REACTION TO STAINS. 29 



To determine whether bacteria are properly stained examine with the diaphragm 

 of the condenser wide open. If they can not be seen distinctly with this flood of 

 light they are not well stained. The bacteria should be well separated on the cover 

 and deeply stained, while the background should be very free from stain. 



Dr. Weigert seems to have been the first to use nnilin stains for the demon- 

 stration of bacteria in tissues. This was about 1875. Since that time staining in 

 tissues has been worked up carefully for bacteria causing animal diseases, but very 

 little is known respecting best methods of staining bacteria in vegetable tissues. 

 The difficulty lies in the fact that the tissues of the higher plants often take the 

 basic anilin stains as readily as the bacteria and retain them even more tenaciously. 

 Special remarks may be looked for under particular diseases. 



CULTURE MEDIA. 

 NUTRIENT GELATIN. 



() Plate Cultures. Colonies, young and old, buried and superficial, crowded 

 and wide apart, should be examined for color, translucency or opaqueness, shape, 

 thickness of the surface growth, and character of the margin. They should also 

 be studied under low powers of the compound microscope for lobes, branches, 

 granulations, wrinkles, flecks, concentric rings, radial filaments, arrangement of the 

 dividing rods on the margin of the colony, iridescence, etc. The microscopic 

 appearance of the surface colony during the first 48 hours is often different from 

 that later on. The rapidity of growth should be compared with that of some 

 common and easily accessible organism, e.g., Bacillus coli\ B. ainylovorus, Bacterium 

 campestre. The comparative rate of growth of buried and surface colonies should 

 also be carefully noted. How is the appearance of the colony changed by increasing 

 the amount of gelatin, or varying the brand of gelatin? Are the surface colonies 

 viscid, or can they be lifted bodily in one mass from the substratum ? 



(b) Stabs. The nature of the sxirface growths and of the deeper growths should 

 be carefully examined. Is there any marked tendency of the latter to grow down- 

 ward or outward into the body of the gelatin, either in distinct masses or as a dif- 

 fused cloudiness? Observe effect, if any, on growth when the gelatin is acid or only 

 feebly (litmus ) alkaline. If liquefaction of the gelatin occurs, note its rapidity and 

 whether it is mostly restricted to the surface or is equally rapid along the line of 

 the stab in the depths ; note also whether the liquefied gelatin is clear or cloudy in 

 tubes which have not been shaken, and whether a pellicle has formed on its surface. 

 Liquefaction may be very rapid (taking place within a few hours), may occur after 

 three or four days, may be long-delayed and feeble (only visible after some weeks), 

 or may not occur at all. It is the cases of feeble and long-delayed liquefaction 

 which lead to contradictory statements on the part of different observers, and con- 

 sequently cultures should remain under observation for a considerable time and on 

 a variety of gelatins. Various substances interfere with liquefaction. Determine 

 whether liquefaction can be prevented by the addition of grape-sugar or cane-sugar 

 (10 per cent). Look for gas-bubbles, for crystals, for any fluorescence or staining 

 of the medium (green, brown). Inasmuch as the growth of some bacterial plant 



