 2 silver nitrate was used intravenously in the same way and while it was clear that no direct germicidal effect was possible, some kind of beneficial effect was believed to follow the haemolysis caused by the silver salt. 1 Dakin, Brit. Med. Journ., June 17, 1916. 2 Klinisch-Therapeutische Wochenschrift, No. 33, p. 881, 1908, CHAPTER II ANTISEPTICS OF THE CHLORINE GROUP In the present war, which is distinguished by the frequency and intensity of virulent wound infections, no class of anti- septics has received such extensive employment as those of the chlorine group. When properly applied — and this is an important reservation — it is generally conceded that they have proved of genuine value to the surgeon. All of the antiseptics of this group are characterized by chemical instability in the presence of organic matter, and therefore conditions favorable for their use must include either pro- vision for their frequent renewal, or the use of some im- miscible solvent for the antiseptic so that the active compound may be gradually liberated. The members of the group under consideration are often spoken of as containing " active" chlorine as distinct from inert chlorine such as that in common salt. It must not be inferred, however, that the phrase " active chlorine" neces- sarily implies either that free chlorine is contained in the substance or is liberated from it, as more often it will be found that hypochlorous acid or some such compound is the active agent. From the standpoint of disinfection, it is probably correct to connote with the term " active chlorine " in a compound, the ability of any particular substance to part with chlorine, free or combined, in such a way that it can effect the chlorination of bacterial and other proteins. 1 1 All compounds containing active chlorine possess the property of liberating iodine from an acidified solution of potassium iodide. The iodine may be de- tected by the development of a blue color on adding starch paste. This reaction will often be found useful in testing for the presence of unchanged antiseptic. C 17 18 A HANDBOOK OF ANTISEPTICS This chlorination of bacterial protein seems to be incompatible with the life of the microorganism. It is necessary to refer in outline to the nature of this reaction. All proteins, irre- spective of their origin, contain large numbers of amino-acid groups, which may be represented as shown below : (I) H H I I R— C— CO R— C— CO— I I NH NCI I II These groups are capable of attack by substances containing active chlorine in such a way that the hydrogen attached to the nitrogen atom is replaced by chlorine (II). The com- pounds thus formed contain the (NCI) group and hence belong to the class of chloramines. Their chlorine is still active and they are themselves active germicides. Other concomitant reactions also occur which use up part of the chlorine, converting it into an inert form, e.g. the chlorine becomes united to carbon or forms chlorides. This formation of germicidally active chloramines is of importance in several respects. For example, the proteins and other nitrogenous compounds present in wound secre- tions may be converted into chlorine derivatives of antiseptic value by the action of a sufficient quantity of hypochlorite or similar substance. 1 While preformed chlorine derivatives 1 It is of interest to note that while chlorine, bromine, and iodine have not widely differing germicidal properties, hypobromites and hypoiodites in contrast to hypochlorites have but trivial disinfecting action. Correlated with this fact is the observation that hypochlorites react readily with proteins while hypo- bromites and hypoiodites do not. On the other hand, certain synthetic broma- mines which react readily with amino-acids and proteins have a high germicidal potency. The ability to react with proteins and allied bodies is clearly associated with germicidal activity in members of the halogen group of antiseptics. ANTISEPTICS OF THE CHLORINE GROUP 19 prepared from proteins are powerful germicides, they are not convenient substances for general use, but by using other types of nitrogen compounds for chlorinating, synthetic chloramines, with valuable antiseptic properties, are readily obtainable. For detailed information concerning the action of chlorine antiseptics upon amino-acids, proteins, etc., as well as the preparation of many synthetic chloramines, reference must be made to the original papers. 1 In addition to their disinfecting action, the chlorine antiseptics are strong oxidizing agents and deodorants and moreover possess in high degree the property of decompos- ing toxins. By the regulated action of hypochlorous acid, Dean 2 has prepared a non-toxic dysentery vaccine and it is a common observation that the free use of hypochlorites may reduce the constitutional symptoms arising from septic processes and that they reappear on discontinuing the anti- septic treatment. The antiseptics of the chlorine group which are most com- monly employed in the treatment of infected wounds, are the following : 3 (a) Hypochlorous acid and its sodium and other salts (including "eupad," "eusol" and so-called Dakin's solution). (b) Chloramine-T, the abbreviated name for sodium toluene sulphonchloramide. (c) Dichloramine-T, the abbreviated name for toluene sulphondichloramine. In most respects, the action of these various chlorine compounds is essentially similar, though each possesses certain properties which render it more or less suitable for 1 Brit. Med. Journ., Jan. 29, 1916, June 17, 1916; Proc. Roy. Soc. B, 8q, p. 232, 1916. Biochem. Journ., June, 1917. 2 Brit. Med. Journ., April 29, 1916. 3 Chlorine water has been used to some extent, but it is doubtful whether its use is preferable to that of the more convenient iodine solution, which in most respects it resembles. 20 A HANDBOOK OF ANTISEPTICS particular purposes. As a matter of convenience, it may be desirable to give a short resume of these considerations. I. Hypochlorous acid and hypochlorites are best suited to cleansing septic wounds by irrigation. They markedly assist in the dissolution of necrosed tissue. They are unstable and very reactive, and must be frequently renewed to all parts of a wound, this being best achieved by the method of intermittent instillation (p. 13). They are the cheapest antiseptics of the chlorine group and are much cheaper than other effective germicidal substances. Free hypochlorous acid is more irritating than the sodium salt. The latter in 0.5 per cent neutral or feebly acid (boric acid) solution may be used in large quantities under appropriate precautions for a considerable time without causing irritation. The skin is more susceptible than the deeper tissues and should be protected with vaseline or some similar substance. II. Chloramine-T can be used in stronger solution (up to two per cent) than the hypochlorites. It is more stable and exerts more prolonged antiseptic action and is consider- ably more effective than hypochlorite when acting in the presence of much blood. It is not toxic and is less irritating than the hypochlorites and has but little solvent action on necrosed tissue. It is well suited for use on wounds previously cleansed with hypochlorites or dichloramine-T, and in suitably dilute solutions may be used in the eye and on other sensitive parts. It may be applied in solution, as an impregnation of gauze, or in a sodium stearate cream. III. Dichloramine-T dissolved in oily media may be sprayed upon wound-surfaces or poured into accessible parts of deep wounds. It yields moderate amounts of anti- septic to watery media such as secretions from wounds or mucous membranes. It is suitable for cases requiring pro- longed antiseptic treatment, and for first dressings of recent wounds which do not require irrigation. It is also used for nasal antisepsis (p. 102). Dichloramine-T in oil solution has a ANTISEPTICS OF THE CHLORINE GROUP 21 great advantage over the other chlorine antiseptics in that it may be used in high concentration, and its action is of much longer duration. The application of the oil is extremely simple and it ordinarily need not be renewed more than once in 24 hours. Hypochlorous Acid and its Salts The disinfecting action of these substances has been known for over a hundred years and they have received numerous hygienic applications. An account of their early history and uses will be found in the British Medical Journal, Dec. 4, 1915. In the early part of the present war, several surgeons made use of commercial sodium hypochlorite, "eau de Javel," for the treatment of infected wounds, but there were many accidents owing to its caustic action, which was largely due to the presence of excessive amounts of alkali in the solution. Apart from this objectionable property, the substance appeared to possess desirable qualities and it was for this reason that several workers sought to obtain hypochlorite solutions which were less irritating but w T hich retained their germicidal properties unchanged. These various solutions are all prepared from bleaching powder (chloride of lime) which is the most readily accessible solid source of hypochlorous acid and its salts. Bleaching powder is prepared by the action of chlorine upon slaked lime and in most respects behaves like a mixture of calcium hypochlorite and calcium chloride. It is of variable com- position and slowly decomposes on keeping, especially when exposed to air or light (p. 41). In making some of the anti- septic solutions, the calcium hypochlorite of the bleaching powder is converted into sodium hypochlorite by the action of sodium carbonate. In most of them boric acid is added to counteract the objectionable alkalinity of ordinary hypo- chlorites, and this has also the effect of liberating a certain 22 A HANDBOOK OF ANTISEPTICS amount of free hypochlorous acid, leaving the solution neutral or faintly acid. Sodium hypochlorite may also be prepared by the electrolysis of sodium chloride solution (p. 1 1 6) and by the action of chlorine on sodium carbonate or sodium bicarbonate. It will be impossible to describe the preparation and uses of all the various hypochlorite solutions that have been employed for surgical use and it would appear sufficient to give an account of the two that seem to be most widely used, namely " eusol " and the so-called Dakin solution. All hypochlorite solutions attack metals and hence they should not be used for the sterilization of instruments. Eupad and Eusol. 1 — These are preparations of bleaching powder and boric acid, either dry (eupad) or in solution (eusol). The former is prepared by intimately mixing equal weights of bleaching powder and boric acid, both in fine powder. It has been employed as a dusting powder, and in strands of gauze for drainage or between layers of moistened gauze or lint as a dressing. When moistened, eupad liberates hypochlorous acid partly in gaseous form and in variable amount and this, if excessive, is liable to prove highly irri- tating, so that the quantity of eupad used must be carefully controlled. Eupad forms a rather thick, white coagulum with wound exudates and this may occasion inconvenience. It is used much less extensively than is the aqueous eusol. An almost identical mixture was recommended by Vincent some years ago while Lumiere advocates a mixture con- taining bleaching powder one part with boric acid three parts. Eusol is prepared in either of two ways : (i) Twenty-five grams of eupad are shaken up with one liter of water, allowed to stand for a few hours, then filtered through cloth or filter paper. 1 Lorrain Smith, Drennan, Rettie, and Campbell, Brit. Med. Journ,, July 24, I9I5- ANTISEPTICS OF THE CHLORINE GROUP 23 (2) To i liter of water add 12.5 grams bleaching powder, shake vigorously. Then add 12.5 grams boric acid powder and shake again. Allow to stand for some hours, preferably overnight, then filter off, and the clear solution is ready for use. Eusol prepared in this way from good quality bleaching powder contains the equivalent of about 0.27 per cent hypo- chlorous acid. 1 The solution gives thick precipitates contain- ing calcium with blood or wound exudates and is strongly hemolytic. It is frequently spoken of as a solution of hypochlorous acid but actually the mixture is alkaline to litmus and contains a balanced mixture of calcium hypo- chlorite and borate with an undetermined amount of free hypochlorous acid. The separate estimation of the latter is a difficult problem. A large number of experiments on the germicidal action of eusol will be found in Lorrain Smith's paper, together with suggestions as to methods of use in the treatment of infected wounds. It is recommended by its authors for use (a) as a lotion, diluted if necessary, (b) as a fomentation, (c) as a wet dressing with gauze without a waterproof covering, and (d) as a bath, diluted if necessary. The general principles concerning the use of chlorine anti- septics as germicides apply equally to eusol and the other hypochlorites (cp. pp. 12, 20). Neutral Sodium Hypochlorite Solution ("Dakin's Solution"). — This preparation is essentially a solution of sodium hypochlorite, containing 0.45 to 0.5 per cent NaCIO made in such a way that it is, and remains, substantially neutral. Ordinarily commercial hypochlorite is very variable in composition and commonly contains much free alkali and occasionally free chlorine. Such solutions are very irritating and should not be used for surgical purposes. The original formula 2 for making the neutral solution requires 1 Through an error in calculation, the composition was first given as 0.54 per cent. The other analytical figures given in the original paper also contain errors. 2 Comptes rendus, 161, p. 150, 1915, Brit. Med. Journ., Aug. 28, 1915. 24 A HANDBOOK OF ANTISEPTICS the use of boric acid for neutralization. The reason for this may perhaps be briefly referred to. It is well known that blood and some other body fluids and also certain artificial salt solutions containing mixtures of the salts of polybasic acids — e.g. phosphoric or carbonic acid — ■ are able to retain their essential neutrality even after the addition of limited quantities of acid or alkali. This is due to the fact that the addition of acid or alkali simply changes the relative pro- portion of two or more salts of the polybasic acid present in the solution. Such solutions are often referred to as " bal- anced " and the salts in them are called " buffer salts/' Uti- lizing the same principle and employing the feeble polybasic boric acid, a simple balanced hypochlorite mixture was prepared which maintains essential neutrality under all conditions. It should be understood that the insignificant antiseptic action of boric acid has nothing to do with the em- ployment of this acid nor is the boric acid employed for the purpose of liberating free hypochlorous acid as in Lumiere's or Lorrain Smith's preparations. Preparation of Neutral Sodium Hypochlorite. — One hundred and forty grams of dry sodium carbonate (Na 2 C0 3 ) or 400 grams of* the crystallized salt (washing soda) are dissolved in 10 liters of tap water, and 200 grams of bleach- ing powder containing 24-28 per cent of " available chlorine " are added. 1 The mixture is very thoroughly shaken, both to make good contact and to render the precipitated calcium 1 Bleaching powder varies considerably in its available chlorine content, though when bought in bulk the fresh product is fairly constant in composition. It is advisable to determine the " available chlorine " in each large batch of bleach- ing powder purchased, as described on p. 41. Bleaching powder with less than 23 per cent of available chlorine should be rejected. Exceptional samples may contain as high as 35 per cent available chlorine and in such cases it is well to reduce correspondingly the ingredients taken in the above formula. For pur- poses of rough calculation, one may assume that using 200 grams of bleaching powder for 10 liters of solution, the resulting product will contain as much sodium hypochlorite as is represented by the available chlorine of the bleaching powder divided by 50. Thus 25 per cent " available chlorine " bleaching powder will give 0.5 per cent sodium hypochlorite solution. ANTISEPTICS OF THE CHLORINE GROUP 25 carbonate granular and promote its settling. It is then allowed to stand quietly and after half an hour the clear liquid is siphoned off from the precipitate and filtered through a cotton plug or paper. Forty grams of boric acid are added to the clear filtrate and the resulting solution is ready for use. The boric acid must not be added before filtering but only afterwards. The exact strength should be determined from time to time, as directed on p. 41. It is important that the solution should not be stronger than 0.5 per cent sodium hypochlorite or irritation of the skin may be frequent. On the other hand, it should not be less than 0.4 per cent or its germicidal action is materially diminished. The solution should also be tested for neutrality by adding a little of it to a trace of solid phenolphthalein suspended in water. No red color indicating free alkali should develop or else more boric acid must be added ; this is, however, rarely necessary with the above proportions. The solution should not be kept longer than one week. 1 Daufresne has shown that it is possible to prepare a satis- factory solution of sodium hypochlorite without employing' boric acid for neutralization if sodium bicarbonate be used in conjunction with sodium carbonate for decomposing the bleaching powder. The relative proportions of sodium carbonate and bicarbonate required to furnish a neutral 1 A stronger solution may be prepared by decomposing bleaching powder with dry sodium carbonate in the proportion of 150 gms. to 105 gms., dissolved in 1 liter of water. The mixture is filtered and a measured portion of it (20 cc.) rapidly titrated with a boric acid solution of known strength (31 gms. per liter, \ normal), using phenolphthalein suspended in water as indicator (the usual alco- holic solution of phenolphthalein will not serve, because the alcohol is at once at- tacked) in order to determine the amount of boric acid to be added to the rest of the filtrate. (Each cubic centimeter of N/2 boric acid calls for 3 gms. boric acid to be added.) An excess of boric acid should be avoided as it favors the liberation of hypochlorous acid and renders the solution less stable. It is best to add slightly less than the calculated amount. The concentrated solution thus prepared contains about 4 per cent of sodium hypochlorite and should be mixed with 7 parts of water before use. It can be kept for a month without serious decomposition. 26 A HANDBOOK OF ANTISEPTICS solution depends upon the varying composition of the bleach- ing powder used and cannot simply be deduced from its " available chlorine " content, as is frequently stated. The proportion of free lime in the bleaching powder is obviously as important as its chlorine strength. With some brands of bleaching powder retailed in the United States, the fol- lowing proportions have proved useful : Two hundred grams of bleaching powder (24-28 per cent available chlorine) l is shaken well with five liters of water and allowed to stand for an hour or two. In a separate vessel dry sodium carbonate (94 grams) and sodium bicar- bonate (86 grams) are mixed with five liters of cold water, and when dissolved, the solution is added to the bleaching powder suspension and the mixture well shaken. The pre- cipitate of calcium carbonate is allowed to settle and the clear supernatant solution is syphoned off and filtered. The solution should contain close to 0.5 per cent sodium hypo- chlorite and this should be checked by analysis (p. 41). If too strong it should be diluted with water to 0.5 per cent strength. It is most important to test the solution for free alkali by adding a trace of solid phenolphthalein to a little of it. In case a red color develops indicating free alkali, the solution may still be used if it is previously neutralized either by passing carbon dioxide through the solution or by adding a little boric acid, until the alkaline reaction is abolished. But in making further quantities of the solution, using the same sample of bleaching powder, alkalinity may be avoided by reducing the quantity of sodium carbonate and correspond- ingly increasing the bicarbonate. Sodium hypochlorite, whether prepared according to the preceding formulae or according to other methods that will occur to the chemist, e.g. from salt by electrolysis (p. 116) or from liquefied chlorine gas, when used in neutral solution 1 Or an equivalent amount of stronger bleaching powder. ANTISEPTICS OF THE CHLORINE GROUP 27 at 0.5 per cent concentration is found to be a valuable anti- septic for the treatment of infected wounds. Its action is extremely rapid and then ceases as soon as all the hypo- chlorite is decomposed, hence the methods for using the solu- tion efficiently must provide for its frequent renewal. A short account of the technique advocated by Carrel and Dehelly will be found on p. 13. For further details, reference may be made to their book and to the papers noted below. 1 The hypochlorite solutions possess the valuable property of assisting in the rapid dissolution of necrotic tissue, doubt- less owing to their ability to react with proteins with the formation of soluble products. They possess a slight but definite haemostatic action but are actively hemolytic and should not be injected intravenously. The hypochlorites are extremely reactive substances chemically, and should neither be heated above 37 C. or used with other antiseptics nor with alcohol nor ether. It is rather difficult to give useful figures for the germicidal effects of sodium hypochlorite since so much depends on the capacity of the medium to decompose the hypochlorite before it can complete disinfection. Most pyogenic organ- isms suspended in water are killed at a concentration of less than 1 : 100,000, while in serum about 1 : 1500 is necessary. Blood decomposes the hypochlorites rapidly, so that 1 : 300 or more may be necessary before sterilization is complete. The action of sodium hypochlorite and eusol on a mixture of pyo- genic and other organisms in a blood serum muscle extract mixture is recorded on p. 85. The lethal concentration under the stated conditions is probably a little less than 1 : 1000. m 1 Le traitement des plaies infect es. A. Carrel & G. Dehelly. Masson et Cie., Paris, 1917. Carrel, Dakin, Daufresne, Dehelly, and Dumas. Presse Medicale, Oct. 11, 1915. Turner, Bull, de l'Acad. de Med., 74, No. 38, 1915. Depage, A., Bull, et Mem. Soc. de chir. de Paris, 42, p. 1987, 1916. Lyle, H. H. M., Journ. Am. Med. Assoc, Jan. 13, 1917. 28 . A HANDBOOK OF ANTISEPTICS The extraordinary rapidity of its action in concentrations even lower than those employed for surgical purposes is well illustrated. Chloramine-T Chloramine-T is the abbreviated name for sodium toluene- sulphonchloramide. 1 It is a crystalline, odorless substance containing 12.6 per cent of chlorine. It is readily soluble in water and the solutions, which have a bitter taste, are stable, neither moderate exposure to heat nor light caus- ing appreciable decomposition. In equimolecular solutions its germicidal activity is about four times that of sodium hypochlorite. This may, perhaps, be explained by the fact that the chlorine is already linked to nitrogen and is less rapidly appropriated through reactions with proteins and other substances in the wound secretions. The relatively slight solvent action of chloramine-T on necrosed tissue supports this view. Its antiseptic efficiency is prolonged by this reduction in reactivity. Its germicidal action is rapidly exerted and in most respects it resembles the hypo- chlorites closely, though decidedly less irritating than the latter. An idea of its potency may be gathered frorrl the accompanying tables, although in some respects a better indication is given by the results recorded in a later chapter (p. 86). In another series of experiments note was taken of the speed of disinfection. Horse blood serum (2 cc.) or 0.7 per cent Witte's peptone (2 cc.) was inoculated with staphy- lococcus aureus and subsequently treated with weak chlora- mine-T solutions (1 cc). The results show clearly that the rate of disinfection is very rapid, the maximum effect being observed in a few minutes. In those cases where disinfection was incomplete and no active antiseptic persisted in the mixture, subsequent growth took place (p. 30). 1 Dakin, Cohen, and Kenyon, Brit. Med. Journ., Jan. 29, 1916. ANTISEPTICS OF THE CHLORINE GROUP 29 Table showing the Germicidal Action of Chloramine-T on Several Common Organisms Two drops of a fresh culture of the organisms were suspended in 5 cc. of fluid, either water or 50 per cent horse serum, and the antiseptic was allowed to act two hours at room temperature. The mixtures were then subcultured. In comparison, a few figures for sodium hypochlorite and phenol are added. Chloramine-T Sodium Hypochlorite Phenol Staphylococci in water . . 1 : 500,000 — 1 : 500,000 — 1 : 250 - 1 : 1,000,000 + 1 : 1,000,000 + 1 : 500 + Staphylococci in serum 1 : 1,500 - 1 : 1,500 - 1: 50 - 1 : 2,500 + 1 : 2,000 + 1 : 100 + B . pyocyaneus in water 1 : 200,000 — 1 : 100,000 — 1 : 200 — 1 : 400,000 + 1 : 1,000,000 + 1 : 400 + B. p3 r ocyaneus in serum . 1 : 1,250 - 1 : 2,500 — 1: 25 - 1 : 2,000 + 1 : 5,000 + 1: 50 + Streptococci in water . '. 1 : 1,000,000 — Streptococci in serum . . 1 : 2,500 — 1 : 5,000 + B. capsulatus in water . . 1 : 1,000,000 — B. capsulatus in serum 1 : 2,500 — 1 : 5,000 + Complete sterilization is indicated by survived. -, while + indicates that organisms Chloramine-T may be used for wound treatment in solu- tion, in the dry state as an impregnation of gauze, or in a cream-like paste, all these methods having proved efficient and useful. In Solution. — At first, solutions containing as much as 4 per cent of chloramine-T were used in the treatment of wounds. Although there were no cases reported to show that these were unduly irritating, subsequent experience has shown that there is no occasion for exceeding a concen- tration of 2 per cent. For, as chloramine-T has, weight for weight, as great a germicidal power as sodium hypochlorite, a 2 per cent solution is considerably more potent than the 30 A HANDBOOK OF ANTISEPTICS Medium Concentration of Chloramene-T in the Mixture Time of Action Bacterial Count ( 1 Drop = ^cc.) Blood Serum .... 1 : 1000 1751 5 mm. Blood Serum .... 1 : 2000 15 mm. 45 1831 Blood Serum .... 1 : 3000 5 mm. 15 min. 45 min. 5 min. 15 min. 9 1 15 49 1509 96 1 82 Peptone 0.7 per cent . . 1 : 5000 45 min. 5 min. 211 9360 15 mm. Peptone 0.7 per cent . . 1 : 6000 45 mm. 5 min. 15 min. 1323 13 l 12 45 mm. 14 solution of sodium hypochlorite usually employed (0.5 per cent). The 2 per cent solution may be used for the treat- ment of septic wounds, using the same methods as employed for the hypochlorites. It must be borne in mind that in severe septic conditions much of the chloramine-T is promptly decomposed by the secretions. This is true of all antiseptics of the chlorine group and is even more marked in the case of hypochlorites than with chloramine-T. While this circumstance undoubt- edly exerts a favorable influence through the destruction of substances of a toxic nature, it reduces the germicidal activity of the solution. It follows also that where sepsis is less marked, much weaker solutions may be employed. 1 All antiseptic decomposed. No active chlorine present. ANTISEPTICS OF THE CHLORIXE GROUP 31 In the eye, for example, a solution of one part of chloramine-T in a thousand parts of normal saline solution will exert a satisfactory germicidal action, while i : 500 may prove rather irritating to the inflamed conjunctiva. Here, because of the constant irrigation by tears, there is chance for only a moderate accumulation of septic products likely to reduce the strength of the application. In cystitis, the tolerance often appears to be less than in the eye, and it is advisable to start treatment with a weak solution, increasing the strength according to the degree of tolerance manifested. In chronic urethral infections, 1 : 500 can be used for the initial injections and the concentrations subsequently increased. Similar considerations apply to the irrigation of the pleural cavity in empyema. The use of chloramine-T for the disinfection of meningococcus carriers is referred to on p. 100. From the foregoing statements it will be evident that the choice of strength to be used must be left to the judgment of the surgeon. In practice it is advisable to keep a 2 per cent solution in stock and to dilute this, if necessary, either with water or, in case of considerable dilution, with normal saline solution. A 2 per cent solution is slightly hypotonic and when an approximately isotonic medium is desired, normal saline solution should be used as a diluent. Chloramine-T, like hypochlorites, has a corrosive action on most metals and should not be used for the sterilization of instruments. /;/ Impregnated Gauze. — Chloramine-T is well adapted to this use, for which very few substances of high antiseptic value have proved successful. It is possible, for example, to incorporate as much as 25 per cent of the weight of the gauze. This is a much larger amount than is advisable, 5 per cent being adequate. Obviously the gauze should not be moistened before use lest the antiseptic be washed out because of its ready solubility. It can be used dry for lightly packing and" subsequently moistened if necessary 32 A HANDBOOK OF ANTISEPTICS when in position. Impregnated gauze finds application in wounds of recent origin in which the chief object is to pre- vent progress of infection. It is particularly adapted to use in cases of industrial accident where treatment can be promptly instituted. Where frequent renewals of the anti- septic or irrigation are called for, it is superfluous. In Soap Paste. — None of the chlorine group of anti- septics can be used in ointments containing fats or oils as these rapidly withdraw the active chlorine with the pro- duction of inert compounds. It is possible, however, with the exceptionally stable chloramine-T to obtain a prepara- tion which can be used as an acceptable substitute for oint- ments. A preparation of this sort introduced by Daufresne, 1 which has been extensively used, contains 0.7 per cent to 1 per cent of chloramine-T dissolved in water containing 5-10 per cent of sodium stearate. It is important that the mixture should not contain any substance which can unite with chlo- rine to form an inert compound. For this reason the stearate used must be free from any fatty acid of an unsaturated series. The paste is not oleagenous. It has a creamy consistency and can be spread readily. As the antiseptic is dissolved in the water constituting the chief bulk of the paste, it has ready access to the parts treated. An investigation of the effect of this chloramine-T paste in sterilizing moderately infected wounds and maintaining asepsis in wounds previously sterilized by other means, has been published by Carrel and Hartmann. 2 Its use does not delay the rate of cicatrization. Preparation. — Chloramine-T was first prepared by Chat- taway 3 by the action of sodium hydroxide upon toluene- sulphondichloramine (dichloramine-T, vide infra).* A more 1 Journ. Exper. Med., 26, p. 91, 1917- 2 Ibid., p. 95, I9i7- 3 Trans. Chem. Soc, 87, p. 153, ioo5- 4 Chloramine-T is manufactured by Messrs. Boot, Island Street, Nottingham, England, and several other firms. It is also marketed by the Abbott Laboratories, Chicago, under the name of Chlorazene. ANTISEPTICS OF THE CHLORINE GROUP 33 economical method of preparation consists in dissolving toluene-p-sulphonamide (i mol.) in 5 per cent cold alkaline solution of sodium hypochlorite (1.2 mol.), warming gently if necessary, filtering, and adding 1^ vols, of saturated salt solution. The chloramine-T crystallizes out of solution as a white glistening meal of crystals and is filtered off, washed with salt solution, and dried in the air. The product con- tains three molecules of water of crystallization. If it is desired to free the substance from adherent salt left from the process of preparation, it may be obtained pure from a hot concentrated solution, from which on slow cooling it will separate in large crystals. The purity of a given sample may be determined by titration with decinormal thiosulphate solution, as described on p. 42. The reaction which takes place in the above preparation .may be represented as follows : CH 3 CH 3 + NaCIO =1] + H 2 SO2NH2 S0 2 NaNCl Toluene-p-sulphonamide Chloramine-T DlCHLORAMINE-T Dichloramine-T is the abbreviated name for toluene-p- sulphondichlor amine. It is a yellowish white crystalline substance possessing a sweetish, rather pungent chlorous odor. It is stable in the solid state especially when kept in the dark. Water dissolves only traces of it, though it is readily soluble in most organic solvents except paraffin or petroleum. It has an intense germicidal action (pp. 86, 93) corresponding to its high content of active chlorine, but it is difficult to find perfectly satisfactory solvents for it which will yield stable solutions. Up to the present, the best 34 A HANDBOOK OF ANTISEPTICS medium that we have been able to find is a mixture of euca- lyptol and paraffin oil, both previously treated as described on p. 37 to reduce their avidity for chlorine. A more highly chlorinated eucalyptol prepared according to a formula worked out by Paul Lewis may prove preferable for many purposes. Dichloramine-T was originally used in oil solution for nasopharyngeal disinfection (p. 102), but more recently it has found a wider application in the treatment of infected wounds. The results obtained in the treatment of industrial injuries by W. E. Lee x and his colleagues in Philadelphia and by J. E. Sweet 2 in war wounds, have been extremely satisfactory. It will be well, perhaps, to refer first of all to the way in which the antiseptic action of dichloramine-T in oil-solution is exerted. It is well recognized that antiseptics incorporated with or dissolved in oily substances usually possess little if any antiseptic activity because intimate contact with the infected matter is hindered by the oil. When, however, such oil-solutions of dichloramine-T as will be described are brought in contact with aqueous media, the partition co- efficient between the oil and the water is such that a certain amount of the dichloramine-T passes into the water and there exerts its germicidal action. The amount of dichloramine-T thus passing from the oil is enhanced by the presence in the aqueous medium of substances capable of taking up chlorine. So that the oil solution serves as a store of the antiseptic which is drawn upon to maintain the germi- cidal activity of the aqueous medium with which it is in contact. Thus the amount of active antiseptic leaving the oil solution is, to a considerable extent, dependent upon the rate at which it is used up in the aqueous medium. As illustrating the influence of varying conditions on the passage of the active chlorine from the oil to an aqueous 1 Journ. Amer. Med. Assoc, July 7, 1917. 2 Brit. Med. Journ., Aug. 25, 1917. AXTISEPTICS OF THE CHLORINE GROUP 35 medium, the following experiments may be cited. A 6.5 per cent solution of dichloramine-T, prepared as described later, was mixed (a) with an equal volume of saline, (b) with muscle extract, (c) with blood serum. The oil was then separated completely after 3 hours. The active chlorine stated in terms of dichloramine-T in the saline solution was 1 : 6000, in the filtered muscle extract 1 : 300, and in serum which was mostly coagulated 1 : in. It is thus seen that the dichloramine-T dissolved in the oil is in a readily available form and direct bacteriological tests following its action on bacterial suspensions in blood and muscle extract (p. 186) on the organisms of the nasopharynx (p. 102) and on war w r ounds have clearly shown its germicidal action to be great. Dichloramine-T is employed for surgical purposes in a 6.5 to 10 per cent solution in treated eucalyptol and paraffin oil, or in a 20 per cent solution in specially highly chlorinated eucalyptol. The preparation of the substance and solvents is described on succeeding pages. It is best applied by means of an oil spray, an ordinary hard rubber or all glass " atomizer " being best, as metal is slowly attacked. It may also be poured into wound cavities and it can easily be introduced into sinuses by means of a cotton swab dipped in the solution. The amount of the solution needed for each treatment is extremely small, 1 to 2 cc. being sufficient for most moderate- sized wounds, and it need not be renewed more often than once in 24 hours. A minimum of dressings is required and they do not stick to the granulation tissue. Dichloramine-T, like most other antiseptics of the chlorine group, is an active lymphagogue when placed on fresh wounds. As granulation tissue develops, the lymph discharge decreases and the w r ound becomes comparatively dry. It also possesses in marked degree the property of aiding in the removal of necrotic tissue. The first application of the oil solution causes a smarting sensation, which passes aw r ay in a few minutes. Wounds treated with the oil fill rapidly with granulation 36 A HANDBOOK OF ANTISEPTICS tissue of healthy color which shows no tendency to exuberant growth nor to become sodden. The early reports as to the results of the use of dichloramine-T in oil solution for wound treatment are decidedly encouraging and the method appears to us to be of genuine value and deserving of extended use. 1 Dichloramine-T in oil solution has been tried extensively in the early treatment of injuries, not only of the soft parts but of tendons, bones, and joints. At the primary dressing of these wounds, after the excision of dead tissue and obvious foci of infection, the wound is liberally flooded with the oil and then closed without drainage. In Lee's experi- ence primary union follows in at least 75 per cent of these sutured wounds if treated within three hours of their in- fliction. If signs of infection should appear, one or more stitches are removed and oil is introduced by means of a grooved director to the focus of infection once in 24 hours. With wounds treated after a longer interval the question of their closure becomes a matter of surgical judgment. Di- chloramine-T in oil has also been found decidedly useful in the treatment of boils and carbuncles with minimal incision, in osteomyelitis, non-tuberculous empyema and in the control of post-operative wound infection. More recently it has been used by Lee for the treatment of burns, and in these cases it is advisable to cover the burn with one layer of a coarse-meshed gauze previously soaked in paraffin wax. In this way the exudate easily comes through the open mesh and the dressings do not stick. It is an advantage if such wounds can be exposed to the air. The absence of suppuration and freedom of drainage in these cases is significant. Preparation of Dichloramine-T . — The following details are based on the method employed by Chattaway : Bleach- ing powder (350 to 400 gms.) of good quality (25 per cent 1 At the time of writing more than ten thousand cases have been treated with dichloramine-T. ANTISEPTICS OF THE CHLORINE GROUP 37 or more "available chlorine ") is shaken with two liters of water in a shaker for an hour and then the mixture allowed to settle. The supernatant fluid is siphoned off and the remainder filtered. Powdered toluene-p-sulphonamide (75 gms.) is then added to the whole of the hypochlorite solution and shaken till dissolved. The solution is filtered if necessary, placed in a large separating funnel, and acidified with acetic acid (100 cc.) added in portions. About 100 cc. of chloro- form are then added to extract the dichloramine precipitated by the acid, and the whole well shaken. The chloroform layer is tapped off, dried over calcium chloride, filtered and allowed to evaporate. The residue is powdered and dried in vacuo. It is sufficiently pure for most purposes without recrystallization. An alternate method of preparation is as follows : Toluene- p-sulphonamide (50 gms.), water (500 cc), crystallized sodium acetate (100 gms.), and chloroform (100 cc.) are placed in a flask which is immersed in cold water and the con- tents treated with chlorine gas to saturation. If necessary, more chloroform is added to dissolve the dichloramine com- pletely. The chloroform is separated, dried, filtered, and evaporated as above described. The yield is practically theoretical. The product may be tested by titration with thiosulphate solution (p. 42). Preparation of Chlorinated Eucalyptol for Use as a Solvent for Dichloramine-T. — Eucalyptol (U.S. P. or Brit. Pharm.), and not eucalyptus oil, must be used. Five hundred cc. are treated with 15 gms. potassium or sodium chlorate and 50 cc. concentrated hydrochloric acid for 12 hours or longer. It is then well washed in a separating funnel, first with water and then with a solution of sodium carbonate, to remove all traces of hydrochloric acid. After tapping off the aqueous layer, 15 gms. dry sodium carbonate are added to the oil and the whole allowed to stand for 24 hours. It is then filtered, further dried with a little solid calcium chloride, 38 A HANDBOOK OF ANTISEPTICS which must be allowed to act for a considerable time, and is then ready for use. The degree of chlorination thus achieved does not affect the appearance or limpidity of the eucalyptol. A more complete chlorination converts the eucalyptol into a much heavier oil, which has been used in preparing more concen- trated solutions of dichloramine-T for use in the treatment of wounds. Its preparation is as follows : Dry chlorine is passed into eucalyptol without cooling, allowing the mixture to heat up only moderately. The hydrochloric acid formed is permitted to escape freely. After prolonged chlorination the specific gravity of the mixture rises to about 1.2 and very little more chlorine is taken up at room temperatures. The oil is washed with sodium carbonate solution, then shaken with a concentrated solution of calcium chloride, and finally dried over solid calcium chloride and filtered. Solutions of dichloramine-T in this oil are stable for many weeks. Preparation of Chlorinated Paraffin Oil for Use in Diluting Eucalyptol Solutions of Dichloramine : T. — Paraffin oils derived from different sources vary greatly in their capacity for taking up chlorine. By intensive chlo- rination it is possible to break up all grades of the oil. The following treatment has been found serviceable for a moderate chlorination sufficing to protect the dichloramine-T from rapid decomposition : To 500 cc. of pharmacopceal liquid par- affin add 15 gms. potassium or sodium chlorate and 50 cc. concentrated hydrochloric acid. Expose the mixture to light, preferably sunlight, for several hours. Transfer to a separating funnel and wash successively with water, a solu- tion of sodium carbonate, and again water. The water is tapped off as completely as possible and a small quantity of solid calcium chloride added to the opalescent oiL After standing for several hours about 5 gms. of animal charcoal are added and the mixture well shaken. On subsequent AXTISEPTICS OF THE CHLORINE GROUP 39 filtering through paper a clear, sometimes slightly yellowish oil is obtained, which is ready for use. Preparation or the Dichloramine-T Solution. — The constituents of the solution are all stable and may be pre- served indefinitely, especially in colored bottles, but the solution itself is sensitive to light and should be used only for a few days. A definite crystalline deposit as distinct from a faint opalescence is evidence of decomposition and such solutions should be rejected. Careful protection of the oil from light will do much to prevent decomposition. In preparing the solution it is necessary to dissolve the di- chloramine-T in the chlorinated eucalyptol first and to add the paraffin oil last. To hasten the rate of solution, the dichloramine-T and eucalyptol may be w r armed for a few minutes to 6o°. The mixture as used by Sweet on war * wounds was made by dissolving 10 grams dichloramine-T in 75 cc. of chlorinated eucalyptol and then adding 75 cc. of chlorinated paraffin oil. This mixture contains about 6.5 per cent dichloramine-T. The proportion of paraffin oil may be reduced one half if a stronger (10 per cent) solution is required. Much stronger solutions than this have been used by Lee. The more highly chlorinated eucalyptol referred to on p. 38 was used as sole solvent, no paraffin oil being added. As strong as 20 per cent dichloramine-T solutions are used in this way with excellent results for primary dressings with- out noticeable irritation. The Chemical Determination of the Concentration of Chlorine Antiseptics It is very desirable that the concentration of solutions of the various antiseptics of the chlorine group should be sub- jected to analytical control. This is particularly true in the case of the hypochlorites and other unstable products. 40 A HANDBOOK OF ANTISEPTICS The methods for doing this are extremely simple and the slight extra trouble is well repaid by the resulting certainty as to the strength of the antiseptic solutions. In principle, the same method is used for all the substances mentioned in this chapter. A known quantity of the solu- tion or substance is taken and an excess of potassium or sodium iodide and of acetic acid is added. Iodine is at once liberated in amount equivalent to the active chlorin^ of the antiseptic, and this iodine is measured by determin- ing the amount of a decinormal solution of sodiun thiosulphate necessary to react completely w T ith the iodine. The following solutions are required : Decinormal Sodium Thiosulphate Solution. — This is prepared with sufficient accuracy by dissolving 24.8 grams of the pure crystals in water and diluting to 1000 cc. The solution is moderately stable, especially if protected from light, and will serve for two or three months. Precipitation of sulphur in the bottle indicates decomposition and when this occurs a fresh lot should be made. Each cubic centimeter of this solution is equivalent to : 0.0127 gram Iodine 0.00354 gram Chlorine 0.00262 gram Hypochlorous Acid 0.00372 gram Sodium Hypochlorite 0.01407 gram Chloramine-T 0.006 gram Dichloramine-T 0.00675 gram Halazone Potassium Iodide Solution. — A 10 per cent solution in water is used. Its exact strength is immaterial and sodium iodide may be employed equally well. Acetic Acid. — A 10 per cent solution of the pure acid. Starch Paste. — Prepared by boiling about 0,1 gram starch with 100 cc. of water, cooling, and allowing to sediment. The clear solution is poured off and used as an indicator for AXTISEPTICS OF THE CHLORINE GROUP 41 iodine. It must be made fgresh occasionally as moulds are apt to grow in the solution. Determination of the " Available Chlorine " in Bleaching Powder. — A fair average sample from bulk is taken and of this 10 grams is exactly weighed out into a mortar. The powder is triturated in the mortar with suc- cessive small quantities of water and completely transferred to a liter flask which is filled to the mark with water. The whole is well shaken and allowed to stand for an hour or two. Ten cc. of the supernatant liquid is measured with a pipette and transferred to a small flask. Five cc. each of the iodide and acetic acid solutions are then added. The iodine which is now liberated is determined by adding from a burette the decinormal sodium thiosulphate solution until almost all the iodine has disappeared. A few drops of the starch paste are then added and the addition of the thiosulphate continued until the blue color just disappears. The 10 cc. of bleaching powder solution is equivalent to o.i gram of the solid substance and as each cc. of thiosulphate = 0.00354 gram available chlorine, the percentage of available chlorine in the bleaching powder is found by multiplying the number of cubic centimeters of thiosulphate used by 0.00354 X 1000 = 3.54. Thus if a particular sample of bleaching powder treated as described required 9.4 cc. of N/10 sodium thiosulphate, the available chlorine would be 33.3 per cent. Titration oe Sodium Hypochlorite Solutions or Eusol. — Ten cc. of the solution is treated with 5 cc. each of the iodide and acetic acid solutions and then titrated with sodium thiosulphate, as described above. Each cubic centimeter of thiosulphate used represents 0.00372 gm. sodium hypochlorite or 0.00262 gm. hypochlorous acid. The quantity of hypochlorite or hypochlorous acid in 100 cc. will be given by multiplying the number of cubic centimeters of thiosulphate used by 0.0372 or 0.0262 respectively. 42 A HANDBOOK OF ANTISEPTICS Estimation or Chloeamine-T and Dichloramine-T. — These substances are examined for their content of active chlorine, as described for the hypochlorites with the slight difference that in order to facilitate the reaction of the chloramines with the iodide, it is well to add a little chloro- form (5-10 cc.) or carbon tetrachloride before titrating. The method of calculation follows from the fact that each cubic centimeter of thiosulphate used in the titration is equivalent to 0.01407 gm. crystallized chloramine-T or 0.006 gm. dichlora- mine-T. It will be noticed that one molecule of chloramine-T liberates two atoms of iodine and dichloramine-T liberates four atoms of iodine. The reason for this is that each atom of chlorine in the antiseptic is equivalent to a molecule of hypochlorous acid, each of which liberates two atoms of iodine from an acidified iodide solution. HCIO + 2 HI = I 2 + HC1 + H 2 CHAPTER III THE PHENOLIC GROUP OF ANTISEPTICS Many of the common antiseptics belong to this group and have been long employed for routine disinfection both for surgical and hygienic purposes. It cannot be said that any new properties of value have been observed in this group as the result of their employment in war surgery. Phenol has found steady advocates ever since Lister adopted it as an aid' in obtaining his brilliant successes in antiseptic surgery. Early in the present war the mistaken notion seemed to prevail that a decision as to the general utility of antiseptics could be attained by treating infected wounds receiving a minimum of preliminary surgical treatment with pure phenol and observing whether infection supervened. In the light of present experience it seems hardly necessary to say that wound sterilization cannot often be effected by such methods. The use of destructive coagulants such as phenol in high concentration is undoubtedly undesirable and apt to lead to conditions favorable to the growth of anaerobes. On the other hand, phenol at a concentration of 2.5 to 5 per cent is still in common use and a mixture of equal parts of one of these solutions with hydrogen peroxide is viewed with favor by many. Phenol and camphor when rubbed together in equal proportions give a liquid which is said to give useful results in infected cases of long standing. 1 1 Feldman and Walton, Lancet, Dec. 3, 1916. 43 44 A HANDBOOK OF ANTISEPTICS The stability of phenol solutions and their clean odor are attractive qualities but, while phenol is undoubtedly a good disinfectant for many purposes, it does not seem to give as good results in the treatment of badly infected wouncs as many other antiseptics. The use of alcohol and glycerine l as solvents for phenol has not much to recommend it, viewed simply as regards disinfection, since both of these solvents depress its germicidal activity. Phenol dissolved in vege- table oils is almost devoid of germicidal activity as but little of the antiseptic leaves the fatty solvent. Much weaker solutions dissolved in mineral oil, in which it is sparingly soluble, have been found by Lewis and Richards to be more effective. The fairly strong inhibitory effect of phenol upon phagocytosis has already been referred to, although it may be doubted whether this is as important a matter as it is sometimes regarded. Anthrax spores are remarkably resistant to phenol solu- tions and may be viable after four days' immersion in a five per cent solution. When acting in the presence of blood serum, defibrinated blood, or pus, for reasonable lengths of time, e.g. 2 hours, concentrations below 2 per cent are relatively ineffective against pyogenic cocci. The rather slow but progressive disinfection of a heavily infected mix- ture of blood serum and muscle extract, to which one third volume of 2 per cent phenol had been added, is well illustrated on p. 86. Sterilization was incomplete after 22 hours although less than 1 per cent of the organism sur- vived. The way in which phenol exerts its bactericidal action is not understood. Cooper 2 has produced some evidence tending to show that chemical reaction between the bacterial proteins and phenol is not a sine qua non of phenol disinfection and it is suggested that the absorption of phenols by bacteria is merely the initial stage in the pro- 1 Goodrich, Brit. Med. Journ., May 19, 1917. 2 Biochem. Journal, 7, p. 175, 1913- THE PHEXOLIC GROUP OF ANTISEPTICS 45 cess of disinfection and that the germicidal action which follows is due to a de-emulsifying action upon the colloidal suspension of some constituent protein essential for the stability of the organism. With phenol and its derivatives there appears to be an intimate relation between their germicidal powers and their protein precipitating capacity. Phenol is commonly used as a standard for the measure- ment of the efficiency of disinfectants by the Walker-Rideal method (page 79). Various halogen derivatives of phenol have been proposed as antiseptics and while many of them are highly germicidal against bacteria suspended in water, they are not particularly active in the presence of blood serum or other protein material and hence have little to recommend them for wound treatment. A great many of these compounds have been carefully studied by Bechold and Ehrlich. 1 Cresols, or methylphenols, occur in three isomeric modi- fications. They are more actively germicidal than phenol itself and it is stated that a 1 per cent solution of commer- cial cresol in water is as active as 3 per cent phenol. The commercial mixture goes by the name of " tricresol " and is often employed for sterilization of the hands and of instruments. Cresol paste, made with lanoline and white wax, was recommended by Sir. W. Watson Cheyne 2 and his colleagues for the early treatment of infected war wounds, but the results obtained early in the present war were gener- ally regarded as unfavorable and its use has been discontinued, Lysol is prepared by treating the fraction of tar-oils chiefly composed of cresols with fat and then saponifying with alcoholic soda. Its use for general disinfecting pur- poses is well known, but its employment as a dressing for wounds is limited, except in veterinary practice. It gives 1 Zeitschr. F. physiol. chem., 47, p. 173, 1906. 2 Journ. Royal Naval Medical Service, April, 1915. Lancet, Nov. 21, 1014, Feb. 27, 1915. 46 A HANDBOOK OF ANTISEPTICS a soapy, frothing solution when mixed with water. Creolin is a similar preparation. The interesting suggestion has been made by Miss Mary Davies 1 that wound infections might be limited to some extent by the use of antiseptic substances for impregnating the clothing of soldiers. After studying the effect of various substances she concludes by recommending the use of a 5 per cent solution of "pyxol," a cresol and soft soap preparation analogous to lysol. It appears that some bactericidal power is retained by cloth so treated after a month's exposure to sun and rain and possibly even for a longer period. The practical results of these suggestions will be awaited with interest. Thymol, or propylmethylphenol, has been recommended as an antiseptic for surgical purposes but has been prac- tically discarded, although dentists find its low solubility an advantage in some conditions requiring antiseptic treatment. Di-iododithymol is known under the name of aristol. It is used to some extent as a substitute for iodoform but is unsuited for general use in the treatment of war wounds. Salicylic Acid, or orthohydroxybenzoic acid, has received much attention as an antiseptic for the treatment of war wounds. In particular, a powdered mixture of salicylic acid and boric acid, introduced by Sir W. Watson Cheyne 2 under the name of borsal, was given an ex- tended trial. A mixture of salicylic acid and borax had been tried, with ineffective results, as a wound dressing by the Japanese in the Russo-Japanese war. Borsal does not seem to have achieved much greater success in most surgeons' hands, and after fairly extensive trials in France, either alone or in conjunction with cresol past, its use was abandoned. 3 It appeared that borsal did not effectively 1 Lancet, Sept. 30, 1916, p. 603. 2 Brit. Med. Journ., May 22, 1915, p. 912. 3 Ibid., June 5, 1915, p. 984. THE PHENOLIC GROUP OF ANTISEPTICS 47 check sepsis save in superficial wounds and its use, in the opinion of many, was not unattended by danger. It seems that the coagulating action of salicylic acid on blood and wound exudates impedes free drainage and under these conditions, when sterilization has not been complete, the possibilities for the growth of anaerobes such as the gas bacillus and bacillus of malignant oedema, are considerable. The disinfecting action of salicylic acid alone is not great in the presence of wound exudates and is, moreover, limited by its low solubility, i : 500 in cold water. Addition of borax causes a much larger quantity of salicylic acid to dissolve owing to the formation of a double salt, sodium borosalicy- ]ate, which is freely soluble. Solutions of this salt are used to a small extent as antiseptic lotions. Alcoholic solutions of salicylic acid have been added in small quantity to the last funnel full of saline used for irrigating septic wounds. 1 In this way the salicylic acid is precipitated by the water and may be evenly distributed over the surface of the wound. This method has been found to be preferable to dusting the dry powder on the wound. A large number of halogen derivatives of salicylic acid have been examined without revealing any particularly valuable qualities, although occasionally their germicidal propsrties are found to be markedly greater than those of salicylic acid. /3-Naphthol and Bromonaphthols. — The naphthols have antiseptic properties similar to those of the simpler phenols but have not been used extensively as wound antisep- tics, although /2-naphthol finds some employment as an intes- tinal disinfectant and is regarded as valuable in ointments for the treatment of skin diseases of parasitic origin. Recently /?-naphthol has been used as an antiseptic addition to the paraffin wax mixtures used in the treatment of burns. The composition of "ambrine," one of the best known of these 1 L. Garret Anderson and Helen Chambers, Lancet, June 3, 1916. 43 • A HANDBOOK OF ANTISEPTICS mixtures, has not been disclosed by its proprietary owners but a product made according to the following formula, due to A. J. Hull, 1 is stated to give equally good or better results: /3-naphthol 0.25 per cent, eucalyptus oil 2 per cent, olive oil 5 per cent, hard paraffin 25 per cent, and soft paraffin 67.75 P er cent. The mixture may be applied with a broad camel hair brush or sprayed on at a temperature of about 50 C. An illustration of the form of spray used for this purpose in the naval service will be found in the British Med- ical Journal, August 28, 1917. Resorcinol, 0.25-1 per cent, may be used in place of the /3-naphthol. The preparation of the mixture is as follows .: Melt the hard paraffin, and add the soft paraffin and olive oil. Add the resorcinol dissolved in half its weight of absolute alcohol and lastly add the eucalyptus oil when the w r ax has cooled to about 55°C. Becholdt 2 has carried out experiments with a series of bromine derivatives of /?-naphthol and finds that several of them have germicidal properties of a high order when tested against pyogenic cocci in water suspension. The tribrcm- /3-naphthol in particular was found to be especially active, killing staphylococci at a dilution of 1 : 250,000. Our own experiments have, however, indicated a materially lower germicidal value. This substance has been made commer- cially and endorsed as an efficient antiseptic agent for the treatment of infected wounds. It is, however, not suitable for military surgery as it is very sparingly soluble and is not significantly more active in the presence of blood serum than ordinary /?-naphthol. When dissolved in alcohol and tested against staphylococci suspended in serum, the lethal concentration is reduced to 1 : 800 or less. Picric A cid (trinitrophenol) . — Picric acid is made use of more for the treatment of burns than for ordinary infected 1 Brit. Med. Journ., Jan. 13, igi7. 2 Zeitschr. f. Hyg. u. Infekt. Krankh., 64, p. 113, 1909. Zeitschr. f. Angew Chem., 22, p. 2033, 1909. THE PHENOLIC GROUP OF ANTISEPTICS 49 wounds. The acid is soluble at room temperature in about ninety parts of water and dissolves much more readily in alcohol or ether. A i per cent or saturated aqueous solu- tion is generally employed for surgical purposes. As a first dressing for burns it has proved of the greatest value and does much to relieve pain and reduce the risk of sub- sequent infection. It is employed either in solution or as impregnated gauze or wool. The use of picric acid in the treatment of extensive wounds is probably to be deprecated, not only because other more suitable antiseptics are available but also because the sub- stance is decidedly toxic. The germicidal properties of picric acid tested against staphylococci or B. colt in aqueous media are moderately high ; using the Walker-Rideal method of testing, it is found to have a " phenol coefficient " variously estimated between 4 and 6. 1 But picric acid is an active protein precipitant and it is unlikely to be capable of exert- ing very much germicidal action in the presence of serum or wound exudates. Direct experiments in this point appear to be lacking. The coagulating and hardening effect of picric acid due to the power of precipitating proteins is prob- ably responsible in part for its successful use in the treat- ment of burns. 1 H. L. Tidy, Lancet, Sept. 11, 1915. CHAPTER IV SALTS OF THE HEAVY METALS AS ANTISEPTICS The metallic salts which are used because of their anti- septic properties are mainly those of silver, mercury, bismuth, and zinc. With one exception, namely, Rutherford Morison's bismuth paste, no very extended use of these substances has been made in the present war. In the following chapter, their mode of action will be referred to first of all, then their germicidal effects and, lastly, some of their applications. The germicidal activity of many of these metallic salts, when acting upon bacteria suspended in pure water, is ex- traordinarily high. But this powerful action is enormously reduced as soon as the bacteria are placed in other media than pure water. Most of the soluble salts of these metals, with the exception of some colloidal preparations, suffer from the disadvantage that they are precipi table by pro- teins or some of the constituents of wound exudates such as phosphates, so that their high initial antiseptic potency is soon reduced. It also follows from this fact that the metallic salts find their most useful applications under conditions which do not lead to their rapid precipitation, and indeed under such circumstances they are among the most valuable disinfectants. There is a good deal of evidence pointing to the belief that the metallic ions present in aqueous solutions owing to electrolytic dissociation are the chief disinfecting agents * and 1 Dreser Arch. f. Exper. Path. u. Pharm., 32, p. 456, 1893. 50 SALTS OF THE HEAVY METALS 51 that the undissociated salts as such are of minor importance. Thus Kronig and Paul found that the disinfecting action of equimolecular quantities of mercuric chloride, bromide, and cyanide was in proportion to their ionic dissociation in solu- tion. Miss Chick has shown that the laws found to govern other examples of disinfection are only applicable to the action of mercuric chloride if the concentration of Hg" ions is used as the basis of calculation rather than the total concentration of the salt. The metallic ions are re- sponsible for the ordinary chemical reactions of metallic salts in aqueous solution and those metallic compounds which do not yield the ordinary chemical reactions for the metals, such as many protein and other colloidal prepara- tions of the metals, possess inferior disinfecting properties. An interesting example of the importance of the metallic ions in disinfection is shown by the following observation : Two silver salts were compared as regards their action on staphylococci in water and blood serum. One of these salts, silver fluoride, which undergoes dissociation with formation of silver ions, killed in two hours staphylo- cocci in water at a concentration of less than i : 10,000,000. The other salt was the double cyanide of silver and sodium which, on solution in water, gives few if any metallic ions but remains in solution as a complex aggregate. When tested against staphylococci in water, the lethal concentra- tion was only about 1 : 5000. Thus it will be seen that silver fluoride solution containing metallic ions is, under the con- ditions of the experiment, two thousand times as active as silver sodium cyanide which gives few or no metallic ions. When the same salts were tested against staphylococci suspended in blood serum, the lethal concentrations were much more closely approximated, being about 1 : 7000 and 1 : 3000 respectively. It appears probable that the metallic ions of the salts exert their disinfecting action by reacting chemically with the protein or other constituent of the 52 A HANDBOOK OF ANTISEPTICS bacterial protoplasm, and indeed in some cases this phenom- enon has been actually observed. Salts of the heavy metals, even when present in a nutrient medium in extremely small amount, are capable of exerting an inhibitory influence upon the growth of bacteria, so that in all estimations of the germi- cidal action of these salts it is essential to avoid carrying over any of the antiseptic into subcultures. In addition to the inhibitory action upon the growth of bacteria exer- cised by traces of metallic salts, Miss Chick 1 has noted another phenomenon exhibited by this class of disinfectant. " If bacteria are subjected to the action of i : iooo, i : 10,000 or even weaker solutions of mercuric chloride, there is an in- terval during which some at least of them may be resusci- tated by the timely administration of an antidote (in this case a sulphide solution), but if this antidotal treatment is not employed, no amount of subsequent dilution beyond the limits when inhibition occurs, can prevent the death of the organism. It would seem that the mercuric salt has been already absorbed by the bacteria and possibly formed some combination with its substance, not however to a - sufficient extent to prevent recovery if a large excess of the sulphide solution be employed. - " In one case it was observed that in a 24-hour culture of B. paratyphosiis some individuals at least were able to manifest vitality after contact with 5 per cent mercuric chloride solution for four minutes, pro- vided ammonium sulphide was promptly applied as antidote. There is an extensive literature dealing with the germi- cidal action of the metallic salts, but most of the results are not comparable with one another owing to variations in the technique employed in making the tests. Even slight variations in the composition of the medium in which these substances act produce enormous variations in the results of the experiments. Thus we have found the apparent activity of mercuric chloride against staphylococci in a 1 Journ. of Hygiene, 8, p. 92, 1908. SALTS OF THE HEAVY METALS 53 hard tap water to be only one fifth of that shown when distilled water was employed, while with silver salts the results are even more liable to variation. The following extracts from published experiments are given simply as a rough guide to antiseptic potency, but for details, the original papers must be consulted. The use of mercuric chloride as a disinfectant practically dates from Robert Koch's l experiments published in 1881. Somewhat later, Geppert showed that Koch had overes- timated the antiseptic value of the salt through error in tech- nique involving the carrying over of mercury salts into subcultures. Geppert's experiments were adversely criti- cized by von Behring 2 but have been essentially substan- tiated by subsequent workers. The action of mercuric chloride upon spores, especially those of B. anthracis, has -been most carefully studied by Kronig and Paul, 3 Madson and Nyman, 4 and Miss Chick. 5 A chart from Miss Chick's paper, in which the figures are taken from Kronig and Paul's experiments, gives a good idea of the general character of the results, though many more details will be found in the original papers. As exemplifying the action of mercuric chloride on a vege- tative form, B. paratyphosus, the following chart is repro- duced from Miss Chick's paper. The action of mercuric chloride upon bacteria is not nearly so rapid as is commonly thought to be the case. Miss Chick found that B. paratyphosus could withstand the action of five per cent bichloride for four minutes, and staphylococcus aureus, for fifteen minutes, if at once treated with a sulphide antidote. But these experiments relate only to the action of the antiseptic on suspensions of organisms in an aqueous medium. 1 Ueber Desinfection, Mittheil. Kaiserl. Gesendheitsamt, Vol. I. 2 Zeitschr. f. Hygiene, g, p. 396, 1890. 3 Ibid., 25, p. 1, 1897. 4 Ibid., 57, p. 388, 1907. 5 Journ. of Hygiene, 8, p. 92, 1908. 54 A HANDBOOK OF ANTISEPTICS When working in a blood serum medium, its activity is much reduced, while in the presence of whole blood, pus, or muscle CHART I 4000 c 3500 3000 2500 ° 1 s 1500 No. of bacteria o o o * \ i I rv >■— -0-. -X ' -( h r > ■€ 30 4.0 50 Time in minutes Illustrating the results of Kronig and Paul's experiment. Continuous curve, disinfection of anthrax spores with 2.1 per 1000 HgCb. Dotted curve, disinfection of anthrax spores with 1.1 per 1000 HgCla. extract much higher concentrations are required for effec- tive action. A single new experiment may be quoted to illustrate this (see p. 87 ). Mercuric chloride (1 cc. 1 : 1000) was added to blood serum (1 cc.) and 50 per cent muscle extract (1 cc.) previously heavily inoculated with SALTS OF THE HEAVY METALS CHART II 55 1-0 0-9 08 7 o o w .2 o 1 u si ft 50 100 150 200 Time in minutes 250 Times taken for disinfection of B. paratyphosus with varying concentrations of mercuric chloride, H2S being used as antidote. 56 A HANDBOOK OF ANTISEPTICS staphylococcus aureus. The final concentration of mer- curic chloride was therefore i : 3000. The number of sur- viving organisms was estimated at varying intervals of time. 1 The experiment was conducted at 32 C. Although at the end of five minutes almost 90 per cent of the organisms were killed, the mixture was not sterilized completely at the end of three hours. Among other salts of mercury that are employed for surgical purposes, are mercury potassium iodide, cyanide, oxycyanide and double zinc cyanide. "Mercuric biniodide," i.e. mer- curic potassium iodide, resembles mercuric chloride closely in germicidal properties but is regarded as less irritating. The other salts mentioned are less active under most conditions. The germicidal action of most silver salts closely resembles that of the corresponding mercuric compounds. Silver chloride is insoluble and hence ineffective, but silver nitrate appears to resemble mercuric chloride fairly closely. Its action on B. paratyphosus has been quantitatively studied by Miss Chick with results that resemble the curve given on p. 55. Silver cyanides, colloidal silver, and various organic compounds which yield few silver ions on solution in water all appear to have inferior germicidal properties, although some of them find useful application in civil prac- tice. For their preparation, properties, and uses, reference must be made to textbooks of pharmacology. Experiments on the action of silver nitrate solution and argyrol on considerable quantities of staphylococci and other organisms suspended in equal parts of blood serum and muscle extract are recorded on p. 87. Silver nitrate (1 per cent) added so that the final concentration was 0.33 per cent killed about 95 per cent of the organisms in six hours but failed to sterilize completely in 24 hours. Argyrol with a final concentration of 5 per cent did sterilize 1 Potassium sulphide was added as " antidote " to prevent the carrying over of active mercury salts to the subcultures. SALTS OF THE HEAVY METALS 57 in 24 hours but many organisms were still present at the end of six hours. The salts of bismuth do not appear to have been examined very carefully as regards their bactericidal action, although their action in checking undesirable fermentations in beer worts was discovered long ago. Most normal bismuth salts are more or less rapidly decomposed by water with formation of insoluble basic salts, and some of these have found effec- tive use as mild antiseptics capable of slow but prolonged action. Zinc salts have long been known to have antiseptic prop- erties and the chloride especially has been used by Lister, Kocher, and others. Its germicidal action is far inferior to that of most mercury or silver salts. A 5 per cent solution is ineffective against anthrax spores but a 2.5 per cent solution is reported effective against most vegetative -forms in a reasonably short time, when acting in an aqueous medium. Its activity is however much influenced by the medium in which it acts, since it is very readily precipitated by proteins, phosphates, etc. An experiment in which 3 per cent zinc chloride was added to a mixture of equal parts of blood serum and muscle extract inoculated with staphylococci and other organisms, so that the final con- centration of zinc chloride was 1 per cent, showed that about one sixth of the organisms survived at the end of an hour and a half and that complete sterilization was not quite accomplished after 24 hours (p. 88). Uses or Metallic Salts in War Surgery. — The prac- tical uses of the metallic disinfectants in the treatment of infected war wounds has not been extensive with the ex- ception of a paste containing bismuth subnitrate which will be referred to later. Mercury salts alone have almost no advocates, although a certain amount of gauze impregnated with Lister's double cyanide of mercury and zinc is still em- ployed, with the object of preventing re-infection of the wound 58 A HANDBOOK OF ANTISEPTICS and in the hope of controlling further progress of the existing infection. A preparation of mercuric chloride and malachite green introduced by Fildes, Rajchman, and Cheatle has, however, given useful results, especially in chronic suppurat- ing wounds. An account of this preparation will be found in the section on dyes (p. 61). Silver nitrate at i : iooo was tried for some time, but the darkening of the treated wounds on exposure to light was a drawback and the results appeared to be only moderately good, so that at the present time it is scarcely used at all. Zinc salts have a marked caustic and coagulant action and, as is well known, are frequently employed on account of these effects on torpid ulcerations, fistulous tracts, etc. Lister long ago made use of zinc chloride solution in many infected conditions and noted the fact that the precipitation of zinc compounds in the coagulated surface of the wound was generally sufficient to prevent recurrence of sepsis. Strong solutions of zinc chloride, up to as high as 10 per cent, were believed useful, particularly by Belgian surgeons, when used as wet dressings on wounds which had been freely incised subsequent to the development of gas gangrene infec- tion. This treatment, which is a drastic one, has been largely supplanted by irrigation with some form of hypochlorite solution. By far the most useful metallic salt antiseptic so far em- ployed in the present war is the bismuth paste introduced by Rutherford Morison. 1 This is made by mixing bismuth sub nitrate (i part) and iodoform (2 parts) with sufficient liquid paraffin oil to make a thick paste of such consistence that it may be readily spread in a thin layer with the help of a spatula or spoon. This mixture, which is com- monly known as " B. I. P.," was first of all recommended for the treatment of infected war wounds in which suppura- tion was already established. But it is now used to a con- 1 Lancet, Aug. 12, 1916, p. 268. SALTS OF THE HEAVY METALS 59 siderable extent for the treatment of fresh wounds, partly owing to its ease of application and the fact that frequent redressing is usually unnecessary, although adequate pro- vision must be made for free drainage. When casualties are unusually numerous and speed of treatment becomes of great -practical importance, the paste is said to be particu- larly useful. The method of using the paste for suppurating wounds is essentially as follows : After appropriate surgical treat- ment, the wound cavity and surrounding skin is carefully mopped with alcohol. The wound is then rilled with the paste, and dressed with gauze which is covered with an absorbent pad, which in turn is held in position with stick- ing plaster and a bandage. This dressing requires no change for days or weeks if the patient is free from pain and con- stitutional disturbances. Should, however, discharge come through the dressing, the stained part must be soaked in alcohol and a fresh gauze dressing, wet with alcohol, applied as a further covering. In order to redress the wound, wool soaked in alcohol is used to wipe away the sticky, dirty looking discharge. The wound and a small area of skin is again plastered with paste and this in turn covered with gauze dressing, pad, and bandage, as before. It is claimed that the results following the use of this paste are notably good in fractures of the long bones, and the ease of dressing as well as its infrequency and the absence of pain are valuable features. Precise bacteriological analysis, of the effects of the paste upon infected wounds has not yet been supplied, so that final judgment as to the bactericidal value of the mixture must be delayed. It must not be forgotten that the con- stituents of the mixture are not innocuous and that iodoform poisoning, particularly when the drug is placed in closed cavities, is not uncommon. However, iodoform poisoning seems to be very rarely encountered with the present mixture. 60 A HANDBOOK OF ANTISEPTICS The paste appears to be very slowly absorbed and may cause disturbance long after apparent healing of the wound. A small sinus may form and iodoform suspended in a brownish fluid may escape, but the exit wound heals promptly. In the majority of cases, however, it is slowly absorbed and by means of X-rays the slow disappearance of B.I. P. in bone cavities, etc., can be readily followed. CHAPTER V DYES AS ANTISEPTICS A large number of dyestuffs possess germicidal properties, although until recently they have been employed for the destruction of blood parasites such as trypanosomes rather than bacteria. Malachite green, used in conjunction with mercuric chloride, was recommended early in 191 5 in a report to the Medical Research Committee, by Fildes, Rajchman, and Cheatle, 1 and has been used fairly extensively, especially in the naval service. A 2 per cent solution of malachite green in 80 per cent pure alcohol is mixed with an equal volume of a 2 per cent solution of mercuric chloride in 80 per cent alcohol. The two solutions are best kept apart until needed for use. The mixture, as Micklethwaite has shown, contains a double compound consisting of one molecule of malachite green and two molecules of mercuric chloride. This compound ap- pears to be readily dissociated in contact with the tissues. The malachite green is reduced by living tissues to the leuco- compound and therefore becomes invisible in a wound but may still maintain its activity. Sloughs and necrosed tis- sue, however, do not reduce the dye. The mixture is gen- erally applied by means of a spray and is surprisingly non- irritating when the concentration of the mercury salt is considered. It has been found particularly useful as a skin- disinfectant and for the treatment of superficial wounds, but it has also proved of value in cases of osteomyelitis, septic 1 Lancet, 1915, ii, p. 165. 61 62 A HANDBOOK OF ANTISEPTICS fractures, and burns. Experiments on the germicidal effect of malachite green when acting on organisms either in a blood medium or blood serum-muscle extract (p. 89) lead us to the conclusion that the value of the dye as a wound antiseptic probably has been much overestimated. Media such as those noted, to which malachite green had been added to a 1 : 1000 concentration, readily underwent putrefaction. These conclusions, however, simply refer to the dye itself and not to its compound with mercuric chloride as employed by Cheatle and his colleagues. Malachite green belongs to the group of triphenylmethane dyes and may be represented by the following formula: (CH 3 ) 2 N< >-C— < >N(CH 3 ) 2 OH It may be prepared by a variety of methods, one of which, due to Doebner, 1 consists in heating dimethylaniline (2 mols.) with zinc chloride and benzotrichloride (1 mol.). The zinc salt obtained by this method is commonly con- verted into the oxalate which is the usual commercial form of malachite green. Browning has shown that some oxalates of dyestuffs, including malachite green and brilliant green, are more harmful to phagocytosis than other salts, so that it might be desirable to employ some soluble salt of malachite green other than the oxalate for antiseptic purposes. Certain other members of the triphenylmethane group of dyes are known to possess definite bactericidal action, as shown by Dreyer, Kriegler, and Walker. 2 Hexamethyl violet, 1 Liebigs Annalen, 217, p. 250. 2 Journ. Path, and Bact., 15, p. 133* I 9 IC - DYES AS ANTISEPTICS 63 also known as " crystal violet," hexaethyl violet, and brilliant green are all credited with marked germicidal properties. The latter dye has been used to some extent by Browning and his surgical colleagues. Our own experiments do not indicate that brilliant green is nearly so powerful a germicide as claimed by Browning (cp. pp. 90, 95), although it doubtless may be of value in the treatment of certain types of wounds. A particularly exuberant growth of bright red granulation tissue is observed to follow its use. Brilliant green or tetraethyldiaminotriphenylcarbinol has a constitu- tion similar to that of malachite green but with ethyl groups replacing the methyl groups of the latter. It is prepared like malachite green by heating diethylaniline with zinc chloride and benzotrichloride. Acriflavine, Trypaflavine, or Flavine. — This sub- stance was first prepared by Benda 1 at Ehrlich's instigation in 191 1 and was found to have a marked therapeutic effect on trypanosome infections. The systematic name for the compound is 3-6 d iamino-10-me thy 1-acridinium chloride and is represented by the following formula : H 2 N \/\ /\/ NH 2 /N\ CH 3 CI The preparation of the compound was protected by patents and the registered trade mark " trypaflavine" was assigned to it. In order to avoid this name Browning and his colleagues used the simple term "flavine/' but as a vegetable yellow dye of the same name has long been known this designation was 1 Ber. deutsch. Chem. Gesell., 45, p. 1787, 191 2, 64 A HANDBOOK OF ANTISEPTICS unfortunate. To avoid these and other important technical difficulties the Medical Research Committee has recom- mended 1 that so far as Britain is concerned the substance should be officially known as "acriflavine" and under this name various firms have obtained licenses to manufacture it. 2 In this way monopoly from the exploitation of the substance under fancy names has been obviated. Acriflavine has been claimed by Browning and his associ- ates 3 to be a most powerful antiseptic and it has found application in the prophylactic treatment of fresh wounds as well as in cases where suppuration has developed. It has also been employed in the disinfection of the nasopharynx of carriers of the meningococcus. Our own experiments on the germicidal action of acriflavine lead us to regard it as distinctly more active under most conditions than either mala- chite green or brilliant green (p. 97), though its rate of disinfection is decidedly slow. Direct observations on the bacterial count of infected wounds treated with 1 : 1000 acriflavine confirm our belief that the germicidal action of the substance has been overestimated. One of the most remarkable properties of acriflavine is chat its germicidal action is apparently enhanced by admix- ture with serum, though greatly diminished by pus. Relative to its bactericidal power, the dye is less detrimental to phago- cytosis than most other antiseptics and it has but little in- jurious action on the tissues, but on the other hand its germicidal action is exerted decidedly more slowly than that of some commoner antiseptics. Its solutions may be boiled and can even be heated to 120 in the autoclave. It is generally used in 1:1000 solution in 0.8 per cent salt solution and may be employed* for swabbing or syringing septic wounds once or twice daily according to the acuteness 1 Brit. Med. Journ., June 9, 1917, p. 769. 2 Acriflavine is, we believe, being manufactured by Messrs. Boot, Island St., Nottingham, and doubtless by other firms. 3 Brit. Med. Journ., Jan. 20, 1917, p. 73. DYES AS ANTISEPTICS 65 of the condition. Gauze soaked in the solution may be placed next the wound and a protective covering put over the whole to hinder evaporation. Several ounces of i : iooo acriflavine solution may safely be left in the tissues or peri- toneal cavity. It may also be injected with a serum syringe into inflammatory areas. The opinion has been expressed that the special uses of acriflavine for particular purposes have still to be defined in relation to other antiseptics and to the operative methods of surgery, to which it can be at best only a valuable aid. The following table, taken from Browning's paper, con- tains the results of bacteriological tests made with the various dyes mentioned. In judging of these results it must be borne in mind that relatively very small numbers of bacteria were employed (o.i cc. of a i : 20,000 dilution in saline of a 24-hour peptone water culture) and that the action of the antiseptics was allowed to continue for 24 to 48 hours before examination. 1 A loopful of the mixture before adding the acriflavine is stated to yield twenty or more colonies. Such a low concentration of organisms in an old wound would indicate approaching surgical sterility, and moreover a survival of any number less than 5 per cent 1 In the original paper by Browning and his colleagues certain antiseptics of the chlorine group, e.g. chloramine-T, eusol, chlorine water, and Dakin's solution, are included and an attempt is made to determine what is termed " anti- septic potency" as expressed by the ratio: Lethal concentration in serum of the substance in question Lethal concentration in serum of chloramine-T These experiments are valueless since the chlorine antiseptics were added first to media, either peptone, water, or serum, which in the dilutions employed promptly decomposed most of them. For such experiments it is essential to add the disinfectant last, as in practical use, and there is little value in continuing experiments with rapid-acting unstable chlorine antiseptics for more than a few hours. The experiments as described give a false impression of the relative po- tency of acriflavine and similar dyes. The original statements by Dakin, Cohen, Kenyon, and Daufresne as to the germicidal action of hypochlorites and chlora- mine-T in water and serum have been repeatedly confirmed by ourselves and many others. 66 A HANDBOOK OF ANTISEPTICS of the organisms in the test would have a good chance of being overlooked. As already stated, our own tests with heavily infected mixtures indicate much feebler germicidal action than that shown in the fcllowdng table. Antiseptic Staphylococcus Aureus Lethal Concentration Bacillus Colt Communis, Lethal C oncentr ation Concentra- tion Which Inhibits Phagocy- tosis In Peptone Water 0.7 % In Serum In Peptone Water 0.7 % In Serum Malachite green (oxalate and sulphate) Brilliant green (sulphate) Crystal violet Acriflavine 1 : 10,000,000 1 : 10,000,000 1 : 4,000,000 1 : 20,000 1 : 40,000 1 : 30,000 1 : 400,000 1 : 200,000 1 : 20,000 1 : 130,000 1 : 8000 1 : 1300 1 : 1000 1 : 35oo 1 : 8000 1 : 100,000 1 : 7000 1 : 2000 1 : 7000 1 : 500 The preparation of acriflavine on a moderately large scale requires a considerable degree of chemical skill. Several methods for the synthesis of acriflavine and related acridine derivatives 1 are known but only one of them need concern us here. The working details must be sought in the original communication. 2 Aniline and formaldehyde unite to give a polymeric sub- stance, anhydrof ormaldehy de-aniline (i ) . When heated with aniline hydrochloride it undergoes a curious rearrangement with the formation of p-diaminophenylme thane (2). This substance is nitrated with nitric and sulphuric acids so as to give a dinitro derivative (3) ; which in turn is reduced with 1 Bucherer's Chemie der Tierfarbstoffe, 19 14, may be consulted for informa- tion concerning the synthesis of acridine derivatives. 2 Benda, Ber. deutsch. Chem. Gesel., 45, p. 1787, 1912; D. R. Patents, Kl. 22b, 230412 and 243085, Casella and Co., 1911-1912. DYES AS ANTISEPTICS 67 tin and hydrochloric acid. On heating the reaction mixture in an autoclave to 135 , 3-6-diamino acridine is obtained (4). In order to obtain the methyl derivative the latter compound is first converted into its di-acetyl compound to protect the amino-groups and then treated in nitrobenzene solution at 1 7 5 with the methyl ester of toluene sulphonic acid. The product is then hydrolyzed, when acriflavine (hydrochlo- ride) = 3-6-diamino-io-methylacridine (5) results: ■H 2 N ►H 2 N ^ jNNk, >NH 2 ->H 2 N 2 2 (3) NH 2 ->H 2 N NH 2 Apparently the germicidal properties of acriflavine are shared by a number of other acridine derivatives and one of these, diaminoacridine, (4) is easier and cheaper to manufac- ture and appears to be equally desirable. It will be known as " proflavine." A detailed publication on this substance is expected shortly. Experiments on its germicidal action are included in Chapter VII. CHAPTER VI MISCELLANEOUS ANTISEPTICS In this section brief reference will be made to a few anti- septics which cannot be included in preceding sections. It will be impossible to treat these substances systematically, and instead brief notes as to the chief characteristics of the substances is all that will be attempted. Hydrogen Peroxide and other Peroxides. — Hydro- gen peroxide is not held in very high repute as a germicide but it has certain other qualities which render it decidedly valuable. When tested against relatively small quantities of staphylococcus aureus or B. pyocyaneus in water, a concentration of 0.03 per cent actual H 2 2 , i.e. a 1 per cent dilution of the pharmacopoeal product, may suffice to sterilize in two hours, while in blood serum about double this concentration may be necessary. But these conditions are entirely artificial and unrelated to what happens when hydrogen peroxide is applied to a septic wound. Blood, pus, and muscle juice contain an enzyme "catalase" which rapidly brings about the decomposition of hydrogen peroxide with liberation of gaseous oxygen. This rapid decomposition with evolution of gas soon decomposes all the peroxide and its disinfecting action comes to a speedy end. The mechanical effect of the disengagement of gas is often a valuable prop- erty and is made use of in loosening sticky secretions, washing away pus, or loosening adherent dressings. For such purpose it is of great value but it is important that its transient germicidal effect should be recognized. 68 MISCELLANEOUS ANTISEPTICS 69 A great variety of other peroxides have been put forward as disinfectants, but most of them are of doubtful practical value. Two of the most active of these substances are the benzoyl hydrogen peroxide and benzoyl acetyl peroxide dis- covered by Baeyer and studied by Freer and Novy. The latter compound has been used to some extent but the un- stable character of both the substance and its solutions has prevented its extensive employment. Dibenzoyl per- oxide has been recommended as an antiseptic but it is a sparingly soluble, practically indifferent compound of no significant germicidal value. Ozone. — This substance has recently been used by Stoker l for the treatment of infected wounds, especially cavities and sinuses in bone injuries. An Andreoli ozonizer is used to produce the ozonized air which is allowed to act for about fifteen minutes or such shorter period as pro- duces a superficial glazing of the wound surfaces. De- tails of ozone concentration and bacteriological controls are not yet available, although the results are stated to be good and the formation of excessive granulation tissue is avoided. Iodine. — Extensive use is made of iodine as a germicide- Its action is powerful and prompt when the conditions are such that the antiseptic has free excess to the microorganisms. Ampoules containing tincture of iodine have been used largely in the present war as a first aid treatment to be applied by the wounded soldier. In general the results as regards the prevention of sepsis have been disappointing, mainly it would appear because the injured man is not apt in most cases to put the iodine where it can reach the focus of infection. The presence of much blood and pro- longed oozing are of course inimical to the exercise of germicidal action. Usually little more is accomplished than a fair cleansing of the adjacent skin. lancet, Oct. 21, p. 712, 1916. 70 A HANDBOOK OF ANTISEPTICS The use of iodine as a skin disinfectant introduced by Stretton x in 1909 is widely practiced and undoubtedly it has great value for this purpose. A 2 . 5 per cent solution is usually strong enough and alcohol is generally employed as the solvent. Seventy per cent alcohol is preferable to stronger spirit and it is important to use pure alcohol as otherwise iodoacetone and other products are apt to be formed, which are very irritating to the eyes of the operator. Light petroleum and heavy mineral oils are also used as solvents for iodine and act well. They have the advantage of being stable and cheap. Dichlorethylene (i.e. acetylene dichloride) has also been proposed but is much more ex- pensive. The use of iodine for the antiseptic treatment of large war wounds is now practiced much less frequently than before, although it was given a thorough trial in the early days of the war. It has been found much too irritating for repeated application and not infrequently objectionable after effects such as severe neuritis have been observed. Its strong coagulating action on proteins is also an objection- able feature. In general it may be said that iodine will be found most useful when the conditions are such that rapid and complete sterilization may be effected by a single appli- cation as in skin disinfection or small surface wounds. An experiment illustrating the rapid effect of 2 per cent iodine solution on staphylococci and other organisms sus- pended in a blood serum-muscle extract medium will be found on p. 86. A 1 per cent solution added to an equal volume of blood heavily infected with streptococci did not kill all the organisms in one hour according to Emery. Against staphylococci suspended in water, about ten million per cubic centimeter, 1 : 100,000 iodine is effective in two hours, while in blood serum 1 : 1000 is required, according to our own observations. 1 Brit. Med. Journ., Aug. 14, 1909, May 22, 1915. MISCELLANEOUS ANTISEPTICS 71 Borates, Perborates, and Boric Acid. — These sub- stances while possessing almost negligible germicidal prop- erties find extensive use when a bland, mildly antiseptic lotion is required. Sodium monoborate is a rather strongly alkaline salt but is said to be non-irritating to wounds; borax, or sodium biborate is less strongly alkaline, while boric acid is feebly acid. All of these substances are used for restraining the growth of putrefactive organisms rather than for disinfection in the true sense. Sodium perborate is prepared by adding hydrogen peroxide to borax solutions and is stable when preserved in the dry state. It is often used as a convenient substitute for hydrogen peroxide. Persulphates. — The use of potassium or sodium persul- phate has been advocated but actually their disinfecting action on pyogenic organisms is feeble. A solution of the sodium -salt has been regarded by some as useful for stimulating the rate of cicatrization of wounds, though this action can hardly be regarded as satisfactorily demonstrated. Acids. — Almost all acids possess some germicidal action and generally speaking their activity is proportional to their " strength." Just as metallic " ions " appear to be the actual disinfecting agent when metallic salts are employed, so in the case of most acids it is the hydrogen ions which seem to be effective. Extensive experiments on this subject have been carried out by Bial x and by Winslow and Lockridge. 2 A few experiments on the action of various acid substances on B. typhosus will be found on p. 114. Occasionally acids have been employed in wound treatment and it has been thought that irrigation with weak lactic acid was useful in inhibiting the growth of the gas bacillus in infected wounds, but the treatment has not met with general favor. 1 Archiv. exper. Path. a. Pharm., 38, p. 1, 1897. Zeitschr. f. physiol. Chem., 40, p. 513, 1902. 2 Journ. of Infectious Diseases, 3, p. 547, 1906. 72 A HANDBOOK OF ANTISEPTICS Alcohol and Ether. — Both of these substances are employed in the treatment of infected wounds but they are used as much for the help they afford in the mechanical clean- ing of dirty wounds as for any direct germicidal action. Most vegetative forms of bacteria may be killed fairly readily by 50 per cent alcohol but alcohol of much lower or higher strength is less effective, while most spores are un- affected by alcohol of any strength. The following table contains the results of experiments by Minervini x to deter- mine the time required to kill various organisms in alcohol of varying concentration. Koch's " thread " method of testing the viability of the organisms was used and the results are at least comparable among themselves. Organism Staphylococcus aureus B. pyocyaneus . . . M. prodigiosus . . B. coli B. subtilis (spores) B. anthracis (spores) Dilution of Alcohol 25% 12-24 hours under i hour 1 hour 24 hours So % 70 % 10 mm. 10 mm. living after 10 min. 10 min. 6 hours 10 min. 10 min. 6 hours i hour i hour living after all living after 8 days all living after 50 days 80% 99% 3 days 12 hours 1 2-24 hours 24 hours It is well known that alcohol or glycerol materially reduces the germicidal efficiency of some antiseptics, particularly those of the phenol class. Kronig and Paul found that phenol dissolved in 98 per cent alcohol was devoid of bac- tericidal action when tested against spores. Cooper 2 has correlated this fact with a diminished protein precipitating action of alcoholic phenol when contrasted with aqueous phenol, but whatever the explanation may be it is clear that alcohol is not a desirable solvent for phenolic disinfectants. 1 Cp., Rideal, Disinfection and Disinfectants, p. 322. 2 Biochem. Journ., 7, p. 175, 1913. MISCELLANEOUS ANTISEPTICS 73 The germicidal properties of ether have recently been ex- amined by Topley, 1 who finds that the vapors possess a slight but definite action. An exposure to ether vapors of one to forty-eight hours was necessary to sterilize agar slants on which pyogenic organisms were growing. Liquid ether was irregular in its action and good contact with the organ- isms was difficult to secure. It is. clear, however, from these and from clinical results that any beneficial effects following the use of ether in the treatment of septic infec- tions is not due to direct disinfection. 2 Formaldehyde. — The physical and chemical properties of formaldehyde, which is obtainable in commerce as a 40 per cent solution known as " formalin, " make it valuable for various forms of disinfection, such as the fumigation of rooms, etc. But so far as the treatment of septic wounds is 'concerned it has not proved particularly successful and is now scarcely used for such purposes. It is employed to some extent for the sterilization of the hands and instru- ments but it is less popular than formerly. A 0.5 per cent solution is generally employed for such purposes. It is not a very rapid acting disinfectant and when tested in the or- dinary way under the standard conditions it has a phenol coefficient of about 0.4, but if the time of action is prolonged its activity may be somewhat greater than that of phenol. Hexamethylenetetramine. — This substance, obtained by the action of ammonia on formaldehyde, has but little di- rect germicidal action. In acid solution it may be decom- posed with liberation of formaldehyde which can thus exert its antiseptic action. A large number of derivatives of hexa- methylenetetramine have been prepared by Jacobs and Heidelberger and many of these are more powerful than the parent substance, but their useful application has still to be defined. Hexamethylenetetramine itself is not suit- 1 Brit. Med. Journ., Feb. 6, 191 5. 2 Cp. Distaso and Bowen, Brit. Med. Journ., Feb. 24, 1917. 74 A HANDBOOK OF ANTISEPTICS able for wound treatment since under these conditions its germicidal action is quite inadequate. Iodoform. — The use of iodoform in combination with bismuth subnitrate and paraffin oil in the mixture known as "B. I. P." has already been referred to on p. 58. The sub- stance was once considered to be a powerful antiseptic but this is no longer believed to be the case. Microorganisms may flourish in contact with iodoform but, on the other hand, it is not disproved that in contact with living tissues iodoform may be slowly decomposed with the formation of products of genuine antiseptic value. Apart from the paste referred to, its use in surgery is diminishing, probably on account of occasional unpleasant poisoning effects that may follow its free use, especially in confined cavities. Many odorless sub- stitutes for iodoform are known under various trade names, but in general their action appears to be similar to that of iodoform itself. Permanganates. — The potassium salt is principally used for irrigation, 1 : 1000, in gonorrhoea. It is an active germi- cide under conditions not involving rapid decomposition , by excess of organic matter. All the permanganates are strong oxidizing agents and as soon as they are reduced to manganese salts their disinfecting action ceases, so that their maximum germicidal effects are transitory. They are unsuitable for septic wound treatment on account of their rapid reduction but have many other useful applications. Quinine. — The hydrochloride of this alkaloid has been recommended by Kenneth Taylor l as a dressing for sep- tic wounds, especially those infected with the B. cero genes capsulatus. The substance is used in 0.1 per cent aqueous solution with the addition of 0.1 per cent hydrochloric acid or 1 per cent alcohol to inhibit precipitation of the base. Tested in vitro Taylor finds that it is about ten times as ef- fective as phenol against the gas bacillus although, on the 1 Lancet, Sept. 4, 1915. Brit. Med. Journ., Dec. 25, 1915. MISCELLANEOUS ANTISEPTICS 75 other hand, it is used on wounds in much lower concentra- tion than is usual with phenol. Serum or pus is stated to have no very marked action in reducing the germicidal activity of quinine. The solution is used either as a wet dressing or for continuous drip instillation. A curious fact noted by Taylor in connection with the use of quinine is that while there appears to be a decrease in B. cero genes capsulatus infection there was a progressive increase in the appearance of B. pyocyaneus, and laboratory tests showed that the latter organism was the most resistant to quinine of the common pyogenic bacteria. An endeavor has been made to utilize the combined an- aesthetic and antiseptic properties of mixtures of quinine and urea for wound treatment, but this has failed to secure much favor. Chinosol. — This compound was originally considered to be potassium oxyquinoline sulphonate or a double salt of this with potassium sulphate, but is now stated to be neutral oxyquinoline sulphate. It possesses a strong inhib- itory action on the growth of many microorganisms and failure to recognize this fact fully led to exaggerated claims as to its germicidal potency. A full report upon this sub- stance has been made by the Council on Pharmacy and Chemistry of the American Medical Association. 1 The general conclusions arrived at are essentially as follows : As regards staphylococcus, aureus and B. typhosus chinosol is more strongly antiseptic than phenol and about equal to mercuric chloride, but as a germicide in watery solution it is somewhat inferior to phenol and vastly inferior to mercuric chloride. In acid broth the findings were still less favorable to chinosol. These conclusions make it doubtful if much direct germi- cidal effect on wounds can follow its application, though it is possible that the substance might find useful employment 1 Cp. Journ. ; Am. Med. Assoc, May 28, 1910. 76 A HANDBOOK OF ANTISEPTICS as a wound dressing with the object of restraining the growth of organisms and preventing reinfection. It does not appear to have been used extensively in the present war. Acetanilide and its Derivatives. — This substance has often been recommended as an antiseptic, 1 but our own ex- periments have showm its germicidal properties when tested against moderate quantities of staphylococci to be so low that it can hardly exert any effective disinfection in wounds. A half per cent solution failed to sterilize staphylococci in water in two hours, while in blood serum its antiseptic action is negligible. Recently E. F. Greene 2 has revived the ques- tion of its possible use in infected war wounds, applied as a dry powder, but this can hardly be endorsed, not only on account of its poor antiseptic action but also because symp- toms of poisoning have been stated to follow its use. Its free application to extensive wounds would certainly not be without danger to many individuals who are fairly susceptible to this drug. Various derivatives of acetanilide in which the hydrogen of the benzene ring has been replaced by chlorine, bromine, or iodine have been advocated as antiseptics from time to time. Parabromacetanilide is sometimes known under the name of " asepsin " or " antisepsin " and is stated to have anodyne properties. Our own experience indicates that none of these derivatives are sufficiently active disinfectants to be of value in wound treatment. 1 Cp. Beck, New York, Med. Journ., March 19, 1893. 2 Brit. Med. Journ., May 29, 1915, p. 928. CHAPTER VII METHODS OF TESTING ANTISEPTICS The testing of substances for their antiseptic and germicidal power is fraught with innumerable pitfalls. It is possible to take almost any substance and by carefully choosing the conditions under which it acts, make it appear to possess germicidal potency, and conversely, it is equally possible to take valuable germicides and by observing their action 'under unreasonable conditions, make them appear inert. The necessity for choosing methods of testing in some de- gree in conformity with the mode of use of the substance and under conditions not too remote from those under which it is proposed to employ it, is of fundamental importance. The simplest conditions are presented in the determination of the lethal concentration of a germicide acting upon micro- organisms suspended in water. The chief variables in such a determination are the number of organisms taken for each experiment, and the temperature and time of action. With regard to the number of organisms, it is desirable to use a fairly large quantity, partly because spontaneous death of the bacteria then becomes less of a factor and also because the test becomes a more rigid one. In general, some such concentration as a hundred million bacteria to the cubic centimeter will be found appropriate. The effect of tem- perature on the rate of disinfection has already been con- sidered, also the speed of action will be found to vary enormously with different substances and with varying media, 77 78 A HANDBOOK OF ANTISEPTICS One of the oldest methods used for the determination of lethal concentrations is the so-called "thread" method devised by Koch. Sterile raw silk thread cut into portions about a centimeter long are soaked in a broth culture or aqueous suspension of the required organisms and then dried. These threads are soaked for an arbitrary time in varying concentrations of the antiseptic, then removed and trans- ferred to sterile broth, which on incubation will show whether or not the organisms were killed. One of the main objections to this method is the mechanical carrying over of adhering antiseptic into the broth medium, so that inhibition of growth frequently occurs even though the organisms are not killed, and the substance appears to be a more effective germicide than is actually the case. Failure of the antiseptic to promptly penetrate to all parts of the thread may lead to erroneous estimates of its germicidal power. A later method, which is often called the "garnet" method, was worked out by Kronig and Paul. Garnets of equal size are dipped in an emulsion of the organism — usually spore- bearing anthrax bacilli — and then carefully dried so that a thin film of organisms is spread over their surface. The garnets are then immersed in a solution of the antiseptic, which after a definite time is gently washed away and the garnets, if need be, treated with an antidote such as am- monium sulphide when mercury disinfectants are employed. The garnets are then shaken with water to detach a fairly constant proportion of the organisms and an aliquot part of the fluid is plated in solid media for counting. An alternative method using the surface of nutrient agar instead of garnets or thread, is described by Bechold and Ehrlich. 1 But neither the " thread " nor " garnet " method is often used now and the simpler method is usually employed of mixing known but varying quantities of the disinfectant with a constant concentration of organisms and subculturing from 1 Zeit. physiol. Chem., 47, p. 177, 1906. METHODS OF TESTING ANTISEPTICS 79 the mixture into broth or agar at known intervals of time, in order to determine whether disinfection has been completed. An application of this method, which is of great value in standardizing and comparing different substances with regard to their practical uses as disinfectants in hygienic work, is that devised by Walker and Rideal : in 1 903 . By this procedure it is possible to express the value of a disinfectant in terms of a numerical ratio using pure phenol as a standard. The original method is substantially as follows : a definite amount (usually 5 drops) of a 24-hour broth culture of the organism selected, e.g. B. typhosus or B. coli, is added to 5 cc. of solutions of varying concentration of the disinfectant. Subcultures are taken at intervals of 2^ minutes up to 15 minutes to determine the point of complete sterility. A similar series of tests is made with a standard phenol solu- tion of such strength that sterilization of the organisms will be effected within the time limits of 2\ to 15 minutes. The necessary concentration of phenol will usually be about 0.8 per cent with most cultures of B. typhosus. By comparing the concentration of the disinfectant which just effects sterilization in a given time with the phenol concentration which also effects sterilization in the same time, an estimate is formed of the germicidal potency of the substance under investigation. Thus if a concentration of 1 : 600 of a par- ticular substance is as effective under the above conditions and in the same time as 1 : 120 phenol, the " phenol coefficient" will be or 5. It is an essential and important fea- 120 ture of this method that time of action should be constant while the concentration of the disinfectant is varied. The reverse conditions, namely, fixed concentration and variable time, may lead to entirely erroneous inferences as to ger- micidal potency. 1 Journ. Roy. Soc. Inst. 24, p. 424, 1903. Later details concerning the Walker- Rideal method will be found in The Amer. Journ. Pub. Health, Vol. 3, No. 6. 80 A HANDBOOK OF ANTISEPTICS The determination of the " phenol coefficient " of a substance furnishes most valuable information, but chiefly as regards the relative value of disinfectants to be used in fairly homo- geneous aqueous media. Chick and Martin * and many others cited in the paper by these authors have sought to obtain a more practical estimate of the germicidal value of disinfectants by adding organic matter of various kinds to the bacterial suspension. For some types of general disinfectants the addition of a definite quantity of dried feces is recommended, for example, when testing those substances destined to be used for the disinfection of stools. The problem of selecting conditions for studying the action of substances to be used as wound disinfectants is much more difficult. Useful information is obtained by determin- ing the lethal concentration of substances acting on organ- isms suspended in blood serum or in blood, or by studying their action on pus. Certain precautions must be taken in working with these media. With all of them, it is of course essential that, as in practical use, the antiseptic solution should be added to the mixtures last, for in many cases if this is not done totally erroneous results will be obtained. It has happened with extraordinary frequency that small quantities of labile chlorine antiseptics have been added to organic media and then at unstated intervals after all or most of the antiseptic has been destroyed, the mixture is infected with organisms which naturally grow unchecked. Under such conditions, what is really being observed is less the germicidal action of the antiseptic than the rate of chemical reaction which has taken place between the unstable antiseptic and organic media. The results of such a pro- cedure are naturally particularly misleading when high dilutions of antiseptic are employed in experiments to de- termine the lethal concentration. 1 Journ. Hygiene, 8, p. 654, 1908. METHODS OF TESTING ANTISEPTICS 81 A great advantage in the use of serum is the fact that it presents a homogeneous medium of fairly constant com- position ; but it is important to bear in mind that many or- ganisms, e.g. B. paratyphosu's, are very susceptible to the ac- tion of blood serum even though previously heated to 58- 6o° for an hour. To avoid this complication, liberal quanti- ties of an organism such as staphylococcus aureus, which is fairly resistant to serum, should be used. When substances of high germicidal power are examined for the determination of the lethal concentration, and hence low concentrations are employed, it will usually be found sufficient to carry out the tests in a medium containing 50 per cent or even less of serum, since its mass, relative to that of the antiseptic, will be very large. The testing of the germicidal effects of substances acting on organisms suspended in blood would seem the rational procedure for examining compounds which might be used intravenously. A blood medium has, however, been sug- gested as a standard method of testing ordinary antiseptics by Emery. 1 The use of a blood medium as a standard is particularly adverse to the hypochlorite antiseptics which rapidly react with haemoglobin so that a relatively con- siderable amount of the former must be added before any active antiseptic will persist in the mixture. For these tests use may be made of reconstituted blood obtained by mixing serum with cells separated from citrated blood, al- though for most purposes defibrinated blood or citrated blood itself would probably serve. The technique em- ployed by Emery is substantially the following : Nine parts of " reconstituted blood' ' is mixed with one part of a strep- tococcus culture containing about 250,000,000 organisms per cubic centimeter. One volume of the infected blood is mixed on a slide, using a marked capillary pipette, with an equal volume of the antiseptic solution. The mixture is then drawn 1 Lancet, April 15, 1916, p. 817. 82 A HANDBOOK OF ANTISEPTICS into the pipette, which is sealed and incubated and subse- quently examined to determine whether the contents are sterile or whether viable organisms persist. It should be noted that apparently the concentrations of antiseptic quoted in Emery's table refer to the strength of the solution added, so that their actual concentration in the final mixture is half of that given. Antiseptic 15 Minutes 60 Minutes Did not kill Killed Did not kill Killed Phenol Eusol (HCIO) Sodium Hypochlorite .... Mercuric Chloride Mercury Biniodide .... Iodine Lysol Malachite Green 1 : 70 [1 : 400?] [1 : 200?] 1 : 100 1 : 60 1 : 100 1 : 40 1 : 250 1 : 60 1: 80 1 : 40 ? 1: 30 1 : 200 1 : 60 1 : 100 1 : 60 1 : 100 1 : 150 1 : 250 1:50 1:80 1 : 40 ? 1 : 120 1 :2oo The results recorded by Emery do not make the method as de- scribed by him appear particularly accurate, since with only one exception as high a concentration was necessary to steri- lize the mixture in 60 min. as in 15 min., although the act of disinfection is a progressive time reaction. Moreover, a higher concentration of phenol is recorded as necessary to sterilize in sixty minutes than is stated to be effective in fifteen. But the use of blood as a medium for studying the germicidal action of substances in the presence of cellular elements, is of distinct value since, unlike pus, its compo- sition is fairly uniform. The blood should be heavily in- fected with organisms so that its natural bactericidal proper- ties will not effett the end result materially. On pp. 93, 95 a number of experiments with antiseptics of the chlorine group and dyestuffs, acting on staphylococci in blood media, adre recorded. METHODS OF TESTING ANTISEPTICS 83 The determination of the disinfecting action of substances upon pus is obviously important as bearing on the treatment of suppurating wounds. As already stated, the disinfection of pus is difficult of accomplishment and relatively high concen- trations of antiseptic are necessary. But great difficulties are encountered in laboratory experiments on the disinfection of pus owing to the enormous variations in its physical con- dition and the number of organisms it contains and whether these organisms are mainly free or ingested in the leucocytes. Rous and Jones x have shown that living phagocytes are able to protect ingested organisms from the action of substances such as potassium cyanide in the surrounding fluid and even from the action of a strong homologous antiserum, and they obtained evidence that this protection by phagocytes was largely conditioned on their being alive. But even when all the leucocytes are dead, there are considerable mechanical difficulties in securing good contact with the antiseptic solution. An interesting series of experiments on the steri- lization of pus by various antiseptics is recorded in a paper by Parry Morgan, 2 and some of the results have already been cited on p. 8. Thus far, we have considered almost exclusively the ques- tion of the lethal concentration of disinfectants acting under different conditions, but it is becoming constantly more evident that much more than this is needed in judging of the utility of antiseptics. As already stated on p. 6, the act of disinfection resembles in many respects an ordinary chemical reaction in which the two reacting components are represented by disinfectant and bacterial protoplasm. Now the rate of disinfection varies enormously with different germicides, and is of course influenced by the relative mass of bacteria and disinfectant, as well as by temperature and contact between the reacting substances, f The instability 1 Journ., Exp. Med., 23, p. 601, 1916. i 2 B. M. J., May 13, 1916, p. 684. J 84 A HANDBOOK OF ANTISEPTICS of many antiseptics, leading to a reduction or disappearance of the mass of active antiseptic, must also be reckoned with. A knowledge of the speed with which a disinfectant acts is essential to an understanding of the conditions under which it may be appropriately used. For example, the extremely rapid acting aqueous hypochlorites are admirably adapted for intermittent instillation in large quantities into wound cavities, while when the same solutions are applied as wet dressings which are seldom renewed, their action is over as soon as the active chlorine has disappeared, and this may be a matter of only seconds or minutes. For prolonged action when intermittent instillation cannot be practiced, a slower acting but more stable antiseptic is likely to give better re- sults, or they may be attained by using an oil solution of dichloramine-T (p. 34) from which the active antiseptic slowly passes from the oil to the aqueous medium on the surface of the wound. The only satisfactory way to follow the speed of disin- fection is to determine the progressive change in the number of bacteria in a suitable mixture after varying lengths of time. With this end in view, we have made a number of time experiments with various antiseptics, using them in most cases at about the concentration which is recom- mended for wound treatment. The mixtures contained 1 cc. of horse blood serum, 1 cc. of muscle extract obtained by soaking fresh veal with an equal weight of saline and then straining through cloth but not filtering, and 2 drops of a staphylococcus aureus emulsion obtained by shaking a 24-hour agar slant of abundant growth with 8 cc. of saline. The muscle extract doubtless contained organisms of other kinds in fair quantity. After determining the total number of organisms present in the mixture by plating an aliquot part, 1 cc. of antiseptic solution was added and samples with- drawn from time to time and the surviving bacteria esti- mated. In the case of chlorine antiseptics, their further METHODS OF TESTING ANTISEPTICS 85 action after sampling was checked by sodium thiosulphate, while potassium sulphide was used as an antidote with the salts of the heavy metals. No antidote was used with the dyes. The use of the blood serum-muscle extract medium was chosen as bearing some similarity to the composition of wound exudate and it has the further advantage of being easily reproduced in fairly uniform quality. For many antiseptics, the addition of muscle extract makes the tests much more severe than when blood serum alone is used. The experiments were all carried out at 32-35 C. and the results are expressed as the number of bacteria present in one standard drop of the mixture — 1/40 cc. I. Chlorine. Group of Antiseptics (Blood Serum-Muscle Extract Medium ; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count 1 Drop=2 1 oCC. As Added In Mixture I. Sodium Hypochlorite . o.5% 0.17% 2 min. 5 min. 1 966 OOO 405 oi II. do. do. . . o.5% (0.2 cc.) 0.05% • 0.09% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 1 966 OOO 311 200 157 400 98 170 16 120 I 651 1587 1 294 OOO 2 III. Eusol 0.27% 2 min. 5 min. 15 min. 2 I50 OOO 496 2 O 1 1 Active chlorine present. 2 Active chlorine absent. A HANDBOOK OF ANTISEPTICS I. Chlorine Group of Antiseptics — Continued (Blood Serum-Muscle Extract Medium; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count 1 Drop= 5 1 cc. As Added In Mixture IV. Eusol 0.27% (0.2 cc.) 0.025% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 2 129 000 1 310 000 507 900 606 200 641 400 868300 983 000 2 310 000 2 V. Chloramine-T . . . 2% 0.67% 5 min. 1 365 000 o 1 VI. do 0.5% 0.17% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 726 300 998 191 7 4 3 o 2 VII. Dichloramine-T in Oil (v, p. 39) 2% 0.67% 3 0.5 min. 2 020 000 VIII. do 2% 0.67% 4 5 min. 15 min. 45 min. 1 157 000 458 700 294 900 IX. Iodine in Potassium Iodide 2% 0.67% 5 min. 1 463 000 The results shown in Table I in which members of the chlorine group of antiseptics and iodine are considered, il- 1 Active chlorine present. 3 Mixed with platinum wire. 2 Active chlorine absent. 4 Not mixed, left to diffuse. METHODS OF TESTING ANTISEPTICS 87 lustrate their extraordinary speed and completeness of dis- infection when added in adequate amount to the infected mixture. In Experiments I, III, V, VII, and IX, -J volume of the various antiseptic solutions was added at a concen- tration no higher and in some cases lower than that com- monly employed in wound treatment and in every case practical sterility was obtained in less than five minutes. When much smaller amounts of hypochlorites were added, as in Experiments II and IV, their rapid action is seen to cease as soon as all the active antiseptic is decomposed and subsequent growth can then take place. In Experiment VI, in which weak (0.5 per cent) chloramine-T was used, its action is shown to be distinctly more effective than an ap- proximately equivalent amount of chlorine in the form of sodium hypochlorite or hypochlorous acid (Experiments II and IV). II. Metallic Salts, Phenol, Hydrogen Peroxide (Blood Serum-Muscle Extract Medium ; Staphylococcus aureus.) Concentration Antiseptic Used Time op Action Bacterial Count 1 Drop= 5 1 oCC. As Added In Mixture I. Mercuric Chloride . . 0.1% 0.033% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 24 hr. 1 894 000 254 400 44920 12 900 7258 2985 II. Silver Nitrate- . . . 1% o.33% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 786 400 720 900 651 700 530 600 425 900 175 600 38 270 2 643 A HANDBOOK OF ANTISEPTICS II. Metallic Salts, Phenol, Hydrogen Peroxide — Continued (Blood Serum-Muscle Extract Medium; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count 1 Drop= 3 1 cc As Added In Mixture III. Argyrol 15% 5% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 917 500 753 500 655 300 622 500 327 600 9 792 4693 IV. Zinc Chloride . . . 3% 1% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 1 223 OOO 868 300 819 200 49i 5oo 211 900 5i 58o 3667 704 V. Hydrogen Peroxide 2.89% 0.96% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 1 201 000 589 800 819 200 778 200 802 800 1 087 OOO I 136 OOO VI. Phenol 2% 0.67% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 24 hr. 1 409 OOO 1 471 OOO 819 200 192 700 34900 13 700 2985 In the second series of experiments a similar infected medium of blood serum and muscle extract was mixed with solutions of various metallic salts, phenol, and hydrogen METHODS OF TESTING ANTISEPTICS 89 peroxide. Mercuric and zinc chlorides and silver nitrate produced immediate precipitates. The action of mercuric chloride o.i per cent is seen to be more rapid than that of phenol 2 per cent, silver nitrate i per cent, or zinc chloride 3 per cent. Argyrol was tried in very high concentration and while acting slowly sterilized completely in 24 hours. Neither phenol, silver nitrate, nor zinc chloride sterilized completely in 24 hours, although the number of viable organisms was less than 1 per cent of those originally present in the mix- ture. The effect of hydrogen peroxide is interesting as showing an extremely rapid but transitory action which reached its maximum in a few minutes, after which the organisms grew unchecked. III. Dyes (Blood Serum-Muscle Extract Medium ; Staphylo coccus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count 1 Drop= 5 1 oCC. As Added In Mixture I. Malachite Green . . (Griibler) 0.3% 0.1% 5 min. 15 min. 1. 5 hr. 3 hr. 6 hr. 24 hr. 48 hr. 821 600 182 700 60 920 8 920 5 574 78 200 1 015 000 3 706 000 II. do. . . do. . . . 0.1% 0.033% 5 min. 15 min. 45 min. 3 hr. 6 hr. 24 hr. 48 hr. 2 097 coo 1 359 coo 369 100 211 500 272 300 557 coo 1 096 000 6 553 000 90 A HANDBOOK OF ANTISEPTICS III. Dyes — Continued (Blood Serum-Muscle Extract Medium; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count As Added In Mixture iDrop = 3 1 oCC. III. Brilliant Green . . . o.3% 0.1% 810 200 (Griibler) 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 48 hr. 29 950 27 410 20 160 14 310 8871 1 946 256 600 658 900 IV. Acriflavine .... 0.3% 0.1% 5 min. 15 min. 45 min. 1.5 hr. 6 hr. 24 hr. 589 800 358 400 249 300 179 200 78 210 704 V. do 0.1% 0.033% 5 min. 15 min. 45 min. 1.5 hr. 6 hr. 24 hr. 557 100 218 600 121 900 100 800 81 540 474 VI. do 0.03% 0.01% 755 400 524300 5 min. 15 mm. 319 300 45 min. 308 200 1.5 hr. 106 500 3 hr. 94 840 6 hr. 22 780 24 hr. 1 113 OOO VII. Proflavine 0.3% 0.1% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 888 100 794 500 744 500 308 200 104 800 66 560 53 250 1 242 METHODS OF TESTING ANTISEPTICS 91 III. Dyes — Continued (Blood Serum-Muscle Extract Medium ; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count 1 Drop =3^00. As Added In Mixture VIII. Proflavine .... 0.1% 0.033% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 888 100 744 500 655 400 326 100 169 900 154 800 84 900 4 IX. do 0.03% 0.01% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 855 400 761 800 744 5oo 469 500 304 500 196 400 93 750 183 600 In the third series of experiments the action of malachite green, brilliant green, and acriflavine acting in a similar mix- ture of blood serum and muscle extract were examined. The germicidal action of these dyes, in view of claims made for them, proved disappointing. Brilliant green has been recom- mended for use by Browning and his colleagues at a concen- tration of 0.1 per cent but addition of one third volume of brilliant green or malachite green at this concentration gave but poor evidence of much germicidal action. Subsequent experiments with these two dyes were made at a higher concentration, 0.3 per cent. In no case was sterility reached and after some hours the organisms grew unchecked . It was noted that similar mixtures of blood serum and muscle 92 A HANDBOOK OF ANTISEPTICS extract mixed with these dyes so that the final concentration was o.i per cent, on exposure to air readily underwent thorough putrefaction. It would appear that under the con- ditions of these experiments the dyes in question are readily inactivated and that the presence of apparently unchanged coloring matter in the mixture is no evidence of the continua- tion of any antiseptic action. Similar experiments with acriflavine, o.i per cent, showed a slow initial effect but anti- septic action was more sustained, and while sterility was not attained in six hours, after 24 hours disinfection was com- plete. When the concentration of the acriflavine solution added was 0.03 per cent more organisms were present at the end of 24 hours than at the commencement of the experi- ment. The results with proflavine were not widely different from acriflavine, although the higher concentrations did not effect perfect sterilization in 24 hours. The effect of these dyes appears more pronounced when acting in blood or blood serum than when muscle extract is present. We have also made some experiments in the progressive change in the bacterial count when various chlorine antiseptics and dyes were added to defibrinated blood. With the excep- tion of the sodium hypochlorite and eusol tests these experi- ments were made by adding one volume of antiseptic to two volumes of freshly drawn sterile rabbit blood heavily inocu- lated with staphylococcus aureus. The general conditions of the experiments were identical with those preceding. METHODS OF TESTIXG ANTISEPTICS 93 IV. Chlorine Group of Antiseptics (Defibrinated Blood Medium ; Staphylococcus aureus.) Antiseptic Used CONXENTRATION Time of Action Bacterial Count 1 Drop= 4 1 u cc. As Added In Mixture I. Sodium Hypochlorite . o.5% o.33% 5 min. 15 min. 45 min. 573 400 563 282 2 432 II. do. do. . -. . . C5% 0.3% 5 min. 15 min. 45 min. 573 400 1 774 1485 19 656 III. do. do 0.5% 0.25% 45 min. 232 oco 89 600 IV. Eusol ...... 0.27% 0.18% 5 min. 15 min. 45 min. 578 600 1 562 2 330 36 600 V. do 0.27% 0.16% 5 min. 15 min. 45 min. 8ll OOO 36 280 48 250 51 580 VT. do 0.27% 0.13^ 5 min. 260 80O 143 300 VTI. Chloramine-T . . . 2% 0.67% 5 min. 15 min. 224 50O 125 O VII. do •1% o.5% 45 min. 235 OOO O VIII. do 0-75% 0.25% 5 min. 15 min. 45 min. 3 hr. I 178 OOO 20 l6o 5 451 4 032 806 IX. Dichloramine-T in Oil 2% 0.67% » 2 min. 59 900 O Mixture stirred with platinum wire. 94 A HANDBOOK OF ANTISEPTICS The results of the action of 0.5 per cent sodium hypochlorite and of eusol on staphylococci in a blood medium show that a relatively large addition of these must be made before any marked germicidal effect is produced. Even when two volumes of the antiseptic were added to one of infected blood, complete sterilization was not quite accomplished, although considerably over 99 per cent of the organisms were killed. As already stated, the reason for the apparent low germicidal action of hypochlorites in a blood medium is due to their ready decomposition by the haemoglobin and other substances in the blood. From a practical standpoint this is not of much importance when methods of intermittent instillation of hypochlorite solution into wounds are practiced but it serves to emphasize the necessity for the frequent renewal of the solution. Chloramine-T and dichloramine-T give materially better results than the hypochlorites when acting on organisms in a blood medium. A comparison of Experiments I and IX shows that a concentration of 0.25 per cent of chloramine-T in the mixture was as effective as 0.33 per cent sodium hy- pochlorite, although the active chlorine in the latter was about five times as much as that in the chloramine-T. The reason for this difference is to be sought largely in the slower rate of reaction between chloramine-T and haemoglobin and other proteins, compared with the hypochlorite solutions. METHODS OF TESTING ANTISEPTICS 95 V. Dyes (Defibrinated Blood Medium ; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bactertal Count 1 Drop=3 1 oCC As Added In Mixture I. Malachite Green . . (Grubler) 0.3% 0.1% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 72 hr. 1 754 OOO 107 88 66 82 35 36 13 II. do 0.1% 0.033% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 1 693 OOO 1 310 oco 819 200 208 OOO 146 900 86 010 89 600 1 219 OOO III. Brilliant Green . . . (Grubler) 0.3% 0.1% 5 min. 1 966 OOO IV. do. do 0.1% 0.033% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 1 630 OOO 1 261 563 755 717 857 2 592 1 171 OOO V. Acriflavine .... 0.3% 0.1% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 654 600 358 400 108 800 98 180 I 472 203 17 96 A HANDBOOK OF ANTISEPTICS V. Dyes — Continued (Defibrinated Blood Medium ; Staphylococcus aureus.) Antiseptic Used Concentration Time of Action Bacterial Count As Added In Mixture 1 Drop= ¥ \)CC. VI. Acriflavine .... 0.1% 0.03% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 654 700 411 900 225 800 138 100 30 640 1 152 32 VII. do 0.03% 0.01% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 900 600 819 200 508 900 190 OOO 83 860 32 260 768 3 VIII. Proflavine .... 0.3% 0.1% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 819 200 4710 1638 474 304 113 12 IX. do 0.1% 0.033% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 24 hr. 819 200 201 300 208 OOO 122 5OO 53 220 29 670 2 995 2 X. do 0.03% 0.01% 5 min. 15 min. 45 min. 1.5 hr. 3 hr. 6 hr. 819 200 753 70c 852 OOO 591 400 394 200 448 OOO 48380 24 hr. 282 METHODS OF TESTING ANTISEPTICS 97 The action of malachite green and brilliant green in blood heavily infected with staphylococcus aureus was dis- appointing. In only one case, namely with brilliant green, did a concentration of o.i per cent in the mixture bring about apparent sterility. It is by no means certain that even in this case all the organisms were killed, since it was not pos- sible to avoid carrying over of some of the dye to the agar subcultures, and in the concentration referred to we showed that reproduction of living staphylococci actually was in- hibited, although they were not killed. It is significant that with the exception of the two experiments in which brilliant green and malachite green were added in 0.33 per cent solu- tion, an initial fall in the number of bacteria was followed by practically unrestricted growth. These results are in marked contrast to those of Browning and his colleagues referred to in Chapter V. The action of acriflavine and proflavine in blood, while slow, is seen to be distinctly superior to that of the other dyes. In judging of the suitability of substances for wound treatment, there are of course other factors to be considered besides germicidal efficiency. Prominent among these is the determination of the concentration at which irritation of the skin and other tissues becomes noticeable, also the effect of germicidal substances upon phagocytosis and their influence if any on the rate of dissolution of necrotic tissue. All of these factors may best be investigated in vivo. Ref- erence may be made to Colonel Bond's ingenious experiments noted on p. 11 with regard to phagocytic activity as influenced by antiseptics. Laboratory experiments serve primarily for the sorting out of substances which are likely to be of value for wound antisepsis, and they are particularly important in indicating the mode of use for the selected substance which is most likely to give successful clinical results, but the final decision of the utility of any germicide will necessarily follow from a 98 A H AN D BOOK OF ANTISEPTICS study of its effects upon actual wounds. The treatment of comparable infected wounds with various antiseptics, accom- panied by daily bacteriological estimations of the degree of infection gives most useful results. This method has been practiced extensively in the present war and gives valuable indications as to the earliest date at which wounds may be safely closed by suture or otherwise. The influence of antiseptics on the rate of cicatrization of wounds may also be followed with the aid of a formula worked out by Du Xotiy. 1 The technique of these experimental methods is beyond the scope of this book and reference should be made to original sources. 1 Journ., Exper. Med., 24, pp. 451, 461, 1916; 25, p. 721, 1917. CHAPTER VIII CERTAIN SPECIAL APPLICATIONS OF ANTISEPTICS I. The Disinfection of Carriers The problem of destroying pathogenic organisms in the nasal and upper air passages by direct disinfection is a diffi- cult one. There are, undoubtedly, many carriers with anatomical abnormalities of the nose, pharyngeal vault and tonsils precluding immediate contact with solutions used either as sprays or gargles. In such cases there is little pros- pect of any antiseptic proving effective. Those organisms which are exposed to direct contact with the antiseptic solution, may be destroyed, but in the course of time those which have preserved their vitality in protected situations regenerate the original condition and only a temporary, although to that extent beneficial, result is attained. This difficulty is encountered in chronic diphtheria carriers, in whom deep tonsilar crypts are frequently encountered form- ing inaccessible regions in which the bacilli are afforded pro- tection. The pneumococcus, existing in the pulmonary air passages, is, in those parts, wholly beyond reach. In con- sidering the applicability of treatment to individual cases. these inevitable limitations must be taken into account. In spite of these limitations, chiefly imposed by anatomical conditions, valuable results have been obtained, notably by Gordon and Flack, with the use of antiseptics in the treatment of meningococcus carriers among soldiers. By a suitable choice of antiseptic, properly applied, there is no doubt that much can be accomplished in limiting the risks 100 A HANDBOOK OF ANTISEPTICS connected with carriers of pathogenic organisms, both to themselves and those coming in contact with them. The methods that have been employed with most success are the following : (i) Chamber disinfection using a steam spray and a solution of chloramine-T or zinc sulphate (Gordon and Flack). Hypochlorites have also been used for chamber disinfection, but they have not been found as satisfactory. 1 (2) Local application of various antiseptics including iodine, with either menthol, guaiacol or glycerine, axgyrol and hydrogen peroxide, zinc salts, acrinavine, chloramine-T and especially dichloramine-T in oil solution. Ferric chloride and potassium permanganate solutions have given only mod- erate results while formalin appears to be distinctly harmful. Gordon and Flack found that many of the antiseptic solutions employed as gargles and in hand sprays at the time when they commenced their experiments gave un- satisfactory results when used on heavily infected chronic carriers. This appeared to be due as much to lack of per- fect contact as to germicidal inefficiency. They therefore employed an inhalation chamber with a steam pressure spray or atomizer capable of converting the antiseptic solution into a fine mist. The chamber was about 750 to 1000 cubic feet capacity, and the apparatus was capable of spraying a liter of antiseptic solution in the course of tw T enty minutes. A working diagram of two forms of these sprays will be found in Gordon and Flack's original paper, 2 in which there will also be found details of the successful treatment of numbers of chronic meningococcus carriers. The prepared antiseptic solution used for spraying by Gordon and Flack was either chloramine-T, 0.5 per cent, or zinc sulphate, 1.2 per cent. The chloramine-T gave the better results, especially with persistent carriers, but is somewhat less well 1 Kuster, Deutsch. Med. Woch, 41, p. 11 16, 1915. 2 Brit. Med. Journ., p. 673, Nov. 18, 1916. THE DISIXFECTIOX OF CARRIERS 101 tolerated than the zinc salt. The inhalation was carried out once daily and on each occasion the carriers were in the inhaling room from fifteen to twenty minutes during which time they vigorously inhaled the spray-laden atmosphere through the nostrils. Using a different apparatus with a high pressure air spray,, the authors l have examined the antiseptic action of chlor- amine-T solution on the mixed bacterial flora of the normal nasopharynx. The results are recorded in the following table in which, for comparison, the effect of spraying with salt solution is included. The figures, which are the aver- age of six similar experiments, indicate the total number of organisms obtained from plating a swab taken under approxi- mately constant conditions and in which any antiseptic adhering to the swab was immediately destroyed with sterile sodium thiosulphate solution. Aqueous Chloramine-T and Neutral Salt Solution Number of Colonies Derived from Sample First Period Second Period Time of Treatment 5 min. 10 min. 15 min. 5 min. 10 min. 15 min. 0.5% chloramine-T (control, 43.240) Normal salt solution (control, 56.738) 12,352 707 121,620 ■ 526 13,587 439 2,137 153 10,055 17 34,339 From these and other results, it appeared that exposure to the chloramine-T spray for less than fifteen minutes was not very effective and that frequent repetition of the treat- ment was desirable. Repetition several times daily of the 1 Brit. Med. Journ., June 23, 1917. 102 A HANDBOOK OF ANTISEPTICS chloramine-T spray for 15 or 20 minute periods is rather a severe procedure, so that as an alternative we have em- ployed an oily solution of the related dichloramine-T (see p. 33), which has the great advantage that its action is much more prolonged, owing to the slow diffusion of the antisep- tic into the aqueous nasal secretions. The oil solution is sprayed into the nose and throat from an ordinary oil spray, preferably one made entirely of glass, thus avoiding the necessity of an inhalation chamber. Appli- cations Time of Test, Number of Treatment Employed of Oil from Beginning Colonies on between of Treatment Agar Plate Tests I. o-5% aq. chloramine-T 42,240 followed by 2 % di-chlor- 1 30 min. 9 amine-T in oil. 60 min. II. 1.5% dichloramine-T in 16,104 oil. 1 1 hour 175 1 3.5 hours 1 6 hours 9 III. o-5% aq. chloramine-T 6,129 followed by 1.3% di- 1 1 hour 8,960 chloramine-T in oil. 4 4 hours 87 20 hours 1,980 1 22 hours 59 1 24 hours 3 1 26 hours 1 1 27.5 hours 20 Experience in the treatment of meningococcus carriers with 2 per cent dichloramine-T dissolved in chlorinated eucalyptol and paraffin (1:4) has as yet been limited, but those cases treated have been promptly freed from the me- ningococcus and the method appears likely to be used ex- tensively. The above table shows the effect of spraying the oil solution of dichloramine-T on the normal nose. In such experiments it must be recalled that many of the bac- THE DISINFECTION OF CARRIERS 103 teria are much more resistant to antiseptics than the menin- gococcus and that spore forms were probably present. In the above experiments, the strongest solution was 2 per cent and the result was very striking. It should be understood, however, that the concentration of the antisep- tic in the oil bears no simple relation to the strength ac- tually applied to the organisms in the aqueous secretions. For example, a sample of the oil containing 2 per cent di- chloramine-T, when shaken with an equal volume of clear nasal secretion, showed that the filtered aqueous portion had a concentration of active chlorine corresponding to about 0.1 per cent dichloramine-T. The chief advantage of using a concentrated oil solution lies in prolonging the period through which it can serve as a store of active antiseptic. In the normal nose, a 2 per cent solution is not exhausted until about two hours have elapsed; but when foul discharges are present, this period would be curtailed. The experience hitherto gained in the treatment of carriers with dichloramine-T indicates that the following procedure may be advantageously followed : First cleanse the nose with normal salt solution, with or without the addition of 0.25 per cent chloramine-T, by spray- ing, or irrigation. The same chloramine-T solution should be used as a gargle. After this preliminary treatment and when the augmentation of nasal, secretion has subsided, apply the oil solution of dichloramine-T l (2 per cent) with an oil " atomizer," endeavoring to reach all the parts with an abundant supply of oil. It is not certain that the oil so introduced is ever active for more than two hours, so that for intensive treatment it should be renewed at the end of 1 This solution is best prepared by dissolving 0.2 gram of dichloramine-T in 2 cc. chlorinated eucalyptol and when all dissolved, adding 8 cc. chlorinated paraffin. The solution should be discarded as soon as signs of a definite crys- talline sediment appear. Ordinarily it is stable for about three days. It should be carefully protected as far as possible from direct exposure to sunlight, as this hastens its decomposition. 104 A HANDBOOK OF ANTISEPTICS that time. In any case, it appears important to repeat spraying with the oil so that four treatments a day are given at about equal intervals. The first few applications of oil sometimes occasion sneezing, but the nose appears to acquire a tolerance for the treatment, and subsequent applica- tions occasion no inconvenience. In cases of acute coryza, dichloramine-T applied as described is too irritating to the inflamed membrane and its use is not advised. In the way of prophylaxis, spraying of the upper air pas- sages with a 0.25 per cent solution of chloramine-T in saline, or an oil solution of dichloramine-T appears to be followed by good results in reducing the incidence of coryza, measles and mumps, although it is not possible to cite convincing statistical evidence. Colonel Parke's x report on army trans- ports covering long voyages in which an inhalatorium of the Gordon and Flack type was used, lends support to the view that such treatment is useful. The simplicity of these methods and the apparent value of the results indicate that more extended trials are desirable. Among the other antiseptic solutions employed in the treatment of meningococcus carriers, reference may be made to an iodine and menthol preparation frequently employed in England, while Vincent in the French army advocated the following mixture as an inhalation: iodine 12 grams, potassium iodide 6 grams, guaiacol 2 grams, thymol 0.35 gram, alcohol 200 grams, used five or six times a day. For disinfection of the pharynx, swabbing with a 3 to 5 per cent solution of glycerine and iodine is advocated. This would appear a decidedly drastic treatment. Sophian has recom- mended hydrogen peroxide 1 per cent and argyrol 9 per cent, used as a spray, as of value for freeing carriers of me- ningococci, although the mixture does not seem to have been used extensively in military centres. The antiseptic treatment of carriers has been much more 1 Brit. Med. Journ., Feb. 24, 1917. THE DISINFECTION OF WATER 105 successful in meningococcus cases than with most other infec- tions. The reasons for this are doubtless due in large meas- ure to the fact that the meningococcus is very readily killed by antiseptics and is usually found in situations accessible to treatment. The treatment of diphtheria carriers has been less successful, though encouraging, while pneumococcus carriers give disappointing results. The reasons for this are obvious when the pathological conditions are considered. II. The Disinfection of Water The chemical disinfection on a large scale of contaminated or suspected water supplies for civilian military or naval use is now almost exclusively effected with substances belonging to the chlorine group. 1 When working with considerable volumes of water, the choice of reagent is prac- tically limited to free chlorine supplied from cylinders of the liquefied gas, sodium hypochlorite solutions of known concentration, or bleaching powder (chloride of lime). The action of these substances is essentially similar, and their usefulness depends upon the fact that their addition in such quantity that about 0.5 to 1 part of active chlorine per million parts of water persists for a short period, is followed by an almost complete disappearance of organisms of the coli group. Spore forms which are of little hygienic impor- tance are not much affected. Preliminary filtration of turbid waters is desirable, and occasionally in the case of extreme turbid waters, clarifying with some precipitant, such as alum followed by alkali, is resorted to. The choice of reagent will depend largely upon the actual conditions of its employment. When large volumes of water are to be treated and liquefied chlorine is available, 1 An old chemical method of purifying water depended on the use of potassium permanganate. Except against the cholera spirillum, it is not very effective, and the cost of the material, and the disagreeable appearance and taste of the treated water, are serious drawbacks. 106 A HANDBOOK OF ANTISEPTICS this may be the most convenient method, and especially if a gas gauge such as the Wallace-Tiernan is employed by means of which the desired chlorine concentration in a known quantity of water may be easily regulated. Sodium hypochlorite solution is preferable to bleaching powder in that it is more readily distributed through the water, but its varying composition and instability make it less satis- factory for field use. For the sterilization of moderate volumes of water such as those contained in army water carts, e.g. no gallons, bleaching powder has given most satisfactory results. The successful use of chlorine compounds for the disin- fection of water depends largely upon adequate mixing with the water; the use of an effective concentration of disinfectant such as that already indicated ; and lastly the avoidance of excess. The taste and smell of unnecessarily highly chlorinated water is objectionable to most people. Not infrequently a reducing substance such as sodium thiosulphate is added to remove the excess of chlorine, but with careful chlorination this should not be necessary. The exact amount of disinfectant to be added to water can only be fixed with regard to individual supplies of constant com- position. The reason for this is that different waters con- tain such varying amounts of organic matter and some- times ferrous salts or sulphides as well, and the chlorine used up in oxidizing these substances will not effect steriliza- tion. A slight excess only of the disinfectant must therefore be employed, and the proper amount is most readily deter- mined by adding a little more {e.g. 0.5 to i per million) than the minimum quantity necessary for obtaining a positive reaction for active chlorine in the water. The test for active chlorine is most easily carried out by adding a few drops each of 10 per cent potassium iodide solution and starch, paste to about 200 cc. of water and noting whether a blue color de- velops. Since starch paste is apt to become mouldy, it is THE DISINFECTION OF WATER 107 often convenient to use zinc iodide (7.5 per cent) in place of the potassium salt and to add this directly to the starch paste together with two per cent of alcohol. This mix- ture keeps well. Various simple devices have been used for carrying out this test in the field so that accurate dosage may be rapidly determined. It should be remembered that the sensitiveness of the starch-iodide reaction for active chlorine rapidly diminishes with rise in temperature of the water. The speed with which the reaction for active chlorine disappears from a treated water depends upon many factors, such as the degree of surface exposure, carbon dioxide con- centration, the presence or absence of much ammonia in the water, and the nature and amount of the organic matter. Under most circumstances, an initial concentration of 0.5 part per million of active chlorine will vanish in a very few hours. It has been observed that if an addition of ammonia is made to the hypochlorite to be used for water sterilization, the resulting product monochloramine, NH 2 C1, is still more active as a disinfectant than the original hypochlorite, 1 and its action is more prolonged. Use of this fact has been made in the sterilization of the water supplies of several large communities with excellent results. A concentration of 0.6 part per million of active chlorine, used as mono- chloramine, appears to give a considerable margin of safety. Further details concerning the large scale disinfection of water should be sought in books dealing primarily with questions of hygiene. The Disinfection of Small Quantities of Water. — Chlorine gas, bleaching powder or sodium hypochlorite, which are so effective for the disinfection of relatively large volumes of water, are not suitable for the individual sterilization of small quantities such as the contents of an army water bottle. 1 Rideal, Journ. Roy. Sanitary Inst., 31, p. 33, 1910. 108 A HANDBOOK OF ANTISEPTICS The problem of sterilizing small individual quantities of water such as are needed by cavalry or rapidly moving troops is a difficult one, for the use of the chlorine antiseptics just mentioned is generally impracticable owing to the instability of small tablets containing the required minute quantity of active disinfectant. In their place acid sulphates of the alkali metals have been used to a considerable extent, and these will be considered later ; but the superior potency of the chlorine antiseptics induced us to try and find suitably stable compounds, and one of these which will now be de- scribed has met with some success as the result of practical trials. A number of compounds which at first sight seemed likely to be useful had to be discarded either because they were ineffective in hard waters or because of difficulties concern- ing solubility or stability. The most suitable substance that we have as yet found is p-sulphondichloramino- benzoic acid, — Cl2N0 2 SC 6 H 4 COOH. It is easily pre- pared from cheap, readily available materials, and appears to be effective and reasonably stable. The presence of the COOH group confers a slight but definite degree of solu- bility in water, which is increased by dispensing it with alkaline salts such as sodium carbonate or borax. For- mulae for the tablets are appended together with details of the preparation of the substance and an estimate of its cost. Since the systematic name of the disinfectant is inconveniently long for ordinary use, we have adopted the name "halazone." The abbreviated name gives some indi- cation of the character of the compound. In the following table are given some of the bacteriological results. The technique employed was that in general use and requires no special description. Five or ten standard drops of the treated w r ater were generally used for plating on agar for counting the surviving organisms. Suitable controls were invariably carried out. THE DISINFECTION OF WATER 109 Experiments with p-Sulphondichloraminobenzoic Acid in Tablet Form. No. Water Treated Concentra- tion of Dis- H H < P '- - Surviving Organisms Q W > infectant per c.c. s^ as ►J i Tap water B. coli. - 112,525 2 Tap water B. coli. 1 : 400,000 15 178,528 IO 1 : 500,000 15 987 IO 3 Tap water 5% sewage B. coli. 1-: 500,000 30 1,119,000 IO 1 : 175,000 15 6,000 9 4 Tap water 5% sewage B. coli. 30 60 1,158,500 9 9 1 : 330,000 15 120,064 7 1 : 330,000 30 9,146 7 5 Deep yellow polluted river water B. 1 : 330,000 60 7 coli. — 33,152 1 : 250,000 20 10,940 13 1 : 250,000 40 852 6 Tap water B. typhosus. 1 : 250,000 60 1,155,400 7 Hard water B. typhosus. 1 : 225,000 20 85,400 ' 17 8 Tap water 5% sewage B. typhosus. 1 : 333,333 20 66,017 14 1 : 333,333 20 242 14 9 Tap water B. paratyphosus A. 1 : 333,333 40 112,000 1 : 225,000 20 17 io Tap water 5% sewage B. paraty- phosus A. . 29,400 1 : 333,333 20 15 12 ii Tap water B. paratyphosus B. 1 : 333,333 40 322,500 1 : 225,000 20 10 17 1 : 225,000 40 110 i A HANDBOOK OF ANTISEPTICS Experiments with p-Sulphondichloraminobenzoic Acid in Tablet Form. — Continued. No. Water Treated Concentra- tion or Dis- < Surviving Organisms Q w > ft infectant PER C.C. pa 5 12 Tap water 5% sewage B. paraty- phosus B. 130,776 1 : 333,333 20 290 12 13 Tap water V. cholerae. 1 : 333,333 40 13,706 14 Tap water 5% Sewage V. Cholerae. 1 : 450,000 20 11,170 I3J 13 15 Tap water B. dysenteriae (Flexner). 1-333,333 20 66,998 1 : 450,000 15 25 13 16 Tap water 5% sewage B . dysenteriae (Flexner) . 1 : 450,000 30 164,864 1 : 333,333 20 38 13 17 Tap water B. dysenteriae (Shiga). 1 : 333,333 40 31,200 1 : 333,333 20 10,934 II 18 Tap water 5% sewage B. dysenteriae (Shiga). 1 : 333,333 40 2,108 1 : 333,333 20 13 From the results in the table it appears that a concentra- tion of 1 : 300,000 is sufficient to sterilize an ordinarily heavily contaminated water in about thirty minutes. Such a con- centration could be relied upon to remove coli, typhoid, or cholera organisms. Special experiments showed that the substance in tablet form was efficacious when acting on water contained in aluminium bottles, although a very trifling action on the metal may be observed if tablets are allowed to remain undisturbed in long contact with the metal. We THE DISINFECTION OF WATER 111 believe such action to be of no practical moment. The concentration of disinfectant given above is just perceptible to the taste, especially in warm water containing little or- ganic matter, but the water is perfectly palatable. Tea brewed with or without the addition of halazone to the water cannot be distinguished. One point of advantage possessed by the present disinfectant over most hypo- chlorite preparations is the fact that the active chlorine is less rapidly used up, so that the process of disinfection con- tinues for a longer period. 1 Even in large doses the substance is not toxic (250 milli- grams per kilo) and is excreted by rabbits in the urine as p-.sulphonamidobenzoic acid. Preparation and Properties of Halazone. — The starting point in the preparation of the substance is p- . toluenesulphonamide, a product which is readily obtained by the action of ammonia on p-toluenesulphonic chloride. The latter substance is a cheap waste product in the manu- facture of saccharine, and is available in relatively large quantities. It is also used for the manufacture of chloramine- T, and dichloramine-T. Toluenesulphonamide is oxidized to p-sulphonamido- benzoic acid, and the latter substance on treatment with chlorine under suitable conditions gives the desired dichlor- amino acid. The reactions may be expressed as follows : CH 3 COOH COOH S0 2 NH 2 S0 2 NH 2 S0 2 NC1 2 The experimental details are as follows: Add 250 grams commercial sodium dichromate to a mixture of 200 cc. 1 It may be noted that " halazone" is unsuited for use as a preservative for milk or other foodstuffs, or as a general antiseptic. 112 A HANDBOOK OF ANTISEPTICS sulphuric acid and 600 cc. water contained in a 2 liter round flask. Then add 100 grams crude toluene-p-sulphonamide and heat on a sand bath with reflux condenser for one hour, using a small flame *at first as the reaction is vigorous. On cooling wash the separated crystals well with cold water, and then dissolve them in hot dilute sodium hydroxide in slight excess. Filter hot, and add excess of hydrochloric acid and when cold filter off the precipitated sulphonamido- benzoic acid, wash well with water and dry. The yield is about 80 per cent of theory. Twenty grams of p-sulphonamidobenzoic acid are dis- solved in 200 cc. approximately normal sodium hydroxide (2 mols.) warming if necessary. About 200 grams of crushed ice is then added, and the mixture saturated with a rapid current of chlorine. The reaction is most conven- iently carried out in a fairly wide-mouthed flask, which may be shaken while the gas is being introduced. If the tempera- ture should rise more ice can be added. A white, rather chalky precipitate of the dichloramino acid is at once precipitated. The acid is filtered off, using suction, well washed with cold water, and dried in vacuo on a porous plate. The dry sub- stance is practically pure, and may be powdered and preserved apparently indefinitely. Prepared by this method, the yield of the dichloramino acid is practically the theoretical amount. The substance is sparingly soluble in water and in chloro- form and insoluble in petroleum. It readily dissolves in glacial acetic acid, crystallizing in stout prisms, which melt at 213 C. The substance explodes feebly when rapidly heated on platinum foil, but, compared with most members of the group, is remarkably stable. The purity of the compound may be checked by titration as follows : 0.1 gram is weighed out, dissolved in 50 per cent acetic acid and potassium iodide added. The liberated iodine is titrated with decinormal sodium thiosulphate, of which 14.8 to 14.9 cc. will be required (see p. 40). THE DISINFECTION OF WATER 113 The dichloramino acid dissolves, apparently without change, in excess of cold sodium hydrate solution, and may be reprecipitated on addition of acids. With smaller quan- tities of sodium hydroxide or with feebly alkaline salts, such as phosphates or borates, hydrolysis occurs, with liberation of disagreeably smelling compounds of nitrogen and chlorine. Dry sodium carbonate or dry borax appear the most satis- factory alkaline salts for making tablets containing halazone. A convenient formula for tablets weighing ioo to 105 mg. is to use sulphondichloraminobenzoic acid 4 per cent, sodium carbonate 4 per cent (or dried borax 8 per cent) sodium chloride (pure) 92 per cent. The acid should be ground up with the dry salt and the sodium carbonate or borax added subsequently. The mixture, which must be kept perfectly dry, may be passed through a 40 mesh sieve. No lubricant or other addition is necessary and should be avoided. The strength of the tablets should be tested by dissolving in 50 per cent acetic acid and potassium iodide solution, and titrating with decinormal sodium thiosulphate as al- ready described (p. 40) (1 cc. of N/10 thiosulphate = 0.00675 gram of the dichloramino acid). They must not be allowed to dissolve in water and subsequently titrated, as then de- composition occurs. The tablets should be stored in small amber glass bottles, to protect them from the action of light. Tablets so prepared of the weight mentioned contain about 4 mg. of the disinfectant, and are suitable for the steriliza- tion of a liter or quart of reasonably heavily contaminated water. In the case of extreme contamination a second tablet may be necessary. 1 The practical success of the disinfectant depends very largely on the stability of the tablet. It appears, as judged by several months' observation, that the tablets are quite stable enough for practical use. They are certainly more 1 Halazone tablets can be obtained from Boots, Island Street, Nottingham, England, or Abbots of Chicago and doubtless other firms. 114 A HANDBOOK OF ANTISEPTICS permanent than other similar compounds with which we are acquainted, and when kept in amber bottles under or- dinary conditions, and at temperatures not exceeding 3o°C, less than 5 per cent decomposition was observed in 150 days. It is rather difficult to give precise estimates of the cost of the finished product, but it is safe to say that the tablets could be sold at such a price that 100 gallons of water could be sterilized at a cost of one penny. Sodium Bisulphate and Other Acid Substances. — The injurious effect of acids upon the cholera vibrio and typhoid bacillus was pointed out long ago by Koch and Kitasato, Stutzen, and others. From Kitasato's experiments it ap- peared that from about 0.2 to 0.5 per cent of most acids was necessary to free water from typhoid bacilli in a few hours, but that a notably lower concentration of sulphuric acid was effective. The following table has been compiled by Colonel Horrocks, illustrating the effect of various acid substances upon typhoid bacilli. Per Cents of Reagent Acids Growth Growth Restrained No Growth Sulphuric Acid Hydrochloric Acid Nitric Acid Sulphurous Acid Phosphoric Acid Acetic Acid Formic Acid Oxalic Acid Lactic Acid Tartaric Acid Citric Acid Malic Acid Tannic Acid ....... Boric Acid 0.049 O.I O.I 0.09 0.15 0.2 0.22 0.23 0.27 0.338 0.338 0.338 1-3 i-5 0.065 0.158 O.I57 0.2 0.224 0.255 0.278 0.285 0.36 0.384 0.384 0.384 2.0 0.08 0.2 0.2 0.28 0.3 0.3 o.34 0.366 0.4 0.476 0.476 0.476 . 1 66 2.7 THE DISINFECTION OF WATER 115 In these experiments broth and gelatine were carefully neutralized before adding the substances, then inoculated with B. typhosus and kept at room temperature, subcul- tures being taken at 4-5 and 10-15 hours. Considerably greater disinfecting action was obtained with waters contain- ing a minimum of organic matter. The possibility of using an acid mixture for the sterilization of small quantities of water was put into practical form by the suggestion that solid tablets of acid sodium sulphate might be employed. From the experiments of Rideal x and Parkes it appears that one gram of sodium bisulphate per pint of water is adequate for the sterilization of waters moderately heavily contaminated with typhoid bacilli in fifteen minutes, while very heavily infected waters may require forty-five minutes. Under similar conditions B. enteritidis is destroyed . about as readily as B. typhosus while the V. cholera is more sensitive than either. Sodium bisulphate tablets were used by the New Zealand contingent in the South African War and subsequently to some extent by cavalry and mounted police and other mo- bile forces. They are frequently compounded with lemon oil and saccharin so that the resulting solution has some resemblance to lemonade. The taste of this acid mixture is actively disliked by some individuals but well tolerated by others. The acid sulphates have the disadvantage of attacking metallic water bottles to a marked extent and while undoubtedly useful, do not possess the potency or security afforded by a disinfectant of the chlorine group. The tablets apart from mechanical disintegration have the advantage of being indefinitely stable. 1 Rideal, Disinfection and the Preservation of Food, John Wiley & Sons, 1903, P- 376. 116 A HANDBOOK OF ANTISEPTICS III. The Disinfection of Hospital Ships, etc., with Sodium Hypochlorite Prepared by Electrolysis of Sea Water or Brine In the following section an account is given of a relatively simple and cheap apparatus for the preparation of consider- able quantities of sodium hypochlorite by the electrolysis of sea water. The results obtained by the use of hypochlorite disinfectant in reducing secondary infections on large hos- pital ships carrying many infectious cases, have been suffi- ciently striking to justify, perhaps, the inclusion of an abridged report on the use of this apparatus. Opportunities for the employment of the apparatus are by no means restricted to hospital ships for the only requi- sites are salt water or brine and a supply of direct current electrical energy. The preparation of sodium hypochlorite by the electrolysis of salt solutions is an old process, and since Watt's initial discovery in 1859 innumerable modifications in its mode of production have been introduced, chiefly for industrial purposes. The deodorizing and germicidal properties of the hypochlorites have been known for more than a century, and it is impossible to refer to many of the applications for hygienic purposes which these substances have found. Reference may be made, however, to the well-known Hermite process for the sterilization of sewage by electrolytic hypochlorite, introduced about 1893 ; to the wide employ- ment of hypochlorites for the purification of suspected potable waters, and to the many uses as disinfectant which hypochlorites have found in sanitary work. Elec- trolytic hypochlorite for general disinfection purposes has been successfully employed by a great many sanitary authorities. The apparatus to be described was originally employed on the Hospital Ship "Aquitania" and since then has been ELECTROLYTIC HYPOCHLORITE 117 adopted on a number of other ships and coast stations. 1 It was first of all necessary to devise an apparatus suitable for use on shipboard, then to determine favorable conditions for carrying out the electrolysis and to decide on suitable methods for applying the product. Nothing essentially new is embodied in the design or use of the apparatus but many existing electrolyzers are either unsuitable or subject to patent restrictions which limit their usefulness. Description of Apparatus. — The necessary apparatus comprises an electrolytic cell, a reversing switch capable of carrying a hundred amperes and some ordinary insulated electric cable. A scale drawing of the electrolytic cell is given on the following pages* The electrolyzer consists of a rectangular box made of teak or cedar securely bolted together. It may be coated internally with marine glue to protect the wood and to reduce the risk of leaking. The interior of the box is divided into twenty or preferably twenty-five cells by means of carbon plates placed parallel to one another. The plates situated at both ends of the box project above the level of the other plates to receive the terminals for the introduction of the current. The terminal electrodes for convenience are made in four pieces placed side by side, while the intermediate carbons are made in three parts superimposed vertically one upon the other. A copper plate attached to the four binding screws at each end insures an even distribution of current. The carbon plates are separated from each other by strips of vulcanite or wood, or glass tubes, and they are kept in position by means of a " making up block" and wedges placed at one end of the tank. A grooved channel is cut along one side of the floor of the wooden tank underneath the carbon plates in order to facilitate emptying by means of a wooden or vulcanite plug tap inserted in a hole con- 1 Dakin and Carlisle; Jour. Roy. Army Medical Corps, Feb., 1916. 118 A HANDBOOK OF ANTISEPTICS nected with the floor of the tank. A word must be added as to the necessity of employing suitable carbon plates. These should be of Acheson graphite prepared by treating amorphous carbon in the electric furnace. Ordinary carbon plates rapidly deteriorate and cannot be used satisfactorily. The tank should be mounted on a rubber mat or on glass or porcelain insulators, and securely fixed to a low table so that its contents can be run conveniently into a tub placed beneath it. The necessary electrical connections are made as follows : — two wires leading the ship's current (one hundred to one hundred and ten volts, direct current) are connected re- spectively with the middle pair of binding screws in the reversing switch. The two binding screws on both sides of the switch are each provided with leading wires which are attached to the electrolyzer in such a fashion that the two wires leading to either side of the switch are attached to opposite ends of the electrolyzer. Alternating current cannot be used for the electrolysis but must be transformed. Most ships furnish direct current of suitable voltage, namely, one hundred to one hundred and ten volts. In the case of the voltage being two hundred to two hundred and twenty volts, two electrolyzers can be placed in series. The appara- tus is extremely simple and there is nothing to get out of order. It can be easily operated by an untrained person after receiving a few minutes' instruction. The wear and tear of the apparatus is extremely small. The carbon plates are capable of giving many months' service before renewal is necessary. The apparatus as described above was made by Messrs. Mather and Piatt, Park Works, Manchester. We are in- debted to Dr. Edward Hopkinson, a director of the company, for much valuable assistance in constructing a practical and efficient apparatus. 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