Bacteria / Especially as they are related to the economy of nature, to industrial processes, and to the public health

Influenza. In 1892, during the pandemic of influenza, Pfeiffer discovered a bacillus in the bronchial mucus of patients suffering from the disease. It is one of the smallest bacilli known, and frequently occurs in chains not unlike a streptococcus. Carron obtained the same organism from the blood. In the bronchial expectoration it can retain its virulence for as long as a fortnight, but it is quickly destroyed by drying. The bacillus is aërobic, non-motile, and up to the present spores have not been found. It grows somewhat feebly in artificial media, and readily dies out. Blood serum, glycerine agar, broth, and gelatine have all been used at blood-heat. It does not grow at room temperature. Pfeiffer's bacillus appears most abundantly at the height of the disease, and disappears with convalescence. It is said not to appear in any other disease.

Yellow Fever. Sternberg and Havelburg have both isolated bacilli from cases of yellow fever; but the organism discovered by Sanarelli, the Bacillus icteroides, is now accepted as the causal agent of the disease. It is a small, short rod, with round ends, and generally united in pairs; it has various pleomorphic forms; it grows well on all the ordinary media; it is killed in sea-water at 60° C., and also by direct sunlight in a few hours.

Diarrhœa of Infants. From time to time different organisms have been isolated in this diseased condition. Bacillus coli and B. enteriditis sporogenes (Klein) have been held responsible for it. W. D. Booker, of Johns Hopkins University, sums up an extended research into the question as follows:

Actinomycosis. This disease affects both animals and man. As Professor Crookshank points out, it has long been known in this country,100 but its various manifestations have been mistaken for other diseases or have received popular names.

Here we can only mention the most outstanding facts concerning the disease. It is caused by the "ray fungus," or Streptothrix actinomyces, which, growing on certain cereals, often gains entrance to the tissues of man and beast by lacerations of the mucous membrane of the mouth, by wounds, or by decayed teeth. Barley has been the cereal in question in some cases. The result of the introduction of the parasite is what is termed an "infective granuloma." This is, generally speaking, of the nature of an inflammatory tumour composed of round cells, epithelioid cells, giant cells, and fibrous tissue, forming nodules of varying sizes. In some cases they develop to large tumours, in others they soon break down. Actinomycosis in some ways closely resembles tuberculosis in its tissue characters.

In the discharge or pus from human cases of the disease small sulphur-yellow bodies may be detected, and these are tufts of "_clubs_" which are the broken-down rays of the parasite; for in the tissues which are affected the parasite arranges itself in a radiate manner, growing and extending at its outer margin and degenerating behind. In cattle the centre of the old ray becomes caseated, like cheese, or even calcified, like a stone. In the human disease abundant "_threads_" are formed as a tangled mass in the middle of the colony. As clubs characterise the bovine actinomycosis, so threads are a feature of the human form of the disease. But in both there is a third element, namely, small round cells, called by some spores, by others simply cocci. Authorities are not yet agreed as to the precise significance and rôle of these round cells. The life-history of the micro-organism may be summed up thus:

Possibly these clubs represent organs of fructification, and produce the spores. These latter are, it is believed, set free in the vicinity of the ray, and create fresh centres of disease.

In man the disease manifests itself in various parts according to the locality of entrance. When occurring in the mouth it attacks the lower jaw most frequently. In one recorded case the disease was localised to the bronchi, and did not even extend into the lungs. It was probably contracted by inhalation of the parasite. The disease may spread to distant parts by means of the blood stream, and frequently the abscesses are apt to burrow in various directions.

In the ox the disease remains much more localised, and frequently occurs in the lower jaw, palate, or tongue. In the last site it is known as "wooden tongue," owing to the hardness resulting. The skin and subcutaneous tissues are also a favourite seat of the disease, producing the so-called wens or clyers so commonly seen in the fen country (Crookshank). Actinomycosis in cattle is specially prevalent in river valleys, marshes, and on land reclaimed from the sea. The disease occurs at all seasons, but perhaps more commonly in autumn and winter. It is more frequently met with in young animals. The disease is probably not hereditary nor readily communicated from animal to animal.

Actinomyces may be cultivated, like other parasitic diseases, outside the body. Gelatine, blood serum, agar, glycerine agar, and potato have been used for this purpose. After a few days on glycerine agar at the temperature of the blood little white shining colonies appear, which increase and coalesce. In about ten days' time the culture often turns a bright yellow, though it may remain white or even take on a brown or olive tint. The entire mass of growth is raised dry and crinkled, and composed almost exclusively of threads. In its early stage small bacillary forms occur, and in its later stage coccal forms. True clubs never occur in pure cultures, although the threads may occasionally show bulbous endings.

Glanders in the horse and ass, and sometimes by communication in man also, is caused by a short, non-motile, aërobic bacillus, named, after the old Roman nomenclature (malleus), Bacillus mallei. It was discovered in 1882 by Löffler and Schütz. It is found in the nasal discharge of glandered animals. In appearance the bacillus is not unlike B. tuberculosis, except that it is shorter and thicker. The beading of the bacillus of glanders, like that in tubercle, does not denote spores. B. mallei can be cultivated on the usual media, especially on glycerine agar and potato. On the latter medium it forms a very characteristic honey-like growth, which later becomes reddish-brown.

In the horse glanders particularly affects the nasal mucous membrane, forming nodules which degenerate and emit an offensive discharge. From the nose, or nasal septum, as a centre, the disease spreads to surrounding parts. It may also occur as nodules in and under the skin, when it is known as "farcy." Persons attending a glandered animal may contract the disease, often by direct inoculation.

Mallein is a substance analogous to tuberculin, and is made by growing a pure culture of Bacillus mallei in glycerine-veal broth in flat flasks, with free access of calcined air. After a month's growth the culture is sterilised, filtered, concentrated, and mixed with an equal volume of a .5 per cent. solution of carbolic. The dose is 1 cc., and it is used, like tuberculin, for diagnostic purposes. If the suspected animal reacts to the injection, it is suffering from glanders. Reaction is judged by three signs, namely, a rise of temperature 2–3° C., a large "soup-plate" swelling at the site of inoculation, and an enlargement of the lymphatic glands.

Swine fever, foot-and-mouth disease, chicken cholera, dysentery, rinderpest, and other diseases of animals have micro-organisms intimately related to them.

There is a group of diseases due to the presence in the blood or tissues of hæmatozoa, that is, protozoa which can live and perform their function in the blood. Amongst these are malaria, sleeping sickness, and other tropical diseases in man, and surra and various hæmatozoa in horses, fish, frogs, or rats.

Malaria. Although a Bacillus malariæ has been described as the cause of this disease, it is now almost universally supposed that the true cause is a protozoan parasite. In 1880 Laveran first described this organism, and the discovery was confirmed by Marchiafava, Celli, and others. Laveran claimed that it occurred in four different forms during the progress of its life-history:

(a) Spherical or Irregular Bodies attached to the blood corpuscle, or free in the blood plasma. They are a little smaller than the blood-cells, and may or may not contain pigment. They eventually invade the corpuscles, possess more pigment, and lose their amœboid movement. Within the red blood corpuscles they increase in size until they reach the adult stage.

(b) Segmentation Forms, often assuming a rosette shape, follow next. They are pigmented, are possibly a sporing stage, and are finally set free in the blood.

(c) The Crescents, or Semilunar Bodies, are free in the blood, but motionless. They are colourless, have a distinct membrane, and generally show a little pigment about the middle; they taper towards the poles. They appear in the blood after the fever has existed for some time, occurring chiefly, sometimes only, in the quotidian and malignant types of malaria.

(d) The Flagellated Bodies apparently occur only in the blood outside the body. They are extracorpuscular bodies, and possess several long flagella, and are therefore actively motile. They are derived from the crescents or irregular intracorpuscular bodies.

What is the precise significance of these various forms and modifications of them is not at present known. Possibly the semilune is a resting stage inside the body, and the flagellated body another similar stage outside. Attempts to cultivate the parasite outside the body have failed. There is a good deal of evidence to show that the mosquito is the host outside the human body. There may be different forms and varieties of parasite, if not actually different species, causing the diverse forms of clinical malaria.

The above account of diseases caused by bacteria does not profess to be in any sense exhaustive. It is merely illustrative. It reveals some of the disease-producing powers of micro-organisms. There are a large number of other diseases in which bacteria have been found. They are not the causes, but only accidentally present or associated with "secondary infection." Variola (small-pox), scarlet fever, and measles are excellent examples. It is possible that the danger at the present time is rather in the direction of supposing that every disease will readily yield its secret to the bacteriologist. Such, of course, is not the case. Nevertheless, as in the past, so in the future, constant research and patient investigation is the only hope we have for the elucidation of truth in respect to the causes of disease.

CHAPTER IX

DISINFECTION

The object of modern bacteriology is not merely to accumulate tested facts of knowledge, nor only to learn the truth respecting the biology and life-history of bacteria. These are most important things from a scientific point of view. But they are also a means to an end; that end is the prevention of preventable diseases and the treatment of any departure from health. In a science not a quarter of a century old much has already been accomplished in this direction. The knowledge acquired of, and the secrets learned from, these tiny vegetable cells which have such potentiality for good or evil have been, in some degree, turned against them. When we know what favours their growth and vitality and virulence, we know something of the physical conditions which are inimical to their life; when we know how to grow them, we also know how to kill them.

We have previously made a cursory examination of the methods which are adopted for opposing bacteria and their products in the tissues and body fluids. We must now turn to consider shortly the modes which may be adopted in preventive medicine for opposing bacteria outside the body.

It will be clear at once that we may have varying degrees of opposition to bacteria. Some substances kill bacteria, and they are known as germicides; other substances prevent their development and resulting septic action, and these are termed antiseptics. The word disinfectant is used more or less indiscriminately to cover both these terms. A deodorant is, of course, a substance removing the odour of evil-smelling putrefactive processes. Here, then, we have the common designations of substances able to act injuriously on bacteria and their products outside, or upon the surface of, the body. But a moment's reflection will bring to our minds two facts not to be forgotten. In the first place, an antiseptic applied in very strong dose, or for an extended period, may act as a germicide; and, vice versâ, a germicide in too weak solution to act as such may perform only the function of an antiseptic. Moreover, the action of these disinfecting substances not only varies according to their own strength and mode of application, but it varies also according to the specific resistance of the protoplasm of the bacteria in question. Examples of the latter are abundant, and readers who have only assimilated the simple facts set forth in these pages are aware that between the bacillus of diphtheria and the spores of anthrax there is an enormous difference in power of resistance. In the second place, reflection will enable us to recall what has already been said, when discussing the requirements necessary for bacterial growth, respecting the physical conditions injurious to development. In a cold temperature, as a general rule, bacteria do not multiply with the same rapidity as at blood-heat. Within the limits of a moist perimeter the air is, to all intents and purposes, germ-free. Direct sunlight has a definitely germicidal effect in the course of time upon some of the most virulent bacteria we know. Here, then, are three examples of physical agents—low temperature, moist perimeter, sunlight—which, if strong enough in degree, or acting for a long enough period of time, become first antiseptics and then germicides. Yet for a limited period they have no injurious effect upon bacteria. These are simple points, and call for little comment, yet the pages of medical and sanitary journals reveal not a few keen controversies upon the injurious action of certain substances upon certain bacteria owing to the discrepancies, of necessity arising, between results of different skilled observers who have been carrying out different experiments with different solutions of the same substance upon different protoplasms of the same species of bacteria. We feel no doubt that in these pioneering researches much labour has been to some extent misspent, owing to the neglect of a common denominator. Only a more accurate knowledge of bacteria or a recognised standard for disinfecting experiments can ever supply such common denominator.

Species of bacteria for comparative observation-experiments upon the action of chemical or physical agents must be not only the same species, but cultured under the same conditions, and treated by the agent in the same manner, otherwise the results cannot be compared upon a common platform, or with any hope of arriving at exactly the same conclusions.

Sir George Buchanan laid down, in 1884, a very simple and suitable standard of what true disinfection meant, viz., the destruction of the most stable known infective matter. Such a test is high and difficult to attain unto; nevertheless, it is the only satisfactory one. Obviously many substances which are useful antiseptics in practical life would fall far short of such a standard, yet for true and complete disinfection such an ideal is the only adequate one.

Quite recently three or four workers at Leipzig101 have drawn up simple directions, the adoption of which would considerably assist in securing a common standard for disinfectant research. They are as follows:

1. In all comparative observations it is imperative that molecularly equivalent quantities of the reagents should be employed.

2. The bacteria serving as test objects should have equal power of resistance.

3. The numbers of bacteria used in comparative observation should be approximately equal.

4. The disinfecting solution must be always used at the same temperature in comparative experiments.

5. The bacteria must be brought into contact with the disinfectant with as little as possible of the nutrient material carried over. (This obviously will depend upon the object of the research.)

6. After having been exposed to the disinfectant for a fixed time, they should be freed from it as far as possible.

7. They should then be returned in equal numbers to the respective culture medium most favourable to the development of each, and kept at the same, preferably the optimum, temperature for their growth.

8. The number of surviving bacteria capable of giving rise to colonies in solid media must be estimated after the lapse of equal periods of time.

We may now turn from general principles to mention shortly some of the commoner methods and substances adopted to secure efficient disinfection. They are all divisible, according to Sir George Buchanan's standard, into two groups:

1. Heat in various forms;

2. Chemical bodies in various forms.

It should at the outset be understood that we desire in practical disinfection to inhibit or kill micro-organisms without injury to, or destruction of, the substance harbouring the germs for the time being. If this latter is of no moment, as in rags or carcasses, burning is the simplest and most thorough treatment. But with mattresses and beddings, bedclothes and garments, as well as with the human body, it is obvious that something short of burning is required.

1. From the earliest days of bacteriology heat has held a prominent place as a disinfector. But it is only in comparatively recent times that it has been fully established that moist heat is the only really efficient form of heat disinfection. Boiling at atmospheric pressure (100° C.) is the oldest form of moist heat disinfection, and because of the simplicity of its application it has gained a large degree of popularity. But it must not be forgotten that mere boiling (100° C.) may not effectually remove the spores of all bacilli. Besides, boiling is not applicable to furniture, mattresses, and such-like frequently infected objects. For many of these hot-air ovens were used in the early days. But it was found that such disinfection was no disinfection at all, for not only did it leave many organisms and spores untouched, but the degree of temperature was rarely, if ever, uniform throughout the substance being treated.

The failures following in the track of these methods were an indication of the need of some form of moist heat, viz., steam.

Here it will be necessary to digress for a moment into some of the characters of steam. When water is heated certain molecular changes take place, and at a certain temperature (100° C., 212° F.) the water becomes steam, or vapour, and on very little cooling will condense. But if the vapour is heated, it will become practically a gas, and will not condense until it has lost the whole of the heat, i. e., the heat of making water into vapour plus the heat of making vapour into gas. A gas proper is, then, the vapour of a liquid of which the boiling-point is substantially below its actual temperature. But we know that the temperature at which it boils depends upon the pressure to which it is subjected (Regnault's law). Hence in reality "steam at any temperature whatever may be a vapour proper, provided the pressure is such as prevents the liquid from boiling below that temperature." In such a condition of vapour it is termed saturated steam. But if it is at that same pressure further heated, it becomes practically a gas, and is called superheated steam. The former can condense without cooling; the latter cannot so condense at the same pressure. Saturated steam condenses immediately it meets the object to be disinfected, and gives out its latent heat; superheated steam acts by conduction, and not uniformly throughout the object. Its advantage is that it dries moistened objects. As a disinfecting power, superheated steam is much less than saturated steam. There is one further term which must be defined, namely, current steam. This is steam escaping from a disinfector as fast as it is admitted, and may be at atmospheric or higher temperatures. The disinfecting temperature which is now used as a standard is an exposure to saturated steam of 115° C. for fifteen minutes.

A number of different kinds of apparatus have been invented to facilitate disinfection to this standard on a large scale. Most sanitary authorities of importance are now supplied with some form of steam disinfector, though many are unable to go to the expense of high-pressure disinfectors. Professor Delépine has pointed out102 that a current of steam at low pressure may completely disinfect. Whilst such simple current-steam machines have thus been demonstrated as efficient bactericides, for all practical purposes it is important to have disinfectors capable of giving temperatures considerably above 100° C., of simple construction, having steam power of uniform temperature and rapid penetration, and containing, when in action, a minimum of superheated steam. In addition to these characters of a first-rate steam disinfector, two other important points should be borne in mind, namely, the air must be completely ejected from the disinfection chamber before the results due to steam are obtained, and some sort of automatic index giving a record of each disinfection is indispensable.

We may turn from these general principles to mention shortly some of the types of steam disinfectors most commonly in use. They are four, namely, the Washington Lyon, the Equifex (Defries), the Thresh, and the Reck.

Washington Lyon's apparatus consists of an elongated boiler having double walls, with a door at each end. The body of the apparatus is jacketed. The whole is large enough to admit of bedding and mattresses, and generally is so arranged that one end opens into one room, and the other end opens into another room. This convenient position admits of inserting infected articles from one room and receiving them disinfected into the other room. Possible reinfection is thereby prevented. Steam is admitted into the jacket at a pressure of between twenty and twenty-five pounds, and is generally twenty pounds in the interior of the cylinder. At the end of the operation a partial vacuum is created, by which means much of the moisture on the articles may be removed. In some cases a current of warm air is admitted before disinfection in order to diminish the extent of condensation.

The Equifex (Defries) contains no steam jacket, but coils of pipes are placed at the top and bottom of the apparatus, with the object of imparting to the steam as much heat as is lost by radiation through the walls of the disinfecting chamber, and at the same time of preventing undue condensation. The air is first removed by a preliminary current of steam, after which steam at a pressure of ten pounds is intermittently introduced and allowed to escape. The object of this proceeding is to remove air from the pores of the articles to be disinfected by the sudden expansion of the film of water previously condensed on their surface. The apparatus introduced by Dr. Thresh was constructed with a view of overcoming the objection to some of the other machines that bulky articles retained a large percentage of moisture, thus necessitating the use of some additional drying apparatus. A central chamber receives the articles to be disinfected, and is surrounded by a boiler containing a solution of calcium chloride at a temperature of 225° F. This is heated by a small furnace, and the steam given off (218–300° F.) is conducted into the central chamber. The steam is not confined under any pressure except that of the atmosphere. When the steam has passed for a sufficient length of time, it is readily diverted into the open air. Hot air is now introduced, and at the expiration of an hour the articles may be taken out disinfected and as dry as they were when inserted. The apparatus is comparatively inexpensive, and not of a complicated nature. The current steam is saturated, and at a temperature a few degrees above the boiling-point. Many experiments have been performed with this apparatus, and there is now a large amount of evidence in favour of it and current steam disinfection.

Reck's apparatus is another kind of saturated steam disinfector, which resembles the Equifex, but differs from it in employing steam as a current.

It is probable that many other forms of steam disinfector will be invented, and each will have its enthusiastic supporters. Even at the time of writing some excellent results are announced from America.

2. The effects of chemical substances as solutions, or in spray form, upon bacteria have been observed from the earliest days of bacteriology. To some decomposing matter or solution a disinfectant was added and sub-cultures made. If bacteria continued to develop, the disinfection had not been efficient; if, on the other hand, the sub-culture remained sterile, disinfection had been complete. From such rough-and-ready methods large deductions were drawn, and it is hardly too much to say that no branch of bacteriology contains such a vast mass of unassimilated and unassimilable statements as that relating to research into disinfectants. Most of the tabulated and recorded results are conspicuous in having no standard as regards bacterial growth. Yet without such a standard results are not comparable.

Silk threads, impregnated with anthrax spores, were placed in bottles containing carbolic acid of various strengths, and at stated periods threads were removed and placed in nutrient media, and development or otherwise observed. But, as Professor Crookshank103 has pointed out, this method is fallacious, the thread being still wet with the solution when transferred to the medium, and thus modified in culture, possibly even inhibited altogether. It is unnecessary for us here to discuss every mode adopted by investigators in similar researches. We may just mention that the most approved methods at the present time are based upon two simple plans of exposure. In one we use a known volume of recent broth culture of an organism grown under specified conditions. To this is added a measured quantity of the antiseptic. At stated periods loopfuls of the broth and antiseptic mixture are sub-cultured in fresh-sterilised broth, and resulting development or otherwise closely observed. The other method is practicable when we are dealing with volatile bodies. In such cases a standard culture is made of the organism in broth at a standard temperature. Into this are dipped small strips of sterilised linen. When thoroughly impregnated these are removed from the broth and subsequently dried over sulphuric acid in a vacuum at 38° C. These may now be exposed for a longer or shorter period to the fumes of the antiseptic in question, and broth cultures made at the end of the exposure. It is obvious that a very large number of modifications are possible of these two simple devices for testing the bactericidal power of chemical substances. It should be remembered that here, perhaps, more than anywhere else in bacteriological research, careful control experiments are absolutely necessary.

Mineral acids (nitric, hydrochloric, sulphuric), especially concentrated, are all germicides.

The halogens—chlorine, bromine, iodine, and fluorine—are, all four, disinfectants, but not used in practice. They are named in their order of power as such.

A number of separate bodies, such as chloroform and iodoform, have been much advocated as antiseptics. The cost of the former and odour of the latter have, however, greatly militated against their general adoption.

Chloride of lime is a powerful disinfectant. Professor Sheridan Delépine and Dr. Arthur Ransome have demonstrated its germicidal effect as a solution applied directly to the walls of rooms inhabited by tuberculous patients.104 It may also be used in solid form for dusting decomposing matter.

Mercuric chloride (corrosive sublimate) has been an accepted germicide for some time. But the experiments of Behring, Crookshank, and others have proved that the weaker solutions cannot be relied upon. This is, in part, due to the fact that it forms in albuminous liquids an albuminate of mercury which is inactive. Dilute solutions have the further disadvantage of being unstable. Various authorities recommend a solution of 1–500 as a germicide, and much weaker solutions are, of course, antiseptic. An ounce each of corrosive sublimate and hydrochloric acid in three gallons of water makes an efficient disinfectant.

Potassium permanganate is, of course, the chief substance in Condy's fluid, as zinc chloride is in Burnett's disinfecting fluid. A 5 per cent. of the former and a 2-1/2 per cent. of the latter are germicidal.

Boracic acid is one of the most useful antiseptics with which to wash sore eyes, or preserve tinned foods or milk. It is not a strong germicide, but an unirritating and effective wash. Many cases of its addition to milk have found their way into the law courts, owing to cumulative poisoning, and it should only be used with the very greatest care as a food preservative.

Carbolic acid has come into prominence as an antiseptic since its adoption by Lister in antiseptic surgery. It is cheap, volatile, and effective. One part in 400 is antiseptic, and 1 in 20 germicidal. As a wash for the hands the former is used, and a weaker solution for the body generally. Carbolic soap and similar toilet combinations are now very common. At one time it appeared as if corrosive sublimate would oust carbolic from the first place as an antiseptic solution, but a large number of experiments have confirmed opinion in favour of carbolic. Professor Crookshank found that carbolic acid, 1 in 40, acting for only one minute is sufficient to destroy Streptococcus pyogenes, S. erysipelatis, and Staphylococcus pyogenes aureus, and in the strength of 1 in 20 carbolic acid completely sterilised tubercular sputum when shaken up with it for one minute.

Creosol, a member of the phenol series, is a good disinfectant, and the active element in lysol, Jeye's fluid, creoline, izal, and creosote.

Sulphurous acid is one of the commonest disinfectants employed for fumigation—the old orthodox method of disinfecting a room in which a case of infective disease has been nursed. It is evolved, of course, by burning sulphur. For each thousand cubic feet from one to five pounds of sulphur is used, and the walls may be washed with carbolic acid. Dr. Kenwood carried out some experiments in 1896105 which appear to support the disinfecting power of sulphur fumes. He found that the Bacillus diphtheriæ was not killed, though markedly inhibited, when the sulphurous gas (SO₂) did not much exceed .25 per cent. But the bacillus was killed where the sulphur fumes exceeded .5 per cent. Both these results had reference to the

SO₂ in the air in the centre of the room at a height of four feet, and after the lapse of four hours. There can be little doubt that fuming a sealed-up room with sulphur fumes in a moist atmosphere, and leaving it thus for twenty-four hours, is generally, if not always, efficient disinfection. It will kill the bacillus of diphtheria, though not always more resistant germs. Moreover, its simplicity of adoption is greatly in its favour. Anyone can readily apply it by purchasing a few pounds weight of ordinary roll sulphur and burning this in a saucer in the middle of a room which has had all its crevices and cracks in windows and walls blocked up with pasted paper. Nitrous fumes may also be used in this way.

Recently formalin has come much in favour as a room disinfectant. Formalin is a 40 per cent. solution of formaldehyde in water, a gas discovered by Hofmann in 1869. This gas is a product of imperfect oxidation of methyl alcohol, and may be obtained by passing vapour of methyl alcohol, mixed with air, over a glowing platinum wire or other heated metals, such as copper and silver. It is the simplest of a series of aldehydes, the highest of which is palmitic aldehyde. Its formula is CH₂O, and it is a colourless gas with a pungent odour, and having penetrating and irritating properties, particularly affecting the nasal mucous membrane and the eyes of those working with it. It is readily soluble in water, and in the air oxidises into formic acid (CH₂O₂). This latter substance occurs in the stings of bees, wasps, nettles, and various poisonous animal secretions. Formalin is a strong bactericide even in dilute solutions, and, of course, volatile. A solution of 1 to 10,000 is said to be able to destroy the bacilli of typhoid, cholera, and anthrax. A teaspoonful to ten gallons of milk is said to retard souring. When formalin is evaporated down, a white residue is left known as paraform. In lozenge form this latter body is used by combustion of methylated spirit to produce the gas. Hence we have three common forms of the same thing—formalin, formic aldehyde, paraform—each of which yields formic acid, and thus disinfects. The vapour cannot in practice be generated from the formalin as readily as from the paraform.

By a variety of ingenious arrangements formic aldehyde has been tested by a large number of observers during the last two or three years. We may refer to three modes of application. 1. The sprayer (Equifex apparatus) produces a mixture of air and solution for spraying walls, ceilings, floors, and sometimes garments. 2. The autoclave (Trillat's apparatus). In this a mixture of a 30–40 per cent. watery solution of formaldehyde and calcium chloride (4–5 per cent.) is heated under a pressure of three or four atmospheres, and the almost pure, dry gas is conducted through a tube passing through the keyhole of the door into the sealed-up room. 3. The paraform lamp (the Alformant). The principle of this lamp is that the hot, moist products from the combustion of methylated spirit act upon the paraform tablets, converting them into gas. Most of the conclusions derived from experiments with these three different forms of apparatus are the same. It is agreed that the gas is harmless to colours and metal and polished wood. The vapour acts best in a warm atmosphere. As for its action on bacteria, it compares favourably with any other disinfectant. In 1 per cent. solution formalin destroys non-spore-bearing bacteria in thirty to sixty minutes.

Many observers have decried formaldehyde on account of its professed lack of penetrating power. Professor Delépine, however, states106 that it possesses "penetration powers probably greater than those of most other active gaseous disinfectants. Bacillus coli, B. tuberculosis, B. pyocyaneus, and Staph. pyogenes aureus were killed in dry or moist state, even when protected by three layers of filter paper." In

Professor Delépine's opinion, the vapours of phenol, izal, dry chlorine, and sulphurous acid have, under the same conditions, given inferior results.

We may now shortly summarise the foregoing facts respecting antiseptics and disinfection in the simplest terms possible to afford facility to the uninitiated in practical application:

To disinfect a room, seal up cracks and crevices, and burn at least one pound of roll sulphur for every 1,000 cubic feet of space.107 Many authorities recommend four or five pounds of sulphur to the same space. Let the room remain sealed up for twenty-four hours.

To disinfect walls, wash with chloride of lime solution (1–100) or carbolic acid (1–40). This latter solution may be used to wipe down furniture. Either or both may be used after sulphur fuming. Formic aldehyde may also be used by lamp or autoclave.

To disinfect bedding, etc., the steam sterilisation secured in a Thresh, Equifex, or Lyon apparatus is the best. Rags and infected clothing, unless valuable, should be burnt.

To disinfect garments and wearing apparel, they should be washed in a disinfectant solution, or fumed with formic aldehyde.

To disinfect excreta or putrefying solutions, enough disinfectant should be added to produce in the solution or matter being disinfected the percentage of disinfectant necessary to act as such. Adding a small quantity of antiseptic to a large volume of fluid or solid is as useless as pouring a small quantity of antiseptic down a sewer with the idea that such treatment will disinfect the sewage. The mixture of the disinfectant with the matter to be disinfected must contain the standard percentage for disinfection. Chloride of lime is a common substance for use in this way. Potassium permanganate (1–100) and carbolic (1–100), and many manufactured bodies containing them, are also widely used. Drs. Hill and Abram recommend108 that the excreta and disinfectant be thoroughly mixed and stand for at least half an hour. For various reasons they particularly advise chinosol as the most convenient disinfectant for this specific purpose.

Antiseptics for wounds. Carbolic acid (1–40) or corrosive sublimate (1–1,000) are commonly used in surgical practice. Boracic acid is one of the most unirritating antiseptics which are known. It may be used in saturated watery solution (1–30) or dusted on copiously as fine powder. It is especially applicable in open wounds, and as an eye-wash.

To disinfect hands and arms. Operating surgeons are those to whom it is a most urgent necessity to cleanse hands and arms antiseptically. Carbolic acid (1–20, or 1–40) is used for this purpose.

It is hardly necessary to add that in a case of infectious disease occurring in a household many of these modes of application, perhaps all of them, must be adopted. Formalin is probably the best gaseous disinfectant which we have, but its use does not, and should not, preclude the simultaneous adoption of other methods.

APPENDIX

It is proposed to add one or two notes on certain technical points in bacteriological work, with a view to assisting those medical men not able to obtain the advantages of a well-equipped laboratory, and yet desirous of occasionally attempting some practical bacteriology.

1. General Examination. All fluids may be examined for bacteria in two chief ways:

(a) A small quantity may be placed on a cover-glass or slide, dried over a lamp or bunsen flame, and stained with aniline dyes for a few minutes. It is then ready for microscopic examination. It is obvious that the result will generally be a mixture of bacteria, for which differentiating stains may be used (Gram, Ziehl-Neelsen, etc.).

(b) A minute drop of the suspected fluid may be added to various fluid media (broth, liquefied gelatine, etc.) and then plated out upon small sterilised sheets of glass. In the course of two or three days the contained bacteria will reveal themselves in characteristic colonies, which may be examined, and if possible sub-cultured, and carefully studied.

Double-Staining Methods. These are various, and are used when it is desired to stain the bacteria themselves one colour, and the matrix or ground substance in which they are situated another colour. Three of the commoner methods are those of Ehrlich, Neelsen, and Gram. They are as follows:

Ehrlich's Method. "Five parts of aniline oil are shaken up with 100 parts distilled water, and the emulsion filtered through moistened filter paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet is added to the filtrate in a watch-glass, drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen to thirty minutes, then washed for a few seconds in dilute nitric acid (one part nitric acid to two of water), and then rinsed in distilled water. The stain is removed from everything except the bacilli; but the ground substance can be after-stained brown if the bacilli are violet, or blue if they have been stained red" (Crookshank, Bacteriology and Infective Diseases, p. 89).

Gram's Method. The primary stain in this method is a solution of aniline gentian-violet (saturated alcoholic solution of gentian-violet 30 cc., aniline water 100 cc.), which stains both ground substance and bacteria in purple. The preparation is next immersed in the following solution for half a minute or a little more:

In this short space of time the iodine solution acts as a mordant of the purple colour in the bacteria, but not in the ground substance. Hence, if the preparation be now (when it has assumed a brown colour) washed in alcohol (methylated spirit), the ground substance slowly loses its colour and becomes clear. But the bacteria retain their colour, and thus stand out in a well-defined manner. Cover-glass preparations decolourise more quickly than sections of hardened tissue, and they should only be left in the methylated spirit until no more colour comes away. The preparation may now be washed in water, dried, and mounted for microscopic examination, or it may be double-stained, that is, immersed in some contrast colour which will lightly stain the ground substance. Eosin or Bismarck brown are commonly used for this purpose. The former is applied for a minute or two, the latter for five minutes, after which the specimen is passed through methylated spirit (and preferably xylol also) and mounted. The result is that the bacteria appear in a dark purple colour on a background of faint pink or brown. Carbol-thionine blue, picro-carmine, and other stains are occasionally used in place of the aniline gentian-violet, and there are other slight modifications of the method.

Ziehl-Neelsen Method. Here the primary stain is a solution of carbol-fuchsin:

It is best to heat the dye in a sand-bath, in order to distribute the heat evenly. The various stages in the staining process are as follows: (a) The cover-glass with the dried film upon it is immersed in the hot stain for one to three minutes. (b) Remove the cover-glass from the carbol-fuchsin, and place it in a capsule containing a 25 per cent. solution of sulphuric acid to decolourise it. Here its redness is changed into a slate-grey colour. (c) Wash in water, and alternately in the acid and water, until it is of a faint pink colour. (d) Now place the cover-glass for a minute or two in a saturated aqueous solution of methylene-blue, which will counter-stain the decolourised ground substance blue. (e) Wash in water. (f) Dehydrate by rinsing in methylated spirit, dry, and mount. A pure culture of bacteria will not necessarily require the counter-stain (methylene-blue). Sections of tissue may require twenty to thirty minutes in the primary stain (carbol-fuchsin). This stain is used for tubercle and leprosy. With a little practice the staining of the bacillus of tubercle when present in pus or sputum becomes a very simple and accurate method of diagnosis. A small particle of sputum or pus is placed between two clean cover-glasses and thus pressed between the thumb and finger into a thin film. This is readily dried and stained as above, the bacillus of tubercle appearing as a delicately-beaded red rod with a background of blue.

Bacteriological Diagnosis. The following points must be ascertained in order to identify any particular micro-organism:

(1) Its morphology, bacillus, coccus, spirillum, etc.; the presence or absence of involution forms.

(2) Motility by the unstained cover-glass preparation ("hanging drop"); note presence of flagella.

(3) Presence of spores, their appearance and position.

(4) Whether or not the organism stains with Gram's method.

(5) The character of the growth upon various media (gelatine, agar, milk, potato, broth); the presence or absence of liquefaction in the gelatine culture; its power of producing acid, gas, or indol.

(6) Whether it is aërobic or anaërobic.

(7) Its colour in cultivation.

(8) If it is a disease-producing organism under examination, its effect upon the animal tissues and the course of the disease should be observed.

There are other points of importance, but the above are essential to a right conclusion.

Diagnosis in Special Diseases:

(1) Diphtheria. This disease may be bacteriologically diagnosed with a minimum of apparatus and equipment. By means of a swab a rubbing from a suspected throat is readily obtained. This may be examined by the microscope, or sub-cultured on favourable medium. Blood serum is perhaps the best, but, as Hewlett remarks, "If no serum tubes can be had, an egg may be used. It is boiled hard, the shell chipped away from one end with a knife sterilised by heating, and the inoculation made on the exposed white surface; the egg is then placed, inoculated end down, in a wine-glass of such a size that it rests on the rim and does not touch the bottom. A few drops of water may with advantage be put at the bottom of the glass to keep the egg-white moist. The preparation is kept in a warm place for twenty-four to forty-eight hours and then examined." The examination, of course, consists in staining and preparing for the microscope and observing the form, arrangement, and characters of the organism or organisms present. A small piece of the membrane may be detached, washed in water, and stained for the bacilli.

(2) Tubercle (Ziehl-Neelsen's stain, vide supra).

(3) Typhoid (Enteric Fever).

Widal's Reaction. This diagnostic test depends upon the effect which the blood of a person suffering from typhoid fever has upon the Bacillus typhosus. The effect is twofold. In the first place, the actively motile B. typhosus becomes immotile; and secondly, there is an agglutination, or grouping together in colonies, of the B. typhosus. Neither of these features occurs if healthy human blood is brought into contact with a culture of the typhoid bacillus. There are various ways in which this "serum diagnosis" can be carried out. The simplest and quickest method is as follows: To ten drops of a twenty-four or forty-eight-hours-old neutral broth culture of the typhoid bacillus one drop of the blood serum to be tested is added. The serum and culture are rapidly mixed in the trough of a hollow ground slide (such as is used for the "hanging drop"), and a single drop is taken, placed upon an ordinary clean slide, and a cover-glass superimposed. The positive reaction of agglutination and immotility, if the blood comes from a case of typhoid fever, will probably appear within fifteen or twenty minutes. The fluid culture of typhoid may be taken from an agar culture as well as from broth. In both cases it may be desirable to filter through ordinary filter paper to remove any normally agglutinated masses of bacilli before commencing the test.

In his first experiments Widal used a test-tube in the following manner: The blood to be tested is diluted by one part of it being added to fifteen parts of broth in a test-tube. The mixture is inoculated with a drop of a typical Bacillus typhosus culture. The tube is then incubated at 37° C. for twenty-four hours, after which it is examined. If the reaction be positive, the broth appears comparatively clear, but at the bottom of the test-tube a more or less abundant sediment will be found. This is due to the clumps of bacilli having fallen owing to gravity. If, on the other hand, the reaction is negative, the broth will appear more or less uniformly turbid.

For the apparatus required to carry out the simpler methods of bacteriological work reference should be made to the standard laboratory text-books, which furnish all necessary details. A good microscope, with a 1/12 oil immersion lens, is, of course, essential. This can now be obtained for about £16 (Beck, Swift, Baker, Watson, etc.), and the other necessary apparatus is readily obtainable of Baird and Tatlock, Hatton Garden, E. C., and other makers.

FOOTNOTES: