Preparation of Nutrient Media—Separation, and Cultivation of Bacteria.
Fig. 260.—Plate Cultivation Showing Colonies.
To cultivate micro-organisms artificially they must be supplied with the proper nutrient material, perfectly free from pre-existing organisms. The secret of Koch’s methods greatly depends upon the possibility, in the case of starting with a mixture of micro-organisms, of being able to isolate them completely one from another, and to obtain an absolutely pure growth of each cultivable species. When sterile nutrient gelatine has been liquefied in a test-tube and inoculated with a mixture of bacteria in such a way that the individual micro-organisms are distributed throughout it, and the liquid is poured out on a glass plate and allowed to solidify, the individual bacteria, instead of moving about freely as in a liquid medium, are fixed to one spot, where they develop their own species. In this way colonies are formed, each possessing its own biological characteristics and morphological appearances ([Fig. 260]).
To maintain individuals isolated from each other during growth, and free from contamination, it is only necessary to thin out the cultivation to protect the plates from the air, and to have facilities for examining them from time to time, and observing the characteristic microscopical appearances. The colonies on nutrient gelatine examined with a low power ([Fig. 260]), if micro-organisms such as Bacillus anthraces and Proteus mirabilis, the naked eye appearances in test-tubes of the growth of the bacilli of anthrax and tubercle, and the brilliant growth of micro-coccus prodigiosus, may be given as examples in which the appearances are often very striking and sometimes quite characteristic. I must, however, first direct attention to a well-recognised fact, that bacteriology only touches animal pathology at a few points, and that so far from bacteria being synonymous with disease germs, the majority of these remarkable organisms appear to be beneficent rather than inimical to man. This is of immense importance to science, as I shall attempt to show further on; although even a brief description of all the useful ferments due to bacteria and brought into use would occupy a volume to themselves, and call for a school of bacteriology quite apart from that involved in the medical aspect of the question, for the purpose of fully investigating problems raised by the agriculturist, the forester, the gardener, the dairyman, brewer, dyer, tanner, and other industries, which open up vistas of practical application, and to some extent are already being taken advantage of in commerce.
The Preparation of Nutrient Gelatine and Agar-agar.—Take half a kilogramme (one pound) of beef as free as possible from fat, chop finely, transfer to a flask or cylindrical vessel, and shake up well with a litre of distilled water. Place the vessel in an ice-pail, or ice-cupboard, or in winter in a cold cellar, and leave for the night. Next morning commence with the preparation of all requisite apparatus. Thoroughly wash and rinse with alcohol about 100 test-tubes, and allow them to dry. Plug the mouths of the test-tubes with cotton-wool, place them in their wire cages in the hot-air steriliser, to be heated for an hour at a temperature of 150°C. In the same manner cleanse and sterilise several flasks, and a small glass funnel. In the meantime, the meat infusion must be well shaken, and the liquid portion separated by filtering and squeezing through a linen cloth or a meat press. The red juice thus obtained must be brought up to a litre by transferring it to a large measuring glass and adding distilled water. It is then poured into a sufficiently large and strong beaker, and set aside after the addition of ten grammes of peptone, five grammes of common salt, and 100 grammes of best gelatine.
In about half an hour the gelatine is sufficiently softened, and subsequent heating in a water bath causes it to be completely dissolved.
The next process requires the greatest care and attention. Some micro-organisms grow best in a slightly acid, others in a slightly alkaline, medium. For example, for the growth and characteristic appearances of the comma bacillus of Asiatic cholera a faintly alkaline soil is absolutely essential. This slightly alkaline medium will be found to answer best for most micro-organisms, and may be obtained as follows:—With a clean glass rod dipped in the mixture, the reaction upon litmus-paper may be obtained, and a concentrated solution of carbonate of soda must be added drop by drop until red litmus-paper becomes faintly blue. If it is too alkaline, it can be neutralised by the addition of lactic acid.
Finally, the mixture is heated for an hour in the water-bath. Ten minutes before the boiling is completed the white of an egg beaten up with the shell is added, and the liquid is then filtered while hot.
During filtration the funnel should be covered over with a plate of glass, and the process of filtering must be repeated, if necessary, until a pale straw-coloured, perfectly transparent filtrate results. The sterilised test-tubes are filled to about a third of their depth by pouring in the gelatine carefully and steadily. The object of this care is to prevent the mixture touching the part of the tube with which the plug comes into contact; otherwise, when the gelatine sets, the cotton-wool adheres to the tubes and becomes a source of embarrassment to subsequent procedures. As the tubes are filled they are placed in a basket, and then sterilised. They are either lowered into the steam steriliser, when the thermometer indicates 100 cc., for twelve minutes, for four or five successive days, or they may be transferred to the test-tube water-bath, and heated for an hour or two for three successive days.
If the gelatine shows any turbidity after, it must be poured back into a flask, boiled for ten minutes, and filtered again, and the process of sterilisation repeated.
Nutrient Agar-agar is a substance prepared from seaweed which grows on the coasts of Japan and India, and is supplied in long crinkled strips. It boils at 90° C., and remains solid up to a temperature of about 45° C. It is therefore substituted for gelatine in the preparation of a jelly for the cultivation of those bacteria which will grow best in the incubator at the temperature of the blood, and also at ordinary temperature for bacteria which lignify gelatine. The preparation is conducted on much the same principles as those already described. Instead, however, of 100 grammes of gelatine, only about twenty grammes of agar-agar (1·5 to 2 per cent.), and to facilitate the solution it must be allowed to soak in salt water overnight. Flannel is substituted for filter paper. The hot-water apparatus is invariably employed. The final results, when solid, should be colourless and clear; but if slightly milky, it may still be employed.
Wort-gelatine is used in studying the bacteria of fermentation. It is made by adding from five to ten per cent. of gelatine to beer-wort.
Glycerine Agar-agar.—This is made by adding five per cent. of glycerine to nutrient agar-agar, after the boiling and before the filtration.
Fig. 261.—Pure Cultivation in Tubes (Crookshank).
Test-tube Cultivations.—To inoculate test-tubes containing nutrient jelly, the cotton-wool plug is removed. A sterilised needle, charged, for example, with blood or pus containing bacteria, is thrust once in the middle line into the nutrient jelly, and steadily withdrawn. The tube should be held horizontally or with its mouth downwards, and the plug replaced as quickly as possible, and an india-rubber cap fitted over the mouth of the tube.
The appearance produced by the growths in the test-tubes can be in most cases sufficiently examined with the naked eye ([Fig. 261]). In some cases the jelly is partially liquefied, while in others it remains solid. The growths may be abundant or scanty, coloured or colourless. When liquefaction slowly takes place in the needle tracts, the appearances which result are often very delicate and in some very characteristic. The appearance of a simple white thread with branching lateral filaments, of a cloudiness, or of a string of beads in the track of the needle, may be given as examples. In some cases much may be learnt by means of a magnifying-glass.
Beneke recommends that gelatine culture tubes should be inoculated by making a puncture quite at the side of the medium, close to the glass. The advantage of this method over the plan of inoculating the mass in the middle is that the growing culture can be microscopically examined from the outside, and various details made out, such as the nature of the growth, the comparative appearance of colonies near the surface and those situated more deeply, and the presence of one or more distinct organisms. If the tubes used have the opposite sides flat and parallel, such examinations will be still further facilitated.
Plate Cultivations.—By this method a mixture of bacteria, whether in fluids, excreta, or in cultivations on solid media, can be so treated that the different species are isolated one from the other, and perfectly pure cultivations of each of the cultivable bacteria in the original mixture established in various nutrient media. We are enabled also to examine under a low power of the microscope the individual colonies of bacteria. The same process, with slight modification, is also employed in the examination of air, soil, and water.
In order to spread out the liquid jelly evenly on the surface of a glass plate, and to hasten its solidification, it is necessary to place the plate upon a level and cool surface. The glass plates are sterilised in an iron box placed in the hot-air steriliser, at 150° C., from one to two hours.
The damp chambers for the reception of the inoculated plates are prepared by cleansing and washing out with one in twenty carbolic acid the shallow glass dish and bell-cover ([Fig. 253]). A piece of filter-paper should cover the bottom of dish, moistened with the same solution.
“In a glass-beaker with pad of cotton-wool at bottom place tube containing cultivation, the three tubes to be inoculated, three glass rods which have to be sterilised, and a thermometer. Liquefy the gelatine in the three tubes by placing them in a beaker containing water 30° C. Keep the tubes, both before and after the inoculation, in the warm water to maintain the gelatine in a state of liquefaction. Remove the plug from the culture and also the plug of test-tube with liquefied jelly. With the needle take up a droplet of the cultivation and stir it round in the liquefied jelly. Replace both plugs, and set aside the cultivation. Hold the freshly-inoculated tube almost horizontally, then raise it to the vertical, so that the liquid gelatine gently flows back. By repeating this motion, and rolling the tube, the micro-organisms which have been introduced are distributed throughout the gelatine. Any violent shaking, and consequent formation of bubbles, must be carefully avoided. Inoculate the second tube, and also third, in the same way, but with three droplets from a sterilised needle. The next process consists in pouring out the gelatine on glass plates and allowing it to solidify.
“Remove cover of box containing sterilised plates, withdraw a plate with sterilised forceps, and rapidly transfer it to the filter-paper under the bell-glass and quickly replace cover of box. Remove plug from the test-tube which was first inoculated, and the contents are poured out on the plate. With a glass rod the gelatine must be then rapidly spread out in an even layer within about half an inch of the margin of the plate, the bell-glass is replaced, and the gelatine is allowed to set. Meanwhile a glass bench is placed in damp chamber, upon which the plate is placed when the gelatine is quite solid; precisely the same process is repeated with the other tubes.
“The colonies will be found to develop in the course of a day or two, the time varying with the temperature of the room. The lower plate will contain a countless number of colonies, which, if the micro-organisms liquefy gelatine, speedily commingle, and produce in a very short time a complete liquefaction of the whole gelatine. On the middle plate the colonies will also be very numerous, but retain their isolated positions for a longer time; while on the uppermost plate the colonies are completely isolated from one another, with an appreciable surface of gelatine intervening.
“The microscopical appearances of the colonies are best studied by placing the plate on a slab of blackened glass, or on a porcelain slab if the colonies are coloured. A small diaphragm is used, and the appearances studied principally with a low power. A much simpler method of plate-cultivation is to pour the liquefied jelly into shallow flat dishes; they take up much less room, and in many ways are more convenient.
“Nutrient agar-agar can also be employed for the preparation of plate-cultivations, but it is much more difficult to obtain satisfactory results.”