ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 
663 
ments in the technique of flagella staining. Three mordants are de- 
scribed, — iron, alum, and antimony. Iron mordant is prepared by boiling 
for 5 minutes 20 grm. of tannic acid aud adding iron oxide in slight ex- 
cess. The solution is filtered, and when cold the mordant is ready for use. 
Pure iron oxide is prepared by precipitating iron chloride with ammonia. 
Better results are obtained with alum-mordant, prepared by heating 
10 grm. tannin with 200 ccm. in a water-bath at 55°-60°, and adding 
aluminium acetate. The mordant should be quite clear, but if cloudy, 
some more tannin solution should be added with the aid of gentle heat. 
Antimony mordant is still stronger, and is prepared by dissolving 
1 grm. of tartar emetic in some water, and then, together with 500 ccm. 
of a freshly prepared 5 per cent, solution of tannin, heating the mixture 
in a water-bath at 35°-40°. This mordant is opalescent when cold, but 
becomes clear on heating. Its action is increased (1) if the preparation 
be allowed to cool in the heated mordant until the latter begins to turn 
turbid ; (2) if caustic soda be carefully added until the reaction be- 
comes amphoteric or faintly alkaline. The cultures, 10-24 hours old 
agar or bouillon, are killed with 4 per cent, formalin ; the latter 
immediately, while the former are washed off the agar with water 
and then killed. The solution is allowed to sediment for 24-72 hours, 
when it is decanted, and replaced by 1 per cent, formalin, and this in 
turn by pure water. The films are fixed with heat, washed with water, 
and then hot-mordanted for 5-10 minutes. 
The films are stained with gold or silver. For the gold method the 
preparation is treated with gold chloride and heated until the solution 
vaporises. The silver solution is made from a solution of 4 grm. silver 
sulphate left in 500 ccm. of water for an hour. A quantity of this satu- 
rated silver solution is mixed with 30 per cent, aqueous solution of 
ethylamin, until a clear colour solution, which does not contain ethylamin 
or silver in excess, is obtained. A few (4-5) drops are placed on the 
mordanted preparation and heated until it steams. The bacteria and 
flagella are brownish-black. If not sufficiently stained, the process may 
be repeated or the colour further developed by means of gold chloride 
or mercury chloride. The latter gives the best results. The prepara- 
tion is treated with 4-5 drops of 1 per cent, sublimate, and then reduced 
with a soda-pyrogallol solution. Soda solution (soda 2 grm., water 
100 ccm.) 4 drops ; pyrogallol solution (1 grm. pyro in 20 ccm. alcohol 
+ 2 drops of acetic acid) 1-2 drops. 
Injection-staining of Vascular Systems of Plants.* — Mr. R. A. 
Robertson has found the following simple method useful for class pur- 
poses and for private work. To the end of a large glass funnel a length 
of india-rubber tubing is securely wired. The funnel is fixed at a 
convenient height, and the lower end hangs free, eight feet or more in 
length; at the lower end is fixed a compressor clip. The stem (air- 
dried, preserved in spirit or fresh) has its end cut smooth and circular, 
and securely wired into the lower free end of the tube. In the case 
of delicate stems it is preferable to lute with balsam or asphalt. A 
beaker is placed beneath to catch the escaping fluid. The funnel and 
tube are filled with weak aqueous solution of fuchsin, the clip removed, 
* Trans, and Proc. Bot. Soc. Edinburgh, xxi. (1897) pp. 54-6. 
