482 BACTEBIOLOGIC TECBNIC. 



5. Gram-Weigert method (stain for fibrin, also good for bacteria) : 

 The sections are stained with anilin or carbol gentian-violet, washed 

 in 0.6% sodium chlorid solution, and dried upon the slide with filter- 

 paper. The iodin solution is then applied and the excess again removed 

 with filter-paper. The section is then decolorized with anilin oil until 

 no more color is given up, placed in xylol, and mounted in Canada 

 balsam. 



6. Kutscher's modification of Gram's method : There is prepared 

 a concentrated solution of gentian-violet in a mixture of anilin water 

 1 part, alcohol 1 part, 5 % carbolic acid water 1 part. Of this concen- 

 trated solution a drop at a time is added to a watch-glassful of water 

 until a shimmering layer forms upon the surface. In this the sections 

 are placed, and after ten to fifteen minutes are washed in water, then 

 placed in the iodin solution for a minute, then in alcohol, xylol, and 

 mounted in balsam. By this method malignant edema and symptom- 

 atic anthrax are also stained. 



7. If tubercle bacilli are to be sta,ined in sections, carbol-fuchsin or 

 anilin gentian-violet are used as for cover-glass preparations, except that 

 the heating is omitted, and instead the stain is allowed to act from 

 fifteen to thirty minutes. 



4. Preparation of Sections. 



At the autopsy small pieces of the organs are placed in an abun- 

 dance of absolute alcohol and allowed to remain there two or three 

 days, the alcohol being changed twice. Usually they can then be cut. 

 For this purpose the firmer parts of the kidneys, liver, muscle, etc., 

 are stuck on corks with liquelied commercial gelatin, ^ and then, with 

 the cork, again immersed in absolute alcohol. After another twenty- 

 four hours the objects may be cut by means of a microtome. In order 

 to obtain sections of more delicate organs, they must be embedded in 

 celloidin or paraiSn. Before staining the paraffin is completely 

 removed by several changes of oil of turpentine or xylol, and the 

 preparation carried from xylol to absolute alcohol. 



II. Cultivation of Bacteria. 



I. Nutrient Media. 



(A) Non=albuminous (according to C. Frankel and Voges). 



Sodium chlorid, 5 gm. ; neutral commercial sodium phosphate, 

 2 gm. ; ammonium lactate, 6 gm. ; asparagin, 4 gm., are dissolved in 1 

 liter of distilled water. "We may add 10% gelatin or 1% agar, and 

 thus obtain a non-saccharine nutrient medium which is suitable for 

 most bacteria. By the addition of milk-sugar a milk-sugar nutrient 

 medium is obtained which is free from dextrose. (Lehmann and 

 Neumann. ) 



To produce bouillon free from sugar according to Th. Smith'g 



^ One part of gelatin is dissolved in two parts of water. 



