204 



METHODS IN PLANT HISTOLOGY 



acid, and 90 c.c. water). Fix 24-48 hours and then follow the usual 

 schedule for imbedding in paraffin. Cut 2-5 fx thick and stain in 

 Haidenhain's iron-alum haematoxylin, or try the following schedule, 

 which is good for Anthrax and many other bacteria: 



1. Gentian violet, 5 minutes. 



2. Rinse in water a few seconds. 



3. Gram's solution (iodine 1 g., potassium iodide 2 g., water 300 c.c.) until 

 the color is almost or quite black ; tliis will generally require 1 or 2 min- 

 utes. 



4. Ninety-five per cent alcohol until the color has nearly disappeared. 



5. Rinse in water and examine. If the bacteria are well stained, a counter- 

 stain may be added. 



6. Light green or erji^hrosin, 5 seconds; 

 or Bismarck brown, 5 or 10 seconds. 



7. Ninety-five and 100 per cent alcohol, 

 dehydrating as rapidly as possible. 

 Not more than 5 or 10 seconds can 

 usually be allowed. 



8. Xylol, 1-5 minutes. 



9. Balsam. 



After the rinsing in water of stage 5, 

 the preparation may be dehydrated 

 rapidly in 95 per cent and 100 per cent 

 alcohol, and then stained for 5 or 10 

 '^y '^Xv' 6 seconds in orange dissolved in clove oil. 

 _ ^ %>4, " ''"'^ From the clove oil, transfer to xylol and 

 mount in balsam (Fig. 37). 



The bacteria are the only plants in 

 which a nucleus has not been conclu- 

 sively demonstrated, and some claim 

 that a nucleus is present even in bac- 

 teria. In determining the presence or 

 absence of a nucleus in bacteria, the 

 crude method, just given, would be of 

 no value, and even the most critical methods of the bacteriologist, 

 who mounts the organisms whole, would be entitled to only scant 

 consideration. The presence or absence of a nucleus will have to be 

 determined by a study of thin, well-stained sections of perfectly fixed 

 material. 



r 



Fig. 37. — Bacillus anthracis, from a 

 paraffin section cut from the liver of a 

 mouse and stained in crystal violet; w, 

 white blood corpuscle; r, red blood cor- 

 puscle. X580. 



