1 88 



BACTERIA IN RELATION TO PLANT DISEASES. 



minute or so into acid alcohol (c. p. nitric acid 

 5 cc., 80 per cent alcohol 100 cc.). It is then 

 washed in water, stained about 5 minutes (for 

 contrast) in an aqueous solution of methylene 

 blue, dried, and mounted in cedar oil or balsam. 



LoefRer's Alkaline Methylene Blue. 

 Alcoholic solution of methylene 



blue (saturated) 30 



Caustic potash i ) JQO 



Distilled water 10,000 j 



This fluid retains its valuable properties for a 

 considerable time and is an excellent stain. 



Kuhne's Carbol-Methylene Blue. 



(1) Methylene blue i-S 



Absolute alcohol 10.0 



(2) After triturating the above in an agate or 

 porcelain mortar, or in a watch glass, add grad- 

 ually 100 cc. of water containing 5 per cent car- 

 bolic acid. Methylene blue is not the same as 

 methyl blue. (See Pregl, Bibliog., XIV.) 



Gram's Stain. 



This is a method of differential bleaching 

 after a stain. The cover-glass preparations or 

 sections are passed from absolute alcohol into 

 Ehrlich's anilin gentian violet or into a watery 

 solution of methyl violet, where they remain 

 i to 3 minutes, except tubercle bacilli prepara- 

 tions, which remain commonly 12 to 24 hours 

 (Gram). They are then placed for i to 3 min- 

 utes (occasionally 5 minutes) in iodine potas- 

 sium iodide water (iodine crystals I, potassic 

 iodide 2, water 300), with or without first wash- 

 ing lightly in alcohol. In this they remain i to 3 

 minutes. They are then placed in absolute alco- 

 hol until sufficiently bleached, after which they 

 are cleared in clove oil and mounted in Canada 

 balsam. By this method the stain is removed 

 from some kinds of bacteria and not from 

 others. 



Too much confidence must not be placed in 

 this method, since in some cases the removal or 

 non-removal of the stain from the organism de- 

 pends on the length of exposure to the iodine 

 water. It would be better, therefore, to expose 

 all for the same period, e. g., 2 minutes. 



Gabbett's Stain. 



Used mostly for tubercle bacteria in sputum. 

 Stain first with carbol-fuchsin, then place the 

 cover-glass for i to 2 minutes in acid methylene 



blue (methylene blue 2 grams, 25 per cent sul- 

 phuric acid water 100 cc.). When washed in 

 water and dried it may be mounted in cedar oil 

 or in balsam. The ordinary bacteria of sputum 

 are decolorized ; the tubercle organism retains 

 the red stain. 



The Ehrlich-W eigert Stain. 



Used for detecting the tubercle organism in 

 sputum. The prepared cover is floated face 

 down on anilin methyl violet, which is heated 

 until steam rises. After 2 to 5 minutes on this 

 hot stain plunge for a few seconds into acidu- 

 lated water (i part nitric acid, 3 parts distilled 

 water), then wash for a few seconds in 60 per 

 cent alcohol, and afterward thoroughly in 

 water. For a contrast stain the cover may be 

 placed for 5 minutes in a saturated aqueous so- 

 lution of vesuvin. It is then washed in water, 

 dried, and mounted in balsam. 



Bacteria which hold the stain after such treat- 

 ment are sometimes called " acid-fast " bacteria. 



Flcmming's Triple Stain. 

 The slide is first placed in (i). 



(1) Safranin O (saturated alcoholic 



solution) 50 



Distilled water 50 



Anilin water 5 



After washing in water, it then goes into (2). 



(2) 'Saturated aqueous solution of 



gentian violet 50 



It is then washed in water and passed into (3). 



(3) Aqueous solution of orange G, 



strong or weak (generally about 

 one-half saturated). 



The slide is then washed quickly in 95 per 

 cent alcohol, dehydrated, cleared, and mounted. 



Pregl's Method. 

 (See '91 Pregl, Bibliog., XIV.) 

 Nicolle's Methods. 

 (See '95 Nicolle, Bibliog., XIV.) 



Benda's Iron Haematoxylin. 

 Mordant the sections for several hours in i 

 part of the following ferric solution* diluted 

 with 2 parts of water: 



Ferrous sulphate 80 



Water 4 



Sulphuric acid 15 



Nitric acid 18 



This solution, known to the German Pharmacopoeia as Liquor ferri sulphuric! oxydati and to the U. S. P. as Liq. 

 f. tersulphatis or sol. persulphate of iron, keeps indefinitely. It is made as follows : Heat in a flask on a water-bath 

 until fluid is brown and clear, and a drop diluted with water is no longer colored blue by potassium ferricyauide, 

 evaporate in a tared porcelain capsule to 100 parts, add a little water and evaporate again. Repeat the dilution and 

 evaporation until the hot fluid is free from the odor of nitric acid. Finally dilute to a weight of 160 parts. 



