180 



Appendices to Fourth Anniml Report 



material, which, as it sinks, carries the bacteria, &c,, along with it, but it 

 is also to be accounted for in part by the fact that near the bottom there 

 is almost invariably a larger quantity of organic matter, in addition to 

 which bacteria devjplop much more rapidly when the fluids in which they 

 are present are at rest. 



Having thus completed the determination of the number of organisms 

 present in each sample of water, the more difficult and prolonged work of 

 isolation and separate investigation of the various species follows. 



In many cases a direct transfer is made of some of the points of growth 

 on the gelatin plate to separate tubes containing nutrient jelly — whence, 

 after further growth, the cultivations can be successively inoculated into 

 various nutrient media, and their properties studied. 



But although the plate method, when it can be employed, is that best 

 adapted for the separation of two or three forms of growth, it is not 

 without serious disadvantages if used alone. Amongst other inconve- 

 niences, the thin layer of jelly is with difficulty kept moist in dry weather, 

 especially in the summer ; and when a large number of plates come to be 

 dealt with, the moist-chamber accommodation is apt to become insufficient. 

 Moreover, it is difficult to maintain an adequate degree of moisture with- 

 out the aid of artificial heat. But there is another and still more serious 

 disadvantage. It is, that when some common forms of bacteria, e.g., 

 bacterium termo, are present, they grow much more rapidly than some 

 other bacteria which do not liquefy the jelly, and the spreading points of 

 liquefaction run together and practically defy all attempts at isolation. 

 There maybe no difficulty in isolating three or four dijfferent species under 

 such conditions ; but when there are many different species, and they have 

 not time to acquire distinctive characters, many must be entirely over- 

 looked. 



It is therefore desirable to employ some additional method which shall 

 allow of a sufficient degree of germination to each species for the produc- 

 tion of distinctive characters. This is obtained by the employment of 

 some more solid soil which does not rapidly deteriorate, and upon which 

 most of the known forms of bacteria and lower fungi grow readily, The 

 cultivating soil which has been found most suitable for this purpose, and 

 most readily prepared, is bread paste. Into flasks of about two inches 

 diameter, carefully sterilised and plugged with cotton wool, bread crumbs 

 are introduced in sufficient quantity to form a layer about half an inch in 

 thickness, the surface being left as even as possible. The crumbs are 

 moistened with distilled water, the flasks plugged, and the whole carefully 

 sterilised by heating for some hours in a steam chamber at 100° C. 

 Several dozen flasks can be prepared in this way at the same time with a 

 small expenditure of labour, and they keep for many months in a moist 

 condition. 



If a very minute quantity of the water under investigation is placed 

 upon the surface of the paste, the various organisms present grow upon 

 and spread over the surface. The growth of some kinds, no doubt, impedes 

 the growth of' others, and in course of time one may entirely overmaster 

 the rest. But during a certain time, in the struggle for existence, several 

 species may go on growing side by side, and show distinctive features by 

 which they can be partially recognised and separated. And in many 

 cases a very long period, it may be months, elapses before the species 

 which have attained a footing on the soil are entirely overcome by any 

 one of their number. 



During this period of growth several points of different colour and 

 aspect may be distinguished side by side, and more or less merged in 

 each other. These difi'erent points indicate difi'erent species, not pure, 



