place the spores in water, and they will germinate at oncefor example, U. segetum, longissima, etc.; but some require a longer period of immersion, e.g. T. tritici, which will not germinate till after being several days in water. The germination of the Tilletia spores can also be conducted in a hanging-drop culture in the following manner :-A piece of glass tube, about half an inch (12 mm.) in diameter and about three-quarters of an inch (18 mm.) long, is cemented on an ordinary glass slide, so as to form a deep cell (Fig. 10).

Fig. 10.-Deep cell for hanging-drop cultures, made by cementing a piece of glass or lead tube upon an ordinary glass slide.

Into this is placed a small quantity of water. The drop containing the spores to be germinated is placed on the centre of a circular cover-glass, which will fit the top of the cell (Fig. 11). If the upper edge of the tube which forms

Fig. 11. Hanging-drop culture, seen in section.

the cell be oiled, no air can get into the culture, and the germination can be watched for months, as the hangingdrop will not evaporate, because the water placed in the bottom of the cell keeps the atmosphere saturated, and any loss which it may sustain from evaporation is made. up from the water at the bottom of the cell.

There is no need to keep these sealed hanging-drop cultures in a moist atmosphere, because the necessary

moisture is contained in the cell itself; all that is required is to place them under a bell-glass to keep them free from dust. The disadvantage of this mode of culture is, that all spores will not germinate normally unless they get a supply of free air. To obviate this hindrance, the glass cover may be fixed to the cell with three tiny fragments of wax. These cells may be readily constructed with lead tubing; an ordinary piece of gas-piping, cut into suitable lengths with a knife, and the ends smoothed on a whetstone, will answer all the requirements of the case.

The germination of the spores in nährlösung, however, requires more care. The nährlösung is prepared by boiling fresh horse-dung in pure water, and filtering first through

Fig. 12.-One of Brefeld's "kämmer" for nährlösung cultures. It consists of a glass tube open at both ends; in the middle a bulb has been blown which has been compressed laterally, so that its sides are parallel to each other, or nearly so. When in use the two open

ends of the tube are closed with cotton wool.

coarse filtering-paper, then through fine; then boiling again for a short time in a flask, the mouth of which is closed, while the steam is issuing from it, by a firm plug of cotton wool. After three or four hours the flask is again boiled for a short time. To sterilize the fluid this boiling requires to be repeated several times, carefully closing the mouth of the flask each time with a plug of cotton wool. The spores to be germinated are put in a small quantity of sterilized nährlösung, and the process watched in camera. The little apparatus necessary consists of a glass tube about eight or ten inches long, open at both ends; in the middle of this tube a bulb has been blown, the sides of which have been compressed laterally (Fig. 12), so that they are flat and parallel to each other. Before using, the apparatus (kammer)

must be sterilized by heat; and after the nährlösung has been introduced, the two ends of the tube are closed by cotton wool; this allows free access of air, but filters out any germs that may be floating in it.

Or the culture may be made in a hanging-drop cell made of lead tubing, in the sides of which two holes have been cut (Fig. 13). I have found it most convenient to

Fig. 13.-Hanging-drop culture cell, made of lead tubing, with two holes for the admis sion of air; when in use with nährlösung these openings are closed with cotton wool.

wrap cotton wool round the cell, and to hold it in its place by a small elastic band. These cells require to be sterilized by washing with a weak solution of corrosive sublimate.

CHAPTER XIII.

THE ARTIFICIAL INFECTION OF PLANTS.

IN order to ensure success in the artificial infection of plants, attention to several little details is absolutely necessary. Of course, if you simply wish to produce Æcidium urtica on a cluster of nettles, you may throw a handful of Carex hirta affected with Puccinia upon the ground where the nettles grow in autumn, and, trusting to chance, you will probably find them bearing the Æcidium the following spring. But such a procedure is open to many objections; the wind may blow away your Carex during the long winter and spring months either before the Puccinia has germinated or before the nettles have appeared above ground. A still greater objection is, that even if a few clusters of acidia happen to be produced on the nettles, you have no proof that they arose from the Puccinia you threw down. Still more important is it to avoid this clumsy method of "laying on," if you are investigating the lifehistory of any particular species of Uredine, for it often happens that more than one species attacks the same hostplant; P. magnusiana, trailii, and phragmitis, on the reed, for instance.

The first thing to be done is to provide suitable plants for infection. These should, it is hardly necessary to state, be healthy, and have had time to become established before

they are infected. It is a good plan to establish a number of plants, say half a dozen, in the autumn; they will then be ready for use in the following spring. It is often convenient to infect every alternate plant, so that the remaining plants may be kept as control specimens. The reason for using established plants is that the young foliage is so much more easily infected by the Uredineæ than the older; in fact, it is by no means uncommon for an old leaf to die off before the Uredine has had time to complete its development.

If

Let us suppose we wish to perform the classical infection of the barberry with P. graminis. In the autumn, six young barberries, small enough to be covered with a bell-glass, having been planted, as soon as their leaves are fully developed in the spring they may be infected in the following manner. A quantity of P. graminis having also been provided in the autumn and kept during the winter in the mode before explained, as soon as the barberry foliage is ready, test the germinative power of the P. graminis by placing a few fragments in water in a watch-glass. it germinate freely and produce a good crop of promycelial spores, as proved by microscopic examination, the contents of the watch-glass may be at once employed. It is best to do your infection experiments in the evening. Water one of the barberries freely through the rose of a watering-can and then cover it with a bell-glass; then water the outside of the bell-glass. By so doing the temperature of the enclosed air is reduced, and the inside of the bell-glass as well as the leaves of the barberry become bedewed with condensed vapour. After leaving it a few minutes, remove the bell-glass, and apply the germinating spores with a camel-hair pencil. As the promycelial spores easily become diffused in the water in the watch-glass, by stirring it with the camel-hair pencil the water becomes equally charged