Chapter XV: Bacteria 107 



B. Bacteria in Tissues 



Tissues may be fixed and. hardened (e. g., Gilson's fluid, Appendix B, 

 reagent 15; or Zenker's, reagent 6; or formalin, reagent 17) in the 

 ordinary way, and sections made by the usual methods. Where practi- 

 cable paraffin sections are preferable to celloidiu sections, because the 

 celloidin tends to hold the stain and thus obscure the bacteria. Sections 

 should be fixed to the slide (paraffin by albumen fixative, celloidin by 

 ether vapor). 



Bacteria which do not stain by the Gram method (memorandum 3/) 

 or the tubercle bacillus method (memorandum 3e) are difficult to demon- 

 strate, because it is hard to stain them so as to differentiate them from 

 the tissues in w r hich they lie; furthermore, most of them easily lose what- 

 ever stain they may have taken up. Loffler's alkaline inethylen blue 

 (memorandum 36) is, perhaps, the most useful stain for these organisms. 



Methylen Blue Stain for Bacteria in Tissues. 1. Stain sections (paraffin) 

 30 minutes to 24 hours. 



2. Acetic acid (1 to 1,000 of water) 10 to 20 seconds. 



3. Rinse in absolute alcohol 20 to 30 seconds. 



4. Xylol. 



5. Xylol-balsam. 



With celloidin sections substitute 95 per cent, alcohol for absolute 

 (step 3), then treat with xylol or, better, carbol-xylol until sections are 

 clear. Mount in xylol -balsam. 



Anilin gentian violet, methyl blue, methyl violet, or fuchsin (memo- 

 randum 3 a), also carbol-fuchsin (memorandum 3c) may be used in the 

 same way. 



Gram's Method for Bacteria in Tissues (Weigert's modification). 

 1. Stain sections (any kind) in lithium carmine 2 to 5 minutes. 



Lithium Carmine (Orth's): 



Carmine 2.5 to 5 grams. 



Carbonate of lithium, saturated aqueous solution 100 c.c. 



Thymol , a crystal or two. 



Filter. 



2. Anilin gentian violet 5 to 20 minutes (celloidin sections should first 

 be dehydrated in 95 per cent, alcohol and affixed to the slide with ether 

 vapor). 



3. Rinse in normal saline. 



4. Gram's solution (memorandum 3/) 1 to 2 minutes. 



5. Rinse in water. 



6. Blot sections with filter paper to remove as much water as possible. 



7. Anilin oil, several changes. The oil dehydrates, and at the same 

 time decolorizes the celloidin. 



8. Xylol, several changes. 



9. Xylol-balsam. 



