THE INFLUENCE OF EXTERNAL CONDITIONS ON GROWTH OF BACTERIA
Nutritive Medium. In the very earliest days of the study of micro-organisms it was observed that they mostly congregate where there is pabulum for their nourishment. The reason why fluids such as milk, and dead animal matter such as a carcass, and living tissues such as a man's body contain so many microbes is because each of these three media is favourable to their growth. Milk affords almost an ideal food and environment for microbes. Its temperature and constitution frequently meet their requirements. Dead animal matter, too, yields a rich diet for some species (saprophytes). In the living tissues bacteria obtain not only nutriment, but a favourable temperature and moisture. Outside the human body it has been the endeavour of bacteriologists to provide media as like the above as possible, and containing many of the same elements of food. Thus the life-history may be carried on outside the body and under observation. By means of cover-glass preparations for the microscope we are able to study the form, size, motility, flagella, spore formation, and peculiarities of staining, all of which characters aid us in determining to what species the organism under examination belongs. By means of artificial nutrient media we may further learn the characters of the organism in "pure culture,"[7] its favourable temperature, its power or otherwise of liquefaction, the curdling milk, or of gas production, its behaviour towards oxygen, its power of producing indol, pigment, and chemical bodies, as well as its thermal death point and resistance to light and disinfectants. It is well known that under artificial cultivation an organism may be greatly modified in its morphology and physiology, and yet its conformity to type remains much more marked than any degeneration which may occur.
The basis of many of these artificial media is broth. This is made from good lean beef, free from fat and gristle, which is finely minced up and extracted in sterilised water (one pound of lean beef to every 1000 cc. of water). It is then filtered and sterilised. It will be understood that such an extract is acid. To provide peptone beef-broth, ten grains of peptone and five grains of common salt are added to every litre of acid beef-broth. It is rendered slightly alkaline by the addition of sodium carbonate, and is filtered and sterilised. Glycerine-broth indicates that 6 to 8 per cent. of glycerine has been added after filtration, glucose-broth 1 or 2 per cent. of grape-sugar. This latter is used for anaërobic organisms. The use of broth as a culture medium is of great value. It is undoubtedly our best fluid medium, and in it may not only be kept pure cultures of bacteria which it is desired to retain for a length of time, but in it also emulsions and mixtures may be placed preparatory to further operations. Gelatine is broth solidified by the addition of 100 grams of best French gelatine to the litre. Its advantage is twofold: it is transparent, and it allows manifestation of the power of liquefaction. When we speak of a liquefying organism we mean a germ having the power of producing a peptonising ferment which can at the temperature of the room break down solid gelatine into a liquid. Grape-sugar gelatine is made like grape-sugar broth. Agar was introduced as a medium which would not melt at 25° C., like gelatine, but remain solid at blood-heat (37·5° C.; 98·5° F.). It is a seaweed generally obtained in dried strips from the Japanese market. Ten to fifteen grams are added to every litre of peptone-broth. Filtration is slow and often difficult, and the result not as transparent as desirable. The former difficulty is avoided by filtering in the Koch's steamer or with a hot-water filter, the latter by the addition of the white of an egg. Glycerine and grape-sugar may be added as elsewhere. Blood agar is ordinary agar with fresh sterile blood smeared over its surface. Blood serum is drawn from a jar of coagulated horse-blood, in which the serum has risen to
Potato in a Roux Tube Prepared for Cultivation Glycerine is placed in the bulb of the tube the top. This is collected in sterilised tubes and coagulated in a special apparatus (the serum inspissator). Potato is prepared by scraping ordinary potatoes, washing in corrosive sublimate, and sterilising. They may then be cut into various shapes convenient for cultivation. Upon any of these forms of solid media the characteristic growth of the organism can be observed. Of the nutrient elements required, nitrogen is obtained from albumens and proteids, carbon from milk-sugar, cane-sugar, or the splitting up of proteids; salts (particularly phosphates and salts of potassium) are readily obtainable from those incorporated in the media; and the water which is required is obtainable from the moisture of the media.
There are two common forms of test-tube culture, viz.: on the surface and in the depth of the medium. In the former the medium is sloped, and the inoculating needle is drawn along its surface; in the latter the needle is thrust vertically downwards into the depth of the solid medium. Plate cultures and anaërobic cultures will be described at a later stage. When the medium has been inoculated the culture is placed at a temperature which will be favourable. Two standards of temperature are in use in bacteriological laboratories. The one is called room temperature, and varies from 18° C.-20° C.; the other is blood-heat, and varies from 35° C.-38° C. It is true, some species will grow below 18° C., and others above 38° C. The pathogenic (disease-producing) bacteria thrive best at 37° C., and the non-pathogenic at the ordinary temperature of the room. The different degrees of temperature are regulated by means of incubators. For the low temperatures gelatine is chosen; as a medium for the higher temperatures agar.
Staphylococcus Pyogenes Aureus
× 1000
Incubator
(Temperature of blood-heat, registered by thermometer, and regulated by thermo-regulator)
Moisture has been shown to have a favourable effect upon the growth of microbes. Drying will of itself kill many species (e. g., the spirillum of cholera), and, other things being equal, the moister a medium is, the better will be the growth upon it. Thus it is that the growth in broth is always more luxuriant than that on solid media. Yet the growth of Bacillus subtilis and other species is an exception to this rule, for they prefer a dry medium.
Culture Media Ready for Inoculation
Temperature. Most bacteria grow well at room temperature, but they will grow more luxuriantly and speedily at blood-heat. The optimum temperature is generally that of the natural habitat of the organism. In exceptional cases growth will occur as low as 5° C. or as high as 70° C. Indeed, some have been cooled to-20° C. and-30° C., and yet retained their vitality,[8] whereas some few can grow at 60–70° C. These latter are termed thermophilic bacteria. The average thermal death-point is at or about 50° C.
Inoculating Needles
Plantinum wire fused into glass handles
Light acts as an inhibitory or even germicidal agent. This fact was first established by Downes and Blunt in a memoir to the Royal Society in 1877. They found by exposing cultures to different degrees of sunlight that thus the growth of the culture was partially or entirely prevented, being most damaged by the direct rays of the sun, although diffuse daylight acted prejudicially. Further, these same investigators proved that of the rays of the spectrum which acted inimically the blue and violet rays acted most bactericidally, next to the blue being the red and orange-red rays. The action of light, they explain, is due to the gradual oxidation which is induced by the sun's rays in the presence of oxygen. Duclaux, who worked at this question at a later date, concluded that the degree of resistance to the bactericidal influence of light which some bacteria possess might be due to difference in species, difference in culture media, and difference in the degrees of intensity of light. Tyndall tested the growth of organisms in flasks exposed to air and light on the Alps, and found that sunlight inhibited the growth temporarily. A large number of experimenters in Europe and England have worked at this fascinating subject since 1877, and though many of their results appear contradictory, we may be satisfied to adopt the following conclusions respecting the matter:
(1) Sunlight has a deleterious effect upon bacteria, and to a less extent on their spores.
(2) This inimical effect can be produced by light irrespective of rise in temperature.
(3) The ultra-violet rays are the most bactericidal, and the infra-red the least so, which indicates that the phenomenon is due to chemical action.
(4) The presence of oxygen and moisture greatly increases this action.
(5) The sunlight acts prejudicially upon the culture medium, and thereby complicates the investigation and after-growth.
(6) The time occupied in the bactericidal action depends upon the heat of the sun and the intrinsic vitality of the organism.
(7) With regard to the action of light upon pathogenic organisms, some results have recently been obtained with Bacillus typhosus. Janowski maintains that direct sunlight exerts a distinctly depressing effect on typhoid bacilli. At present more cannot be said than that sunlight and fresh air are two of the most powerful agents we possess with which to combat pathogenic germs.
Pasteur's Large Incubator for Cultivation at Room Temperature
A very simple method of demonstrating the influence of light is to grow a pure culture in a favourable medium, either in a test-tube or upon a glass plate, and then cover the whole with black paper or cloth. A little window may then be cut in the protective covering, and the whole exposed to the light. Where it reaches in direct rays it will be found that little or no growth has occurred; where, on the other hand, the culture has been in the dark, abundant growth occurs. In diffuse light the growth is merely somewhat inhibited. It has been found that the electric light has but little action upon bacteria, though that which it has is similar to sunlight. Recent experiments with the Röntgen rays have given negative results.
In 1890 Koch stated that tubercle bacilli were killed after an exposure to direct sunlight of from a few minutes to several hours. The influence of diffuse light would obviously be much less. Professor Marshall Ward has experimented with the resistant spores of Bacillus anthracis by growing these on agar plates and exposing to sunlight. From two to six hours' exposure had a germicidal effect.
It should be remembered that several species of sea-water bacteria themselves possess powers of phosphorescence. Pflüger was the first to point out that it was such organisms which provided the phosphorescence upon decomposing wood or decaying fish. To what this light is due, whether capsule, or protoplasm, or chemical product, is not yet known. The only facts at present established are to the effect that certain kinds of media and pabulum favour or deter phosphorescence.
Desiccation. A later opportunity will occur for consideration of the effect of drying upon bacteria. Here it is only necessary to say that, other things being equal, drying diminishes virulence and lessens growth.
Oxygen. Pasteur was the first to lay emphasis upon the effect which free air had upon micro-organisms. He classified them according to whether they grew in air, aërobic, or whether they flourished most without it, anaërobic. Some have the faculty of growing with or without the presence of oxygen, and are designated as facultative aërobes or anaërobes. As regards the cultivation of anaërobic germs, it is only necessary to say here that hydrogen, nitrogen, or carbonic acid gas may be used in place of oxygen, or they may be grown in a medium containing some substance which will absorb the oxygen.
Modes of Bacterial Action. In considering the specific action of micro-organisms, it is desirable, in the first place, to remember the two great functional divisions of saprophyte and parasite. A saprophyte is an organism that obtains its nutrition from dead organic matter. Its services, of whatever nature, lie outside the tissues of living animals. Its life is spent apart from a "host." A parasite, on the other hand, lives always at the expense of some other organism which is its host, in which it lives and upon which it lives. There is a third or intermediate group, known as "facultative," owing to their ability to act as parasites or saprophytes, as the exigencies of their life-history may demand.
Method of Producing Hydrogen by Kipp's Apparatus for Cultivation of Anaërobes (See page [139])
The saprophytic organisms are, generally speaking, those which contribute most to the benefit of man, and the parasitic the reverse, though this statement is only approximately true. In their relation to the processes of fermentation, decomposition, nitrification, etc., we shall see how great and invaluable is the work which saprophytic microbes perform. Their result depends, in nearly all cases, upon the organic chemical constitution of the substances upon which they are exerting their action, as well as upon the varieties of bacteria themselves. Nor must it be understood that the action of saprophytes is wholly that of breaking down and decomposition. As a matter of fact, some of their work is, as we shall see, of a constructive nature; but, of whichever kind it is, the result depends upon the organism and its environment.
Anaërobic Culture
(Buckner's Tube)
with Pyrogallic
Solution in Bulb. This, too, may be said of the pathogenic species, all of which are in a greater or less degree parasitic. It is well known how various are the constitutions of man, how the bodies of some persons are more resistant than those of others, and how the invading microbe will find different receptions according to the constitution and idiosyncrasy of the body which it attacks. Indeed, even after invasion the infectivity of the special disease, whatever it happens to be, will be materially modified by the tissues. When we come to turn to the micro-organisms which are pathogenic parasites we shall further have to keep clear in our minds that their action is double and complex, and not single or simple. In the first place, we have an infection of the body due to the bacteria themselves. It may be a general and widespread infection, as in anthrax, where the bacilli pass, in the blood or lymph current, to each and every part of the body; or it may be a comparatively local one, as in diphtheria, where the invader remains localised at the site of entrance. But, be that as it may, the micro-organisms themselves, by their own bodily presence, set up changes and perform functions which may have far-reaching effects. It is obvious that the wider the distribution the wider is the area of tissue change, and vice versâ. Yet there is something of far greater importance than the mere presence of bacteria in human or animal tissues; for the secondary action of disease-producing germs—and possibly it is present in all bacteria—is due to their poisonous products, or toxins, as they have been termed. These may be of the nature of ferments, and they become diffused throughout the body, whether the bacteria themselves occur locally or generally. They may bring about very slight and even imperceptible changes during the course of the disease, or they may kill the patient in a few hours. Latterly bacteriologists have come to understand that it is not so much the presence of organisms which is injurious to man and other animals, as it is their products which cause the mischief; and the amount of toxic product bears no known proportion to the degree of invasion by the bacteria. The various and widely differing modes of action in bacteria are therefore dependent upon these three elements: the tissues or medium, the bacteria, and the products of the bacteria; and in all organismal processes these three elements act and react upon each other.
A word may be said here respecting the much-discussed question of species in bacteria. A species may be defined as "a group of individuals which, however many characters they share with other individuals, agree in presenting one or more characters of a peculiar and hereditary kind with some certain degree of distinctness."[9] Now, as regards bacteria, there is no doubt that separate species occur and tend to remain as separate species. It is true, there are many variations, due in large measure to the medium in which the organisms are growing,—variations of age, adaptation, nutrition, etc.,—yet the different species tend to remain distinct. Involution forms occur frequently, and degeneration invariably modifies the normal appearance. But because of the occurrence of these morphological and even pathological differences it must not be argued that the demarcation of species is wholly arbitrary.
Means of Sterilisation. As this term occurs frequently in even a book of this untechnical nature, and as it is expressive of an idea which must always be present to the mind of the bacteriologist, it may be desirable to make some passing allusion to it.
Chemical substances, perfect filtration, and heat are the three means at our command in order to secure germ-free conditions of apparatus or medium. The first two, though theoretically admissible, are practically seldom used, the former of the two because the addition of chemical substances annuls or modifies the operation, the latter of the two on account of the great practical difficulties in securing perfection. Hence in the investigation involved in bacteriological research heat is the common sterilising agent. A temperature of 70° C. (158° F.) will kill all bacilli; even 58° C. will kill most kinds. Boiling at 100° C. (212° F.) for three minutes will kill anthrax spores, and boiling for thirty to sixty minutes will kill all bacilli and all spores. This difference in the thermal death-point between bacilli and their spores enables the operator to obtain what are called "pure cultures" of a desired bacillus from its spores which may be present. For example, if a culture contains spores of anthrax and is contaminated with micrococci, heating to 70° C. (158° F.) will kill all the micrococci, but will not affect the spores of anthrax, which can then grow into a pure culture of anthrax bacilli. Fractional or discontinuous sterilisation depends on the principle of heating to the sterilising point for bacilli (say 70°C.) on one day, which will kill the bacilli, but leave the spores uninjured. But by the following day the spores will have germinated into bacilli, and a second heating to 70°C. will kill them before they in their turn have had time to sporulate. Thus the whole will be sterilised, though at a temperature below boiling.
Successful sterilisation, therefore, depends upon killing both bacteria and their spores, and nothing short of that can be considered as sterilisation. The following methods are those generally used in the laboratory. For dry heat (which is never so injurious to organisms as moist heat)[10]: (a) the Bunsen burner, in the flame of which platinum needles, etc., are sterilised; (b) hot-air chamber, in which flasks and test-tubes are heated to a temperature of 150–170°
Koch's Steam Steriliser C. for half an hour. For moist heat: (c) boiling, for knives and instruments; (d) Koch's steam steriliser, by means of which a crate is slung in a metal cylinder, at the bottom of which the water is boiled; (e) the autoclave, which is the most rapid and effective of all the methods. This is in reality a Koch steriliser, but with apparatus for obtaining high pressure. The last two (d, e) are used for sterilising the nutriment media upon which bacteria are cultivated outside the body. Blood serum would, however, coagulate at a temperature over 60° C. (124° F.), and hence a special steriliser has been designed to carry out fractional sterilisation daily for a week at about 55° C.-58° C.
The Association of Organisms. At a later stage we shall have an opportunity of discussing symbiosis and allied conditions. Here it is only necessary to draw attention to a fact that is rapidly becoming of the first importance in bacteriology. When species were first isolated in pure culture it was found that they behaved somewhat differently under differing circumstances. This modification in function has been attributed to differences of environment and physical conditions. Whilst it is true that such external conditions must have a marked effect upon such sensitive units of protoplasm as bacteria, it has recently been proved that one great reason why modification occurs in pure artificial cultures is that the species has been isolated from amongst its colleagues and doomed to a separate existence. One of the most abstruse problems in the immediate future of the science of bacteriology is to learn what intrinsic characters there are in species or individuals which act as a basis for the association of organisms for a specific purpose. Some bacteria appear to be unable to perform their regular function without the aid of others. An example of such association is well illustrated in the case of tetanus, for it has been shown that if the bacilli and spores of tetanus alone obtain entrance to a wound the disease may not follow the same course as when with the specific organism the lactic-acid bacillus or the common organisms of suppuration or putrefaction also gain entrance. There is here evidently something gained by association. Again, the virulence of other bacteria is also increased by means of association. The Bacillus coli is an example, for, in conjunction with other organisms, this bacillus, although normally present in health in the alimentary canal, is able to set up acute intestinal irritation, and various changes in the body of an inflammatory nature. It is not yet possible to say in what way or to what degree the association of bacteria influences their rôle. That is a problem for the future. But whilst we have examples of this association in streptococcus and the bacillus of diphtheria, B. coli and yeasts, tetanus and putrefactive bacteria, Diplococcus pneumoniæ and streptococcus, and association amongst the various suppurative organisms, we cannot doubt that there is an explanation to be found here of many hitherto unsolved results of bacterial action. This is the place in which mention should also be made of higher organisms associated for a specific purpose with bacteria. There is some evidence to support the belief that some of the Leptotricheæ (Crenothrix, Beggiatoa, Leptothrix, etc.) and the Cladotricheæ (Cladothrix) perform a preliminary disintegration of organic matter before the decomposing bacteria commence their labours. This occurs apparently in the self-purification of rivers, as well as in polluted soils.
Antagonism of Bacteria. Study of the life-history of many of the water bacteria will reveal the fact that they can live and multiply under conditions which would at once prove fatal to other species. Some of these water organisms can indeed increase and multiply in distilled water, whereas it is known that other species cannot even live in distilled water, owing to the lack of pabulum. Thus we see that what is favourable for one species may be the reverse for another.
Further, we shall have opportunity of observing, when considering the bacteriology of water and sewage, that there is in these media in nature a keen struggle for the survival of the fittest bacteria for each special medium. In a carcass it is the same. If saprophytic bacteria are present with pathogenic, there is a struggle for the survival of the latter. Now whilst this is in part due to a competition owing to a limited food supply and an unlimited population, as occurs in other spheres, it is also due in part to the inimical influence of the chemical products of the one species upon the life of the bacteria of the other species. Moreover, in one culture medium, as Cast has pointed out, two species will often not grow. When Pasteur found that exposure to air attenuated his cultures, he pointed out that it was not the air per se that hindered his growth, but it was the introduction of other species which competed with the original. The growth of the spirillum of cholera is opposed by Bacillus pyogenes fœtidus. B. anthracis is, in the body, opposed by either B. pyocyaneus or Streptococcus erysipelatis, and yet it is aided in its growth by B. prodigiosus. B. aceti is, under certain circumstances, antagonistic to B. coli communis.
In several of the most recent of the admirable reports of Sir Richard Thorne issued from the Medical Department of the Local Government Board, we have the record of a series of experiments performed by Dr. Klein into this question of the antagonism of microbes. From this work it is clearly demonstrated that whatever opposition one species affords to another it is able to exercise by means of its poisonous properties. These are of two kinds. There is, as is now widely known, the poisonous product named the toxin, into which we shall have to inquire more in detail at a later stage. There is also in many species, as Dr. Klein has pointed out, a poisonous constituent or constituents included in the body protoplasm of the bacillus, and which he therefore terms the intracellular poison. Now, whilst the former is different in every species, the latter may be a property common to several species. Hence those having a similar intracellular poison are antagonistic to each other, each member of such a group being unable to live in an environment of its own intracellular poison. Further, it has been suggested that there are organisms possessing only one poisonous property, namely, their toxin—for example, the bacilli of tetanus and diphtheria—whilst there are other species, as above, possessing a double poisonous property, an intracellular poison and a toxin. In this latter class would be included the bacilli of Anthrax and Tubercle.
Reference has been made to the associated work of higher vegetable life and bacteria. The converse is also true. Just as we have bacterial diseases affecting man and animals, so also plant life has its bacterial diseases. Wakker, Prillieux, Erwin Smith, and others have investigated the pathogenic conditions of plants due to bacteria, and though this branch of the science is in its very early stages, many facts have been learned. Hyacinth disease is due to a flagellated bacillus. The wilt of cucumbers and pumpkins is a common disease in some districts of the world, and may cause widespread injury. It is caused by a white microbe which fills the water-ducts. Wilting vines are full of the same sticky germs. Desiccation and sunlight have a strongly prejudicial effect upon these organisms. Bacterial brown-rot of potatoes and tomatoes is another plant disease probably due to a bacillus. The bacillus passes down the interior of the stem into the tubers, and brown-rots them from within. There is another form of brown-rot which affects cabbages. It blackens the veins of the leaves, and a woody ring which is formed in the stem causes the leaves to fall off. This also is due to a micro-organism, which gains entrance through the water-pores of the leaf, and subsequently passes into the vessels of the plants. It multiplies by simple fission, and possesses a flagellum.
There can be no doubt that these complex biological properties of association and antagonism, as well as the parasitic growth of bacteria upon higher vegetables, are as yet little understood, and we may be glad that any light is being shed upon them. In the biological study of soil bacteria in particular may we expect in the future to find examples of association, even as already there are signs that this is so in certain pathogenic conditions. In the alimentary canal, on the other hand, and in conditions where organic matter is greatly predominating, we may expect to see further light on the subject of antagonism.
Attenuation of Virulence or Function. It was pointed out by some of the pioneer bacteriologists that the function of bacteria suffered under certain circumstances a marked diminution in power. Later workers found that such a change might be artificially produced. Pasteur introduced the first method, which was the simple one of allowing cultures to grow old before sub-culturing. Obviously a pure culture cannot last for ever. To maintain the species in characteristic condition it is necessary frequently to sub-culture upon fresh media. If this simple operation be postponed as long as possible consistent with vitality, and then performed, it will be found that the sub-culture is attenuated, i. e., weakened. Another mode is to raise the pure culture to a temperature approaching its thermal death point. A third way of securing the same end is to place it under disadvantageous external circumstances, for example a too alkaline or too acid medium. A fourth, but rarely necessary, method is to pass it through the tissues of an insusceptible animal. Thus we see that, whilst the favourable conditions which we have considered afford full scope for the growth and performance of functions of bacteria, we are able by a partial withdrawal of these, short of that ending fatally, to modify the character and strength of bacteria. In future chapters we shall have opportunity of observing what can be done in this direction.
[CHAPTER II]
BACTERIA IN WATER
In entering upon a consideration of such a common article of use as water, we shall do well to describe in some detail the process by which we systematically investigate the bacteriology of a water, or, indeed, of any similar fluid suspected of bacterial pollution.
The collection of samples, though it appears simple enough, is sometimes a difficult and responsible undertaking. Complicated apparatus is rarely necessary, and fallacies will generally be avoided by observing two directions. In the first place, the sample should be chosen as representative as possible of the real substance or conditions we wish to examine. Some authorities advise that it is necessary to allow the tap to run for some minutes previous to collecting the sample; but if we desire to examine for lead chemically or for micro-organisms in the pipes biologically, then such a proceeding would be injudicious.[11] Hence we must use common sense in the selection and obtaining of a sample, following this one guide, namely, to collect as nearly as possible a sample of the exact water the quality of which it is desired to learn. In the second place, we must observe strict bacteriological cleanliness in all our manipulations. This means that we must use only sterilised vessels or flasks for collecting the sample, and in the manipulation required we must be extremely careful to avoid any pollution of air or any addition to the organisms of the water from unsterilised apparatus. A flask polluted in only the most infinitesimal degree will entirely vitiate all results.
Accompanying the sample should be a more or less full statement of its source. There can be no doubt that, in addition to a chemical and bacteriological report of a water, there should also be made a careful examination of its source. This may appear to take the bacteriologist far afield, and in point of fact, as regards distance, this may be so. But until he has seen for himself what "the gathering-ground" is like, and from what sources come the feeding streams, he cannot judge the water as fairly as he should be able to do. The configuration of the gathering-ground, its subsoil, its geology, its rainfall, its relation to the slopes which it drains, the nature of its surface, the course of its feeders, and the absence or presence of cultivated areas, of roads, of houses, of farms, of human traffic, of cattle and sheep—all these points must be noted, and their influence, direct or indirect, upon the water carefully borne in mind.
When the sample has been duly collected, sealed, and a label affixed bearing the date, time, and conditions of collection and full address, it should be transmitted with the least possible delay to the laboratory. Frequently it is desirable to pack the bottles in a small ice case for transit. On receipt of such a sample of water the examination must be immediately proceeded with, in order to avoid, as far as possible, the fallacies arising from the rapid multiplication of germs. Even in almost pure water, at the ordinary temperature of a room, Frankland found organisms multiplied as follows:—
| Hours. | No. of Germs per cc. |
| 0 | 1,073 |
| 6 | 6,028 |
| 24 | 7,262 |
| 48 | 48,100 |
Another series of observations revealed the same sort of rapid increase of bacteria. On the date of collection the micro-organisms per cc. in a deep-well water (in April) were seven. After one day's standing at room temperature the number had reached twenty-one per cc. After three days under the same conditions it was 495,000 per cc. At blood-heat the increase would, of course, be much greater, as a higher temperature is more favourable to multiplication. But this would depend upon the degree of impurity in the water, a pure water decreasing in number on account of the exhaustion of the pabulum, whereas, for the first few days at all events, an organically polluted water would show an enormous increase in bacteria.
Furthermore, it is desirable to remember that organisms, in an ordinary water, do not continue to increase indefinitely. There is a limit to all things, even to numbers in bacteriology. Cramer, of Zurich, examined the water of the Lake after it had been standing for different periods, with the following results:—
| Hours and Days of Examination. | No. of Micro-organisms per cc.[12] | |
| 0 | hours | 143 |
| 24 | " | 12,457 |
| 3 | days | 328,543 |
| 8 | " | 233,452 |
| 17 | " | 17,436 |
| 70 | " | 2,500 |
The writer's own experience is entirely in agreement with this cessation of multiplication at or about the end of a week, and the later decline.
Method of Examination. At the outset of a systematic study of a water it is well to observe its physical characters. The colour, if any, should be noted. Suspended matter and deposit may indicate organic or inorganic pollution. If abundant or conspicuous, a microscopic examination of the sediment may be made. The reaction, whether acid, neutral, or alkaline, must be tested, and the exact temperature taken. Any and every fact will help us, perhaps not so much to determine the contents of the water as to interpret rightly the facts we deduce from the entire examination.
Levelling Apparatus for Koch's Plate
At the beginning of the bacteriological work the water should be examined by means of the gelatine plate method. This consists in drawing up into a fine sterilised pipette a small quantity of the water and introducing it thereby into a test-tube of melted gelatine at a temperature below 40° C.[13] It will depend upon the apparent quality of the water as to the exact quantity introduced into the gelatine; about .5 or .1 of a cubic centimetre is a common figure. The stopper is then quickly replaced in the test-tube, and the contents gently mixed more or less equally to distribute the one-tenth cubic centimetre throughout the melted gelatine. A sterilised sheet of glass (4 inches by 3) designated a Koch's plate is now taken and placed upon the stage of a levelling apparatus, which holds iced water in a glass jar under the stage. The gelatine is now poured out over the glass plate, and by means of a sterilised rod stroked into a thin, even film all over the glass. It is then covered with a bell-jar and left at rest to set. The level stage prevents the gelatine running over the edge of the plate; the iced water under the stage expedites the setting of the gelatine into a fixed film. When it is thus set the plate is placed upon a small stand in a moist chamber, and the whole apparatus removed to the room temperature incubator. A moist chamber is a glass dish, in which some filter paper, soaked with corrosive sublimate, is inserted, and the dish covered with a bell-jar. By this means the risks of pollution are minimized, and moisture maintained. In all cases at least two plates must be prepared of the same sample of water, and it is often advisable to make several. They may be made with different media for different purposes, and with different quantities of water, though the same method of procedure is adopted. In a highly polluted water extremely small quantities would be taken, and, vice versâ, in pure water a large quantity.
Moist Chamber in which Koch's Plates are Incubated
When we come to discuss the relation of disease organisms to water, particularly those causing typhoid fever, we shall learn that they are both scarce and intermittent. This point has been dwelt upon frequently by Dr. Klein, and it is clear that such a state of things greatly enhances the difficulties in detecting such bacteria, and he has proposed a simple procedure by which the difficulty of finding the Bacillus typhosus in a large body of water may be met.
Hot Air Steriliser
For the Sterilization of Glass Apparatus, etc.
One or two thousand cubic centimetres of the water under examination are passed through a sterilised Berkefeld filter by means of siphon action or an air-pump. The candle of the filter retains on its outer surface all, or nearly all, the particulate matter contained in the water. The matter thus retained on this outer surface is brushed by means of a sterile brush into 10 or 20 cc. of sterilised water. Thus we have all the organisms contained in two litres of the water reduced into 10 cc. of water. From this, so to speak, concentrated emulsion of the bacteria of the original water, phenol-gelatine plates or Eisner plates (both acid media) may be readily made. In this way we not only catch many bacteria which would evade us if we were content with the examination merely of a few drops of the water, but we eliminate by means of the acid those common water bacteria, like Bacillus fluorescens liquefaciens, which so greatly confuse the issue.
In the course of two or three days the film of gelatine on the plate becomes covered with colonies of germs, and the next step is to examine these quantitatively and qualitatively. We may here insert a simple scheme by which this may be most fully and easily accomplished:—
1. Naked-Eye Observation of the Colonies. By this means at the very outset certain facts may be obtained, viz., the size, elevation, configuration, margin, colour, grouping, number, and kinds of colonies, all of which facts are of importance, and assist in final diagnosis. Moreover, in the case of gelatine plates (it is otherwise in agar) one is able to observe whether or not there is present what is termed liquefaction of the gelatine. Some organisms produce in their development a peptonizing ferment which breaks down gelatine into a fluid condition. Many have not this power, and hence the characteristic is used as a diagnostic feature.
2. Microscopic Examination of Colonies, which confirms or corrects that which has been observed by the naked eye. Fortunately some micro-organisms when growing in colonies produce cultivation features which are peculiar to themselves (especially is this so when growing in test-tube cultures), and in the early stages of such growths a low power of the microscope or magnifying glass facilitates observation.
3. Make cover-glass preparations: (a) unstained—"the hanging drop"; (b) stained—single stains, like gentian-violet, methyl blue, fuchsin, carbol fuchsin, etc.; double stains—Gram's method, Ziehl-Neelsen's method, etc.
The Hanging Drop
This third part of the investigation is obviously to prepare specimens for the microscope. "The hanging drop" is a simple plan for securing the organisms for microscopic examination in a more or less natural condition. A hollow ground slide, which is a slide with a shallow depression in it, is taken, and a small ring of vaseline placed round the edge of the depression. Upon the under side of a clean cover-glass is placed a drop of pure water, and this is inoculated with the smallest possible particle taken from one of the colonies of the gelatine plate on the end of a sterilised platinum wire. The cover-glass is then placed upon the ring of vaseline, and the drop hangs into the space of the depression. Thus is obtained a view of the organisms in a freely moving condition, if they happen to be motile bacteria. As a matter of practice the hollow slide may be dispensed with, and an ordinary slide used.
Drying Stage for Fixing Films
With regard to staining, it will be undesirable here to dwell at length upon the large number of methods which have been adopted. The "single stain" may be shortly mentioned. It is as follows: A clean cover-glass is taken (cleaned with nitric acid and alcohol, or bichromate of potash and alcohol), and a drop of pure sterilised water placed upon it. This is inoculated with the particle of a colony on the end of a platinum needle, and a scum is produced. The film is now "fixed" by slowly drying it over a flame. When the scum is thus dried, a drop of the selected stain (say gentian-violet) is placed over the scum and allowed to remain for varying periods: sarcinæ about thirty seconds; for many of the bacilli three or four minutes. It is then washed off with clean water, dried, and mounted in Canada balsam. The organisms will now appear under the microscope as violet in colour, and will thus be clearly seen.
The "double staining" is adopted when we desire to stain the organisms one colour and the tissue in which they are situated a contrast colour. Some of the details of these methods are mentioned in the Appendix.
4. Sub-culture. The plate method was really introduced by Koch in order to facilitate isolation of species. In a flask it is impossible to isolate individual species, but when the growth is spread over a comparatively large area, like a plate, it is possible to separate the colonies, and this being done by means of a platinum wire, the colonies may be replanted in fresh media; that is to say, a sub-culture may be made, each organism cultivated on its favourite soil, and its manner of life closely watched. We have already mentioned the chief media which are used in the laboratory, and in an investigation many of these would be used, and thus pure cultures would be obtained. Let us suppose that a water contains six kinds of bacteria. On the plate these six kinds would show themselves by their own peculiar growth. Each would then be isolated and placed in a separate tube, on a favourite medium, and at a suitable temperature. Thus each would be a pure culture; i. e., one and only one, species would be present in each of the six tubes. By this simple means an organism can be, we say, cultivated, in the same sort of way as in floriculture. From day to day we can observe the habits of each of our six species, and probably at an early stage of their separated existences we should be able to diagnose what species of bacteria we had found in the water. If not, further microscopic examination could be made, and, if necessary, secondary or tertiary sub-cultures.
5. Inoculation of Animals. It may be necessary to observe the action of supposed pathogenic organisms upon animals. This is obviously a last resource, and any abuse of such a process is strictly limited by law. As a matter of fact, an immense amount of bacteriological investigation can be carried on without inoculating animals; but, strictly speaking, as regards many of the pathogenic bacteria, this test is the most reliable of all. Nor would any responsible bacteriologist be justified in certifying a water as healthy for consumption by a large community if he was in doubt as to the disease-producing action of certain contained organisms.
Types of Liquefaction of Gelatine
By working through some such scheme as the above we are able to detect what quantity and species of organisms, saprophytic or parasitic, a water or similar fluid contains. For, observe what information we have gained. We have learned the form (whether bacillus, micrococcus, or spirillum), size, consistence, motility, method of grouping, and staining reactions of each micro-organism; the characters of its culture, colour, composition, presence or absence of liquefication or gas formation, its rate of growth, smell, or reaction; and lastly, when necessary, the effect that it has upon living tissues. Here, then, are ample data for arriving at a satisfactory conclusion respecting the qualitative estimation of the suspected water.
As to to the quantitative examination, that is fulfilled by counting the number of colonies which appear, say by the third and fourth day, upon the gelatine plates. Each colony has arisen, it is assumed, from one individual, so that if we count the colonies, though we do not thereby know how many organisms we have upon our plate, we do know approximately how many organisms there were when the plate was first poured out, which are the figures we require, and which can at once be multiplied and returned as so many organisms per cubic centimetre. There is, unfortunately, at present no exact standard to which all bacteriologists may refer.
Miquel and Crookshank have suggested standards which allow "very pure water" to contain up to 100 micro-organisms per cc. Pure water must not contain more than 1000, and water containing up to 100,000 bacteria per cc. is contaminated with surface water or sewage. Macé gives the following table:
| Very pure water | 0- | 10 bacteria per cc. |
| Very good water | 20- | 100bac"eria per"eri |
| Good water | 100- | 200bac"eria per"eri |
| Passable (mediocre) water | 200- | 500bac"eria per"eri |
| Bad water | 500- | 1,000bac"eria per"eri |
| Very bad water | 1000- | 10,000 and overeri"eri |
Koch first laid emphasis on the quantity of bacteria present as an index of pollution, and whilst different authorities have all agreed that there is a necessary quantitative limit, it has been so far impossible to arrive at one settled standard of permissible impurity.
Besson adopts the standard suggested by Miquel, and, on the whole, French bacteriologists follow suit. They also agree with him, generally speaking, in not placing much emphasis upon the numerical estimation of bacteria in water. In Germany and England it is the custom to adopt a stricter limit. Koch in 1893 fixed 100 bacteria per cc. as the maximum number of bacteria which should be present in a properly filtered water. Hence the following has been recognised more or less as the standard:
| 0- | 100 bacteria per cc. = | a good potable water, |
| 100- | 500bact"erieri"teria= | a suspicious water. |
| 500- | 1000 or moreeri"teria= | a water which should have further filtration before being used for drinking purposes. |
The personal view of the writer after some experience of water examination would favour a standard of "under 500" being a potable water, if the 500 were of a nature indicating neither sewage pollution nor disease. Miquel holds that not more than ten different species of bacteria should be present in a drinking water, and such is a useful standard. The presence of rapidly liquefying bacteria associated with sewage or surface pollution would, even though present in fewer numbers than a standard, condemn a water. Thus it will be seen that it is impossible to judge alone by the numbers unless they are obviously enormously high.
Wolfhügel's Counter
When we are counting colonies upon a Koch's plate, Wolfhügel's counter may be used. This is a thin plate of glass a size larger than Koch's plates, and upon it are scratched squares, each square being divided into nine smaller squares. The Wolfhügel plate is superimposed upon the Koch's plate, and the colonies counted in one little square or set of squares and multiplied.
Petri's Dish
By using flat, shallow, circular glass dishes, generally known as Petri's dishes, instead of Koch's plates, much manipulation and time is saved, and, on the whole, less risk of pollution occurs. Moreover, these are easily carried about and transferred from place to place. When counting colonies in a Petri's dish it is sufficient to divide the circle into eight equal divisions, and counting the colonies in the average divisions, multiply and reduce to the common denominator of one cc. For example, if the colonies of the plate appear to be distributed fairly uniformly we count those in one of the divisions. They reach, we will suppose, the figure of 60; 60 × 8=480 micro-organisms in the amount taken from the suspected water and added to the melted gelatine from which the plate was made. This amount was .25 cc. Therefore we estimate the number of micro-organisms in the suspected water as 60 × 8=480 × 4= 1920 m.-o. per cc., which is over standard by about 1500. A water might then be condemned upon its quantitative examination alone or qualitative alone, or both. If the quantity were even that of an artesian well, say 4–10 m.-o. per cc., but those four or ten were all Bacillus typhosus, it would clearly be condemned on its quality, though quantitatively it was an almost pure water. If, on the contrary, the water contained 10,000 m.-o. per cc., and none of them disease-producing, it would still be condemned on the ground that so large a number of organisms indicated some kind of organic pollution to supply pabulum for so many organisms to live in one cc. of the water. It is not the number per se which condemns. The large number condemns because it indicates probable pollution with surface water or sewage in order to supply pabulum for so many bacteria per cc.
It should always be remembered that a chemical report and a bacteriological report should assist each other. The former is able to tell us the quantity of salts and condition of the organic matter present; the latter the number and quality of micro-organisms. Neither can take the place of the other and, generally speaking, both are more or less useless until we can learn, by inspection and investigation of the source of the water, the origin of the organic matter or contamination. Hence a water report should contain not only a record of physical characters, of chemical constituents, and of the presence or absence of micro-organisms, injurious and otherwise, but it should also contain information obtained by personal investigation of the source. Only thus can a reasonable opinion be expected. Moreover, it is generally only possible to form an accurate judgment of a water from watching its history, that is, not from one examination only, but from a series of observations. A water yielding a steady standard of bacterial contents is a much more satisfactory water, from every point of view, than one which is unstable, one month possessing 500 bacteria per cc. and another month 5000. It is obvious that rainfall and drought, soil and trade effluents, will have their influence in materially affecting the bacterial condition of a water.
It is perhaps scarcely necessary to add that we have not in the above account of the examination of water included all, or nearly all, the various methods adopted for acquiring a knowledge of the bacterial contents of the water. Many of these are of too detailed and technical a nature to enter into here. Three points, however, we may touch upon. In the first place, as we have said, the particulate matter out of a large body of water should be concentrated in a small quantity. Accordingly it has become the custom to pass 2000 or 3000 cc. of the suspected water through a Berkefeld filter. When this has been accomplished, by means of a
Berkefeld Filter
In Position for Filtration of Water to be Examined. sterile brush the particulate matter on the candle of the filter is brushed off into 10 or 15 cc. of sterilised water. This simple arrangement is analogous to the use of gravity or centrifugal methods of securing the solid matter in milk. The smaller quantity of water is then readily examined, and scanty germs more readily detected. A second point elaborating the scheme of water examination is the choice of media for sub-culturing. Mere examination on gelatine is not sufficient. Even in making the primary plate cultivations it is well to vary the media—agar, carbol-gelatine, Elsner, etc. But when colonies have appeared upon these plates it is important to sub-culture with accuracy and good judgment upon all or any media—gelatine, agar, broth, potato, milk, blood serum, glucose agar, glycerine agar, etc.—that will reveal the real characters of the bacteria present. A method proposed by Professor Sheridan Delépine is to place some of the suspected water in sterilised test-tubes without further treatment, and incubate at 37° C. for twelve or eighteen hours, and then plate out and estimate the number of bacteria as in the ordinary course. "In polluted water, containing an excess of organic matter," he says, "an extremely rapid multiplication of bacteria is observed. In unpolluted water, containing only water bacteria and a very small amount of organic matter, very little or no multiplication takes place, and the growth of the water bacteria liquefying gelatine is checked to a remarkable extent." Thirdly, by none of these methods should we be able to isolate anaërobic bacteria, and to furnish a complete report these also must receive careful attention.
Apparatus for Filtering Water to Facilitate its Bacteriological Examination
The Bacteriology of Water. In many natural waters there will be found varied contents even in regard to flora alone: algæ, diatoms, spirogyræ, desmids, and all sorts of vegetable detritus. Many of these organisms are held responsible for divers disagreeable tastes and odours. The colour of a water may also be due to similar causes. Dr. Garrett, of Cheltenham, has recorded the occurrence of redness of water owing to a growth of Crenothrix polyspora, and many other similar cases make it evident that not unfrequently great changes may be produced in water by contained microscopic vegetation.
With the exception of water from springs and deep wells, all unfiltered natural waters contain numbers of bacteria. The actual number roughly depends upon the amount of organic pabulum present, and upon certain physical conditions of the water. As we have already seen, bacteria multiply with enormous rapidity. In some species multiplication does not appear to depend on the presence of much organic matter, and, indeed, some can live and multiply in sterilised water: Micrococcus aquatilis and Bacillus erythrosporus. Again, others depend not upon the quantity of organic matter, but upon its quality. And frequently in a water of a high degree of organic pollution it will be found that bacteria have been restrained in their development by the competition of other species monopolising the pabulum. Probably at least one hundred different species of non-pathogenic organisms have been isolated from water. Some species are constantly occurring, and are present in almost all natural waters. Amongst such are B. liquefaciens, B. fluorescens liq., B. fluorescens non-liquefaciens, B. termo, B. aquatilis, B. ubiquitus, and not a few micrococci, etc. Percy Frankland[14] collected water from various quarters at various times and seasons, and some of his results may here be added:
RIVER THAMES WATER COLLECTED AT HAMPTON
Number of Micro-organisms Obtained from 1 cc. of Water.
| Month. | 1886. | 1887. | 1888. |
| January | 45,000 | 30,800 | 92,000 |
| February | 15,800 | 6,700 | 40,000 |
| March | 11,415 | 30,900 | 66,000 |
| April | 12,250 | 52,100 | 13,000 |
| May | 4,800 | 2,100 | 1,900 |
| June | 8,300 | 2,200 | 3,500 |
| July | 3,000 | 2,500 | 1,070 |
| August | 6,100 | 7,200 | 3,000 |
| September | 8,400 | 16,700 | 1,740 |
| October | 8,600 | 6,700 | 1,130 |
| November | 56,000 | 81,000 | 11,700 |
| December | 63,000 | 19,000 | 10,600 |
Again, another example:
RIVER LEA WATER COLLECTED AT CHINGFORD
Number of Micro-organisms Obtained from 1 cc. of Water.
| Month. | 1886. | 1887. | 1888. |
| January | 39,300 | 37,700 | 31,000 |
| February | 20,600 | 7,900 | 26,000 |
| March | 9,025 | 24,000 | 63,000 |
| April | 7,300 | 1,330 | 84,000 |
| May | 2,950 | 2,200 | 1,124 |
| June | 4,700 | 12,200 | 7,000 |
| July | 5,400 | 12,300 | 2,190 |
| August | 4,300 | 5,300 | 2,000 |
| September | 3,700 | 9,200 | 1,670 |
| October | 6,400 | 7,600 | 2,310 |
| November | 12,700 | 27,000 | 57,500 |
| December | 121,000 | 11,000 | 4,400 |
"During the summer months these waters are purest as regards micro-organisms, this being due to the fact that during dry weather these rivers are mainly composed of spring water, whilst at other seasons they receive the washings of much cultivated land."—Frankland.
Prausnitz has shown that water differs, as would be expected, according to the locality in the stream at which examination is made. His investigations were made from the river Isar before and after it receives the drainage of Munich:
| No. of Colonies per cc. | |
| Above Munich | 531 |
| Near entrance of principal sewer | 227,369 |
| 13 kilometres from Munich | 9,111 |
| 22kilom"metres"muni" | 4,796 |
| 33kilom"metres"muni" | 2,378 |
Professor Percy Frankland also points out how the river Dee affords another example, even more perfect, of pollution and restoration repeated several times until the river becomes almost bacterially pure.
We cannot here enter more fully into the many conditions of a water which affect its bacterial content than to say that it varies considerably with its source, at different seasons, and under different climatic conditions. An enormous increase will occur if the sediment is disturbed, and conversely sedimentation and subsidence during storage will greatly diminish the numbers of bacteria. Sand filtration, plus a "nitrifying layer," will remove more than 90 per cent. of the bacteria. Sea-water contains comparatively few bacteria, and the deeper the water and the farther it is from shore so much less will be the bacterial pollution.