io 4 MICROSCOPIC METHODS. 



been introduced, but practically only two need be described 

 here as they are the best at present known. One is the 

 original method of Loffler. The other is that of van Ermen- 

 gem, and it has lately attained considerable popularity. 



In all the methods of staining flagella, young cultures on 

 agar should be used, say a culture incubated for twelve 

 to eighteen hours at 37 C. A very small portion of the 

 growth is taken on the point of a platinum needle and 

 carefully mixed in a little water in a watch glass ; the 

 amount should be such as to produce scarcely any turbidity 

 in the water. A film is then made by placing a drop on a 

 clean cover-glass and carefully spreading it out with the 

 needle. It is allowed to dry in the air and then 

 passed twice or thrice through a flame, care being taken 

 not to over-heat it. The cover-glasses used should always 

 be cleaned in the mixture of sulphuric acid and potassium 

 bichromate described on page 84. 



i . Loffler" s Method for Staining Flagella. 



Two solutions must be made up. Loffler gives the following 

 directions for their preparation : 



A. The Mordant. To 10 c.c. of a 20 per cent solution of tannin 

 in water add as many drops of a saturated solution of ferrous sulphate 

 in water as will give the whole fluid a dark-violet tint. To this add 

 3-4 c.c. of a solution made by boiling I gram of logwood with 8 c.c. 

 of water (after boiling, filter and make up to 9 c.c. to compensate for 

 evaporation). The mixture of the tannin solution with the logwood 

 solution appears of a dirty dark-violet colour. If too much logwood 

 is added particles are precipitated which make the fluid useless as a 

 mordant. It is preferable to make up this mordant fresh on each 

 occasion of its use. After standing for a few days it becomes almost 

 black. It still, however, may be used, although it may now have a 

 slight scum on the surface. The addition of 4-5 c.c. of 1-20 carbolic 

 acid solution makes the fluid more permanent without impairing its 

 properties. 



B. The Stain. To 100 c.c. of a filtered saturated solution of 

 aniline oil in water add I c.c. of a I per cent solution of sodium hydrate. 

 The aniline water is ordinarily neutral. The addition of the soda 

 renders it slightly alkaline. To this solution add 4-5 grm. of solid 

 methylene-violet, methylene-blue or fuchsin, and shake well. When a 

 preparation is to be stained, filter a few drops on to the cover-glass. 



