198 



THE AMERICAN MONTHLY 



[October, 



Decolorizing. — The cover-glass, 

 dark-blue on its removal from the 

 coloring fluid, is transferred directly 

 to a mixture of one part nitric acid 

 and three to four parts water, floated 

 about for a few seconds (at the long- 

 est thirty seconds) and then transfer- 

 red for a few minutes to 60 per cent, 

 alcohol, to remove the adherent acid. 

 The preparation need not now appear 

 colorless. Koch emphasizes this 

 point, and believes that the many 

 failures made in the application of 

 the method are very likely due to the 

 prolonged stay in the acid, whereby 

 the color is finally abstracted from 

 the bacilli themselves. 



After-coloration. — From the sixty 

 per cent, alcohol the cover-glass is 

 floated upon a dilute watery solution 

 of the complementary stain, vesuvin, 

 if methyl-violet has been used for the 

 primary stain ; if fuchsin, methylene- 

 blue. The solution can be prepared 

 from a saturated alcoholic solution 

 kept on hand whenever needed, and 

 should be barely translucent at a depth 

 of 2 cm. After a few minutes it is 

 returned to sixty per cent, alcohol to 

 wash away any adherent staining 

 fluid. 



The specimen is now prepared for 

 examination by inverting the cover- 

 glass over a drop of distilled water 

 placed upon a microscopic slide. In 

 general, the after-staining may be 

 dispensed with, as its sole object is 

 to give the various organized sub- 

 stances, including foreign bacteria, 

 epithelial cells, etc., a color which 

 will contrast with the tubercle bacil- 

 lus and bring it into better relief. If 

 the after-stain be omitted, the speci- 

 men is ready for examination after 

 the nitric acid has been washed away 

 in sixty per cent, alcohol. 



Koch regards as indispensable in 

 searching for the bacilli a one-twelfth 

 inch water-immersion objective, es- 

 pecially that made by Zeiss, Jena, 

 giving with proper eye-pieces an en- 

 largement of 500-700 diametei's, and 

 Abbe's substage illuminator, by which 

 the light is not only greatly increased 



in quantity, but the structural details 

 of the histological elements are made 

 to vanish and a pure color picture is 

 obtained. The bacilli amongst masses 

 of detritus, invisible in many cases 

 with ordinary means of illumination, 

 will thus stand out clearly. Oil im- 

 mersions are still better, but not so 

 necessary as in the examination of 

 sections of tubercle. The organisms 

 brought to view appear as blue rods, 

 if methyl-violet has been used for 

 the preliminary stain ; if fuchsin, red, 

 very slender, slightly curved or an- 

 gled, hardly ever straight, from one- 

 fourth to one-half the diameter of a red 

 blood corpuscle (.001 5-. 0035 mm.) 

 in length. The field itself will have 

 the color of the second stain, if that 

 has been employed ; otherwise it will 

 be more or less colorless, the nitric 

 acid having left the primary stain in 

 the tubercle bacilli onty. A syste- 

 matic search of the -whole cover-glass 

 area is to be made by a proper ma- 

 nipulation of the slide-holder arid rec- 

 ord kept of the number of bacilli 

 present in a ceitain number of fields, 

 to serve as a means of comparing with 

 other pi'eparations and of roughly de- 

 termining the relative number pres- 

 ent each day, if a series of examina- 

 tions is being made. 



If the specimen has been found a 

 desirable one, it may be mounted per- 

 manently by removing the cover- 

 glass* and allowing it to dry thor- 

 oughly, sheltered from the dust. It 

 is then mounted in Canada balsam 

 dissolved in turpentine, without heat- 

 ing. Other solvents of balsam are 

 not recommended by Koch, although 

 xylol has been highlv spoken of by 

 some. It is well to re-examine occa- 

 sionally such permanent mounts, as 

 the bacilli do not always retain the 

 stain ; some fade in a few days, others 



*This may adhere to the slide if the water has par- 

 tially evaporated, and unless care be taken the cover- 

 glass will be broken. If a few drops of distilled water 

 be placed along the edge of the cover, it will be drawn 

 under gradually and elevate tne cover- a slight pu^h 

 of the latter towards the water will hasten the process. 

 The cover may then be easily drawn to the edg' ot the 

 slide and removed, with the aid of more water if ne- 

 cessary This should be allowed to accumulate along 

 the edge of the cover and drawn off with filter paper. 



