D. Discovery of germs in the inoculated, diseased tissues, reisolation of the same, and growth on various media until it is determined beyond doubt that they are identical with the organism which was inoculated."

Not one of these steps can be omitted. Possible sources of error beset the investigator at every step, and anything short of a rigid demonstration cannot be accepted as proof. A. is usually quite easy, involving only the careful microscopic examination of abundant material, stained and unstained. B. was made possible by the improvement of methods, i. e. by the use of solid media, and especially by the discovery of the method of isolation by means of plate cultures. C. is quite easy, provided the right organism has been obtained and this be inserted into the proper tissues under the right conditions to insure growth. The fulfillment, however, of these conditions often involves long and vexatious delays, and taxes the acumen of the investigator to the utmost. D. serves as a check on all the preceding work, showing that there has been no unintentional mixing of organisms, and that the results obtained were actually due to the supposed cause. For the sake of brevity these four rules of practice will be referred to in the following pages simply as A. B. C. and D. What weight shall be given any specific statement depends of course on the reputation of the individual. Some men are so careful of their reputation and so little given to making unwarranted statements that their simple word goes a long way even when the statements themselves seem improbable, whereas the elaborate explanations of other men, if the asserted facts are at all out of the ordinary, have to be taken with a grain of salt.

The requirements under the second head, i. e. Description of the organism, are more numerous, and are embraced under two general divisions of very unequal value, namely Morphology and Biology. In the classification of the higher plants and animals morphology has been accepted from time immemorial

14 A series of successful reinoculations is always very desirable and becomes indispensable in case the infections are obtained on plants grown in a locality where the disease prevails naturally. Of course, numerous un-inoculated plants, known "checks" (6 or controls," must always be reserved for comparison.

SB

as answering all the requirements of systematists, but such is far from being the case when it comes to the description of bacteria. These minute organisms, which are among the lowest and simplest forms of living things yet discovered by man, are, within the commonly accepted generic limits, so morphologically similar as very often to be indistinguishable with any certainty even under the highest powers of the microscope. As supplemental, therefore, to morphology, and even in many cases as a complete substitute for it, we must have recourse to Biology, viz. to the behaviour of the living organism under a variety of known, artificially prepared conditions, such for example as the peculiarity of its growth on various culture media, its thermal death point, its ability to ferment various sugars, the chemical products of its growth, its pathogenic power, etc. Morphologically identical organisms often differ so widely and constantly in their biological peculiarities that there can be no question about their being distinct species, or as to the real value of this means of classification. Probably it also has value, hitherto overlooked, for the differentiation of higher plants and animals, especially for determining the limits of polymorphic or closely related species.

It is not my intention in this place to mention all the biological tests which should be applied to any species for its proper characterization. These are being added to constantly by an army of trained workers in all parts of the world, and my own views of what is at present necessary, or at least highly desirable, will be sufficiently evident in what is to follow. Very likely, also, as knowledge increases, some of the tests which are now generally held to be important will be shown to have little specific worth.

This, however, appears to be a good place to insist on accuracy in all the details of bacteriological work, especially in the preparation of culture media, and on explicitness of statement so that other investigators may know just what was done and how it was done, and thus be able to repeat the experiment. When all details of work are suppressed the inference, naturally enough, is that the writer was ignorant or else that he desired to conceal something not specially to his credit, and which if

plainly expressed might millitate against the value of his work. Either horn of the dilemma is equally bad. Some, however, who are desirous of doing good work in this field, or at least appear to be conscientious workers in other lines, do not seem to be aware of the necessity for extreme care in the preparation of culture media and the management of cultures. As a matter of fact, many bacteria are extremely sensitive to slight changes in the composition of the media in which they are grown or to other conditions within the control of the experimenter, and this appears to be true especially of the pathogenic species. Hence the many conflicting statements about the same organism. A few examples will render my meaning plainer. The careless

exposure of cultures to bright sunshine may destroy the organism. An organism supposed to come from diseased tissues or from a culture, and which is being examined in a cover glass preparation, may have been derived actually from a contaminated staining fluid. The apparently simple matter of slightly unclean test tubes or flasks may lead to error, e. g. antiseptic influences may be at work, or preciptates may be thrown down and subsequently mistaken for bacterial growth. Some kinds of glass are unsuited to delicate bacteriological work, the culture fluids being contaminated by substances dissolved out of the walls of the beakers, tubes, and flasks. Tyros, of course, are liable to mistake almost anything for bacteria or to find them anywhere (See a long paper by Bernheim on (12) Die parasitären Bacterien der Cerealen, in Münch. med. Wochenschrift, 1888, pp. 743-745 and 767-770, and comments on the same by Buchner and Lehmann, Ibid., 1888, p. 906, and 1889, p. 110). Boiling culture media, after it has been compounded, in open beakers or in loosely plugged test tubes or flasks may unwittingly lead to its concentration. The use at different times of different peptones, or grades of gelatine, of unlike per cents of gelatine or agar, of varying grades of acidity or alkalinity, of impure chemicals, of different concentrations of the nutrient media, and of different methods in its preparation all tend to render comparative studies impossible. A large source of error in the differentiation of species by means of sugar fermentation experiments has been the employment of bouillon

containing undetected muscle sugar. Even when preliminary tests are made with some gas-producing bacillus there is still an opportunity for error, provided the tests are carried on only for a day or two. No bouillon should be judged free from sugar and safe for use until in fermentation tubes it has been subjected for at least a week to the influence of Bacillus cloaca or some other organism producing an abundance of gas from grape sugar. If at the end of this period no gas has developed, and the transfer of a loop of fluid from such a tube into another fermentation tube containing a dextrose-bouillon sets up an evolution of gas, then the first bouillon may be used with confidence. Again, if cane sugar is sterilized in an acid bouillon at least a part of it is inverted, i. e. changed into dextrose and fructose, and fermentation results obtained therefrom may be due to the presence of any one of three sugars. Bouillon should always be made distinctly alkaline before cane sugar is added.

Many of the older fermentation experiments are worthless on account of neglect of such precautions, to say nothing of some recent ones. Again Bacillus tracheiphilus grows not at all or feebly on nutrient gelatine as ordinarily made, or in media which is acid beyond a determinable slight degree, and if only such media were used the erroneous conclusion might be reached that it could not be grown outside of the host plant, whereas it grows freely in artificial media, even on gelatine, when the right conditions are established. Bacillus amylovorus grows well in some gelatines and refuses to grow in others. Even comparatively slight changes in the acidity or alkalinity of the culture media often have a marked effect on the growth of certain organisms, while others, e. g., Bacillus cloacae, are able to grow in almost any medium. Many bacteria prefer alkaline media, and some are very sensitive to the presence of acids, while a variety of bacteria commonly met with in water will not develop at all if the medium is rendered strongly alkaline. Other organisms grow well in acid media."

14a

14a For a striking illustration of the effect on the growth of water bacteria of comparatively slight charges in the reaction of gelatine, see a recent table by George W. Fuller, in a paper entitled: (13) On the proper reaction of nutrient media for bacterial cultivation.—Journal of the American Public Health Association, Concord, N. H., Oct., 1895, p. 393.

Even the slightly varying acidity of steamed slices from different potato tubers may exert a marked effect on the growth of certain sensitive organisms. On this account some bacteriologists have advised discarding the potato altogether. I have myself found the potato a very useful substratum for the growth of both fungi and bacteria. All comparative tests on potato ought, however, to be made on cylinders or slices cut from the same tuber, and in every case the reaction, acid, neutral, or alkaline, should be carefully recorded. The behavior of the organism on a variety of tubers should also be determined, before deciding that it is something new. It has been thought by some that the best nutrient substance for a parasite must be, unquestionably, the juices of the host plant but this does not follow since there are all grades of parasitism, and even if it did, there are several chances for error in its employment, e. g. the nutrient juices are usually sterilized by steam heat and this may cause a number of chemical changes resulting in a compound very different from the living plant and entirely unsatisfactory as a culture medium, as many have learned by experience. Again, for some particular reason, even the juices of the plant when sterilized at ordinary temperatures by filtration, may be less well adapted to the needs of the parasite than well made beef bouillon or ordinary nutrient agar. In general, the standard culture media of bacteriology should be tried first. Some bacteria can be cultivated only on special media or at special temperatures, or under unusual conditions. Bacillus subtilis will only grow in the presence of free oxygen; Bacillus amylobacter, B. tetani, and B. carbonis will only grow in the absence of oxygen. Winogradsky states that his nitrifying organism obtained from European soils will not grow in the ordinary culture media and thrives best in solutions of inorganic substances, and on silicate jelly. Bacterium tuberculosis can be cultivated only in bouillon and on blood serum and nutrient glycerine agar, and at temperatures above 30°C. Bacterium influenza also flourishes at blood heat and can only be grown, it is said, in the presence of red blood corpuscles or in media containing yolk of eggs; other organisms have thus far refused to be cultivated at any temperature or on any artificial medium, e. g. Bacterium lepra and B. syphilitis. Some bacteria