Dr. KOCH’S NEW METHOD OF PURE CULTIVATION OF BACTERIA
—At the recent meeting of the International Medical Congress in London, during August, Dr. Koch, well known by his researches on the life-history of Bacillus anthracis (see this Journal, Vol. XVII, p. 87), gave a series of demonstrations in the physiological laboratory of King’s College, which were of the greatest interest and importance.
Dr. Koch has recently been appointed to the charge of a laboratory of experimental research connected with the State Department of Public Health in Berlin, and aided by his two assistants, he brought to London material and instruments for the purpose of exhibiting to the members of the Congress the methods of research into the relation of Bacteria to disease, devised by him. The series of photographs of various forms of Bacteria shown by Dr. Koch were valuable, as affording convincing evidence of the necessity of making use of photography as the means of obtaining and preserving a record of the specific form and character of Bacterian growths. Of great interest also were the cultivations of the Bacteria of blue milk, and of those of blue pus, exhibited by Dr. Koch, and of the septic Bacterium of putrid blood, the toxic effects of which were experimentally demonstrated.
Of most general importance, and in our judgment likely to mark altogether a new era in the study of the relations of Bacteria to certain diseases, and to other fermentative processes, was the demonstration by Dr. Koch of a new and yet absolutely simple and obvious method of obtaining pure cultivations of the species of Bacteria.
It is a well-known fact that there are a large number of species of Bacteria differing from one another in the effects which they produce in the medium wherein they are cultivated. It is also well known that Bacteria are so ubiquitous that the examination of any natural medium attacked by them is almost sure to yield evidence of the presence of mote than one species, the varibus species growing together- in inextricable confusion. On this account it has been found a matter of extreme difficulty to determine what effects are due to one species of Bacterium and what to another. And it has indeed been often impossible to determine in such a mixture of forms those which are genetically related to one another, and therefore to distinguish one species from the other forms which are adventitiously associated with it.
To effect the separation of species in a mixture, Mr. Lister employed a method of dilution and division described in his well-known research on the Lactic ferment (see this Journal, vol. xviii, p. 191). Making use of a fluid as the nutrient medium of cultivation (as hitherto has been the almost universal practice in such cultivations), Mr. Lister introduced a drop of sour milk containing possibly twenty kinds of Bacteria, and among them the Bacterium of lactic fermentation, into a large quantity of pure water, the dilution and spacing (so to speak) of the Bacteria thus affected being calculated so to render it probable that a single drop removed frbin the diluted Bacterian mixture would contain a single Bacterium. Such drops were then removed and placed each into a separate culture-tube containing sterilized fluid nutriment, and thus in a certain number of the tubes a pure cultivation consisting of the progeny of a single Bacterium, and, therefore, unquestionably of but one species, was obtained.
This method is tedious and liable to failure owing to the great care necessary to ensure and maintain sterilization of the cultivation fluid whilst exposed for the purpose of inoculation and again for further examination. Dr. Koch was led to this new method of cultivation, which essentially consists in the substitution of a solid for a fluid medium of cultivation, by the use of the method; known to all mycologists of cultivation, upon slices of potato or beet-root. It is readily observed when slices of boiled potato are exposed in a damp condition to the atmosphere that the surface of the slice becomes the seat of development of various Bacteria and of moulds, the spores of which fall from the atmosphere on to the exposed slice, a fact which struck Dr. Koch as of importance in reference to the slices of potato was this—that the various spores falling on to it remain where they fall, and from the spot where each spore or germ originally fell it proceeds to multiply, producing around it a symmetrical hemispherical growth of perfect purity. In fact, owing to the solid character of the nourishing support the germs and spores cannot get mixed as they do in a liquid, each remains distinct from it’s neighbour even though in very close proximity, and without any trouble from the resulting growth, which proceeds in a day or two from each germ— new and perfectly pure cultivations may be started in suitable sterilized fluids.
Dr. Koch’s method consists in substituting for the potato slice a layer of gelatine which is so saturated with water as just to become solid on cooling. The gelatine liquid is readily sterilized by boiling, and into it can be introduced either Pasteur’s salts, peptones, blood-serum, or other nutrient material required by one or other species of Bacterium. The gelatine-medium thus prepared may be kept n a tube and a cultivation thus carried on—on its surface, or (and this is its principal use) it may be spread when liquid on a microscope object-slide and allowed to cool. Then such a gelatine plate may be inoculated by touching its surface with material containing the Bacteria which it is desired to study. The plate is readily protected from the access of accidental atmospheric germs, and maintained at such temperature and degree of moisture (by a glass shade) as the experimenter may desire. The main point of advantage, how’ever, is this—that the point of inoculation on the surface of the gelatine can, owing to its transparency, be readily examined with the highest powers of the microscope and the growth of the Bacteria followed—whilst further, owing to the fact that the medium in which the growth takes place is solid, no mixture of the different kinds which may be present occurs, but each Bacterium produces around it a little spherical nest of its own kind. From these nests, with a sterilized needle-point, individuals can be removed to start new pure cultivations.
But it is obvious that, if the original point of inoculation was very minute, there is no danger of any accidental contamination from atmospheric germs, for these are not likely to fall on the identical spot no bigger than the puncture of a needle’s point, where the experimental culture is going on. As a matter of fact, where they fall on to the gelatine there they remain and grow, and fifty such accidental spores may fall on to the gelatine plate without in the least interfering with the purity of the experimental culture.
There is yet, further, a very simple device which enables Dr. Koch to use this gelatine surface as a means of i! spacing “and dividing the various species in a mixture of Bacteria. Ke dips a sterilized peedlc into such a mixture. and then makes a long shallow streak with the needle’s point upon the surface of the gelatine. The Bacteria which were adhering to the needle’s point are in this way dropped at intervals along the streak, some nearer some further apart, but all (with rare exceptions) in such a way that their subsequent growth keeps clear of that of a neighbour, and can, with the aid of a low power or even without any microscope, be visited by a sterilized needle point, and thus used to start on another gelatine plate a perfectly pure cultivation.
These pure cultivations, such as Lister aimed at by his method of dilution and division, may be called, in order to indicate to what an extent they are known to be pure, “monosporous cultivations,” since the principle which distinguishes them is that all the growth is the offspring of a single isolated germ or spore.
It is only by such monosporous cultivations that we can arrive at solid conclusions in reference to the forms and activities of the Bacteria, e.g. as to whether one form can give rise to progeny of another form when its food and conditions of growth are changed, and again, as to whether special fermentative powers can be lost or acquired in the course of generations derived from one parent germ, but subjected to different conditions as to food, temperature, and oxygen.
The method of gelatine cultivation devised by Dr. Koch, places the means of following out these inquiries in the hands of every careful microscopist. Such methods as Lister’s were too troublesome and too difficult for general and widespread application; but now that monosporous cultivation of Bacteria has been rendered a comparatively simple and certain affair, we may expect immediate and immense advances in our knowledge of the whole series of phenomena to which the Bacteria are related.
Amongst problems which require immediate investigation by the new method are the distinctive properties of the various kinds of Bacteria which may infest the wounds of surgical practice, and their specific susceptibility to the destructive influence of carbolic acid and other antiseptics ; further, the possibility of isolating a specific Bacterium in contagious diseases not yet investigated: and (of great physiological interest) the isolation and investigation of the properties of the specific Bacterium of the ammoniacal fermentation of urine.
Dr. Koch and his assistants will, no doubt, shortly publish a detailed account of the researches which they have been engaged in during the past year, and will give particulars as to the methods of investigation employed by them, which had not (we believe), previously to the meeting of the International Medical Congress, been given to the public.
A remarkable negative result obtained by Dr. Koch, so far as his experiments with the new method of nionosporous culture have yet extended, is, that there is no transition of forms amongst) at any rate, the pathogenous Bacteria—a Micrococcus produces Micrococci, and no other form; a Bacillus produces only Bacilli; a biscuit-shaped form (Bacterium proper) only biscuit-shaped forms; a Spirillum only Spirilla. Moreover, the facies of the discoidal or spherical mass formed by a growth, as seen with a low power excavating its way in the gelatine is characteristic of species, so that a practised observer can, in some cases, recognise a particular Bacillus or Micrococcus by the naked-eye appearance of the growth alone, or, at any rate, without actually observing the individual units of the growth.—L.
