154 PLANT PARASITES. 



added, and mounted in balsam. In specimens prepared in this way the bacteria and 

 some of the nuclei are violet, the remainder of the tissue is red. 



In this as in other methods of staining bacteria in tissue the sections are liable to 

 shrivel and curl. This may in many cases be avoided by fixing the sections on to the 

 cover slip with albumen fixative (see page 51) before the staining begins, carrying 

 cover glass as well as section through the subsequent processes. Since some bacteria 

 are decolorized by Gram's method it is not applicable to the staining of all organisms in 

 tissues. It is often a valuable aid in the differentiation and identification of species with 

 similar morphology and in some instances, as in the staining of gonorrhceal pus, has an 

 especial diagnostic value (see page 194). 



Weigert's Modification of Gram's MetTiod.The sections are laid for half an hour in 

 the anilin gentian-violet solution prepared as above, then rinsed off in three-quarter- 

 per-cent salt solution, and spread on a slide. They are now dried with blotting-paper 

 and covered for two minutes with the iodin solution. The iodin solution is now washed 

 off in the water, the section dried with blotting-paper, and decolorized with anilin oil 

 or a mixture of 2 parts of anilin oil and 1 part of xylol, several times renewed. Finally 

 the sections are cleared in xylol and mounted in balsam. 



In many cases it is well to accomplish a double staining by a preliminary contrast 

 stain. Thus, before the use of Weigert's modification of Gram's stain the sections may 

 be put for half an hour in a solution of picro-carmin, then rinsed in water and stained 

 as above. By this modification of Gram's method fibrin is deeply stained. 



Loffler's alkaline-methylene-blue solution is a very valuable and powerful stain for 

 bacteria, either in fluids or in tissues. 



It consists of 



Saturated alcoholic solution of methylene blue, . . .30 parts. 

 Aqueous solution of caustic potash 1: 10,000, . . . 100 " 



For staining bacteria in tissues the stain is allowed to act for a few minutes. The 

 section is then put for a few seconds in one-half -per-cent acetic acid, then rinsed in 

 water, and the superfluous color removed from the tissue by repeated rinsing in alco- 

 hol, which at the same time dehydrates it. Then it is cleared with oil of cedar and 

 mounted in balsam. Care should be taken not to remove too much color with the 

 alcohol. 



It should always be borne in mind in staining the bacteria that great exactness is 

 not usually necessary either in the strength of the coloring solutions or in the time of 

 exposure of the bacteria to them. We are seeking for certain effects namely, the 

 staining of the germs and this depends not only upon the quality and strength of the 

 dye, the time of exposure, etc., but also upon the nature of the bacterial species and its 

 conditions at the time the staining is undertaken. Thus it not infrequently happens 

 that bacteria which will stain readily and deeply with a given solution when they are 

 in a condition of active growth, may be scarcely at all colored if they have been dead 

 or inactive for a long time, although their outward shape appears to be unchanged. 

 So it should be remembered that, while there is little difficulty in most cases in staining 

 the bacteria, the operation is not one of mere routine, but requires intelligent attention 

 to the particular conditions of the species in hand. 



For the recognition and study of bacteria the best optical apparatus is requisite. 

 An homogeneous immersion lens (at least one-twelfth) and the Abbe condenser are in- 

 dispensable for ordinary work. 



Artificial Cultivation of Bacteria. 



For the complete investigation of the different forms of bacteria, particularly in 

 their relations to disease, we must isolate them so as to be able to study their life his- 

 tory and the effects of their inoculation into healthy animals. It has long been known 

 that bacteria could be cultivated in a variety of artificial, so-called "nutrient media" or 

 soils. Fluids were formerly used for this purpose, but it is very difficult to separate 

 single species in fluids. Robert Koch introduced, some years ago, a technical improve- 



