194 LABORATORY EXERCISES IN BACTERIOLOGY. 



being added in small proportions to a drop of water on a slide or cover. Thus, one 

 should draw the loop filled with a bouillon or milk culture through a large drop of 

 distilled water in a watch-crystal first; then, having flamed the loop, a small amount 

 of this drop is transferred to the drop of water on the slide and again slightly diffused. 

 The organic matter interferes more or less with the even distribution of the film, and 

 when dried prevents a uniform penetration of the stain ; for which reasons the dilution 

 suggested is to be practised. The remaining steps in spreading and fixing the film 

 are the same as above when the material was obtained directly from a mass of bacteria 

 on a solid medium. 



The exudates and fluid secretions and excretions obtained from diseased individuals, 

 as sputum, pus, or urine, are usually spread in films without dilution. When mucus 

 is present in such substance, preventing easy spreading, it is well to warm the slide 

 slightly and insure an even distribution in a thin film by pressing a second slide upon 

 the drop and drawing it across the surface of the first slide. This done, the film is 

 dried in the air and fixed by heat as above. Where, as in case of urine, there is much 

 saline matter present, it is best to remove it before naming the slide for fixation of the 

 film by placing the slide for a few minutes in a vessel with excess of water, by which 

 these substances will be taken up. It is again dried in the air and the film fixed in the 

 flame. It is then ready for immediate application of the stain, or may be kept as 

 above indefinitely. 



(c) Smears from Tissues. After inoculations have been made from foci of disease 

 in tissues obtained by surgical operation or autopsy, a section should be made with a 

 clean knife and the cut surface of the tissue drawn lightly and evenly over the slide. 

 The resulting film is dried in the air and fixed either in the flame or by being immersed 

 for five or ten minutes in a mixture of equal parts of absolute alcohol and ether. It 

 may then be at once stained or kept indefinitely in the fluid or dry. The same pro- 

 cedures are applicable when the material has been obtained on a swab of cotton, as 

 diphtheritic exudate, the swab being wiped lightly over the surface of the slide or 

 cover. 



(d) Impression Films. These are prepared by pressing gently and evenly a clean, 

 dry cover or slide to the surface of a colony growing on the surface of some solid medium. 

 After application, it is raised, without sliding, directly from the colony, a print or im- 

 pression being retained from the adhesion to the glass of the superficial individuals 

 in the growth. As before, the film is dried in the air and fixed in the flame. This pro- 

 cedure is intended for the preservation of the relations of the individual bacteria to 

 each other in the colony, some organisms in their growth producing extremely char- 

 acteristic arrangements, often in marvelous patterns (Bact. mycoides, vel figurans). 



STAINING REAGENTS AND MIXTURES. 



The difficulty of exact appreciation of the isolated bacterium is much diminished 

 by proper staining, for which purpose the basic anilines are most efficient. The ordinary 

 nuclear stains used in histologic work have little penetrative power for bacteria, but 

 if intensely applied are sometimes capable of tinging the bacterial substance. How- 

 ever, their use is attended with such difficulties and the results of such moderate value 

 that they are practically never used, the anilines being employed almost exclusively. 

 Basic fuchsin, gentian violet, methylene-blue, thionine, and a few others are the most 

 commonly used. Aqueous solutions are the most efficient, the dried bacteria in the 

 film preparations permitting their diffusion in the bacterial substance to a far greater 



