192 



BACTERIA IN RELATION TO PLANT DISEASES. 



Loivit's Flagclla Stain. 



Lowit's modification of Loeffler's flagella stain 

 consists in substitnting a copper-tannin mordant 

 for one of iron-tannin. It is made as follows : 



(1) Mordant: 



Distilled water 10 



Tannin 25 



Dissolve and filter tlirough two thicknesses of 

 filter paper, then add : 



Saturated solution copper sulphate... 5 

 Saturated ale. sol. basic fuchsin i 



Filter as before. 



Covers are exposed to this mordant 20 seconds 

 to 3 minutes without heating. Wash thor- 

 oughly. 



(2) Stain: Expose cover to Ehrlich's anilin 

 water gentian violet l to 5 minutes. Wash thor- 

 oughly in water, and if this is not sufficient 

 plunge for a moment into 50 per cent alcohol or 

 into acid alcohol (l drop of 0.3 per cent HCl 

 alcohol in 3 to 4 cc. of 60 per cent alcohol). The 

 mordant and stain must be made up each time. 

 If the mordant has been in use for some hours 

 so that an oxidation film has formed on its sur- 

 face, it is well to stop and remove this by filtra- 

 tion. 



Sclavo's Flagclla Stain. 



Sclavo exposes some minutes in the mordant 

 (tannin I, water 50, alcohol 50) ; washes in aq. 

 dest. ; exposes some minutes in 50 per cent 

 phospho-tungstic acid; washes carefully in aq. 

 dest. ; stains 3 to 5 minutes in gently warmed 

 anilin-water fuchsin ; washes, dries, and mounts 

 in Canada balsam. Some kinds are not stained 

 by this method. 



BoivhiU's flagclla Stain. 



This author used two solutions made as fol- 

 lows : 



( 1 ) Orcein I 



Absolute alcohol 50 



Distilled water 40 



(2) Tannic acid (tannin) 8 



Distilled water (hot) 40 



Equal parts of (i) and (2) are mixed and 

 filtered. Bacteria from a fresh agar culture are 

 suspended in boiled distilled water. This sus- 

 pension is allowed to stand 5 minutes. Drops 

 are taken and .spread and dried on clean covers, 

 which are then taken in the lingers and fixed 

 over the tlame. They are now lloated, film down, 

 on the mordant (gently wanned) bn" from 10 

 to 15 minutes, then washed in water and dried. 



Ehrlich's anilin-water gentian violet is now 

 dropped on the cover, and this is heated over 

 the flame until steam appears. The preparation 

 is then washed, dried, and mounted in xylol 

 balsam. 



Subsequently Bowhill modified the above as 

 follows, using the orcein itself as a stain : 

 (i) ^Saturated solution of orcein (al- 

 lowed to ripen about 10 days). 

 (2) 20 per cent solution of tannin dis- 

 solved in hot water. 

 The stain is prepared by taking of No. I, 15 

 cc. ; No. 2, 10 cc. : and distilled water, 30 cc. 

 After mixing, the fluid should be filtered. Sub- 

 sequent bleaching of the preparation should be 

 avoided. (Hyg. Rundsch., VIII Jahrg., 1898, 

 pp. II and 105.) 



Hintcrbcrgcr's Method for Flagclla. 

 (iSee '00 Hinterberger, Bibliog., XII.) 

 Night Blue Stain for Flagella. 

 (See '99 Morton, Bibliog., XII.) 



Zettnow's llcthod for Flagclla. 



Zettnow fixes with formalin, mordants with 

 tartrate of antimony and tannin, and stains with 

 gold or silver. 



The fixing is done by taking bacteria from a 

 fresh agar or bouillon culture and adding them 

 to water. They are then killed by the addition 

 of 4 per cent formalin. The fixed bacteria set- 

 tle after a day or two, when the sediment con- 

 taining them is pipetted out and washed, first 

 in I per cent formalin water and finally in pure 

 water. The cloudy water is spread and dried 

 on clean covers, and when these have been fixed 

 by gentle heat they arc ready for the mordant. 

 The mordant is made as folows : 



(1) Tannin 5 



Distilled water 100 



Flask and heat to 35° or 40° C. in the water- 

 bath. 



(2) A solution of tartrate of antimony ( i gram 

 dissolved in water in n test-tube) is added drop 

 by drop to solution (i), with shaking until the 

 precipitate which forms is not redissolved. It 

 is then filtered. The filtered mordant should be 

 strongly opalescent, but not cloudy or opaque to 

 transinitted light. This is said to be a perma- 

 nent universal mordant, and one which docs not 

 cause precipitates on the cuvcr. It is used hot 

 (70° to 80° C. ) for 5 or 10 minutes. The cover 

 is then washed and gilded or silvered. After- 

 ward the image may be intensified if desired. 



