then dilute 1 c.c. to 100 c.c. with sterile liquid, and inoculate 10 tubes each with 1 c.c., each tube will contain about 100 microbes; inoculate 10 tubes each with 5 c.c., each tube will contain about 50 microbes; inoculate 10 tubes each with 1 c.c., each tube will contain about 10 microbes. Then dilute 1 c.c. to 1,000 c.c. with sterile liquid, and inoculate 10 tubes each with 1 c.c., each tube will contain about 10 microbes; inoculate 10 tubes each with 5 c.c., each tube will contain about 5 microbes; inoculate 10 tubes each with 1 c.c., each tube will contain about 1 microbe; inoculate 10 tubes each with 05 c.c., each tube will contain about 5 microbes.

Of the last ten tubes, then, about five only would develop growths, and these would, in all probability, be derived from a single microbe each, and thus be pure

cultures.

Now although, in comparison with the gelatine-plate method described below, this dilution process appears tedious and troublesome, yet for the isolation of some micro-organisms it is of the utmost importance, notably in the case of those which, like the bacteria of nitrification, refuse to grow on the ordinary solid culture media. Later on will be found an account of Miquel's application of this dilution process to water-examination.

a

FIG. 5.-HEMATIMETER (after Jörgensen).

a, Glass slide on which the perforated glass square, b, is cemented so as to form an extremely shallow circular cell, the depth of which is accurately determined once and for all. On the glass bottom of this cell some very small squares of known dimensions are etched. A small drop of the liquid in which the number of yeast-cells is to be determined is placed in the cell, and the cover-glass, c, placed on the top so as to be in contact with the liquid in the cell. The volume of liquid resting on each of the little squares can thus be easily calculated, and by counting the yeast-cells visible with the microscope in each square, the number in the particular volume of liquid is determined.

Hansen also successfully employed this dilution method in his first preparation of pure cultures of yeast

in 1882, using the hæmatimeter to approximately estimate the number of yeast-cells contained in the liquid. which was to be diluted.

Gelatine-plate cultures.-Considering, then, what great difficulties attach to the preparation of pure cultures by means of liquid media, it may be imagined how welcome was the introduction by Koch of the new methods of culture on solid media, which greatly facilitated the process of purification.

Ordinary photographic glass plates (quarter-plate size) serve admirably for plate-cultivations. If new, they should be soaked in caustic soda, then washed with water, dilute hydrochloric acid, with water again, and finally rinsed with distilled water. They are then put in a metal box and placed in a hot-air oven and exposed to a temperature of from 150° to 160° C. for two hours. The gas is then turned out and they are allowed to cool, the door of the oven being kept closed from the beginning until the moment when the plates are required for use.

A cylindrical glass dish is filled with ice and water, care being taken that it is quite full, as otherwise in placing the thick glass plate over it bubbles of air become enclosed and thus prevent the uniform cooling of the plate.

The glass plate covering the dish is then carefully levelled by means of a 3-screw levelling-stand (fig. 6) and spirit level. When this is done the condensed moisture which has formed on the surface of the now horizontal glass covering plate is wiped off and a glass bell jar placed upon it. It is convenient to have two such arrangements in use if a large number of platecultivations are to be made, a great economy of time. being thereby secured.

The sterilised glass plate which is to receive the

gelatine is now carefully withdrawn by means of sterile forceps from the box or oven (the door of which is immediately reclosed), and the future upper surface of the plate is held downwards during its transfer to the levelled plate, and only turned up when the bell jar is momentarily raised to admit it. In this way the falling of air-organisms on the culture plate is avoided.

All is now ready for the gelatine-tubes, which should have been previously melted in a beaker of hot water and then cooled down to 30° C., at which temperature the gelatine remains liquid.

The cotton-wool stopper is first singed in a bunsenflame to get rid of any chance organisms which may have fallen upon it, and is then removed very carefully, not pulled straight out, but by gently twisting; the mouth of the tube is then passed quickly through the flame to destroy any organisms which may be present, and the contents are poured on to the sterilised glass plate, the bell jar being again lifted for a moment and held over the plate during the operation. After it has congealed, the gelatine-plate is quickly removed to a damp chamber, where it is placed on a glass bench upon which another glass bench can be placed with its gelatine-plate until the chamber is filled.

The damp chamber consists of an ordinary dinner plate covered with a common glass bell jar which fits into the depression of the plate. The air in this chamber is rendered moist by just covering the bottom of the plate with a little sterilised distilled water.

The damp chamber with its contents is allowed to remain for an hour or two in a cold room and is then placed in a cupboard maintained at a uniform temperature of from 18-22° C. The plates prepared as above would remain sterile excepting in so far as they might be accidentally contaminated by aërial microbes.

or others gaining access during the operation, and in all experiments it is necessary to pour such plates as a

control for comparison with those which have been purposely infected.

The gelatine-plate method as applied to waters will be described later in detail, but it will be convenient here to give an account of how these plate-cultures are employed for the isolation of particular micro-organisms.

For instance, supposing that we take some bouillon which has been either imperfectly sterilised or purposely exposed to contamination, we should probably find that under the microscope a confused mass of forms would be visible, and to separate out some of these different varieties we abstract a small quantity of the liquid by means of a sterilised instrument. For this purpose a piece of platinum-wire about of an inch in thickness and about three inches in length is commonly used, one end of which is fused into a thin glass rod by melting the latter in a bunsen-flame, inserting the wire, and then allowing the junction to cool slowly. The free end of the wire is then twisted into a small oval loop, which will contain quite a sufficient quantity of the liquid, and the wire being readily sterilised by heating immediately before use in the bunsen-flame, lends itself particularly

well for such manipulations. Care must be taken that the wire is permitted to cool before use, and during the minute which elapses in so doing nothing must be allowed to come in contact with it. It is often quite sufficient to simply use a straight piece of platinum wire instead of bending it into a loop, as in the majority of cases enough material can be conveyed even on the point of such a platinum needle.

Having obtained some of the material, we take a melted gelatine-tube, observing in opening it all the precautions previously described, and insert the infected needle into the gelatine, the needle is then rapidly withdrawn and the cotton-wool stopper replaced. The needle should be immediately sterilised; this is of course especially necessary in working with pathogenic microorganisms.

The gelatine-tube thus infected must be carefully shaken to ensure the distribution of the micro-organisms throughout the mass of the liquid gelatine; if we were immediately to pour a plate with this we should in all probability obtain such an enormous number of colonies and so densely crowded together as to prevent their proper individual development. To obviate this we take another gelatine-tube and transfer from the original tube, or first attenuation as it is generally called, one loop to this second tube, and, after thoroughly mixing, several loops from this second tube to a third. It is essential that in each case the gelatine. should be gently but thoroughly shaken, so as to ensure the even distribution of the individual organisms which have been introduced. The amount transferred from one tube to another must be varied according to the judgment of the operator, and after a little practice there is generally little difficulty in procuring successful attenuations or plates in which the colonies are so

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