MICROSCOPIC EXAMINATION. 479 



rations even after twelve days. We usually use freshly prepared 

 mordant. 



According to Loffler, the addition of a very definite amount of acid 

 or alkali to the mordant is required for most kinds of bacteria in order 

 to obtain well-stained flagella. Loffler recommends that to 16 c.c. of 

 the mordant there be added: 



Cholera vibrios J- 1 drop 1 % NaHO. 



Spirillum rubrum 9 drops " " 



Bacterium typhi 20-22 " " " 



Bacillus subtilis 28-38 " " " 



Bacillus oedematis maligni . . 36-37 



Bacterium pyocyaneum ... 5- 6 " equivalent H 2 SO 4 . 



Our results show that, in the majority of cases, very satisfactory 

 pictures are obtained with the original mordant, and that the addition 

 of alkali or acid is by no means essential. Similar results have been 

 obtained by other writers; for example, Lucksch, Giinther, A. Fischer, 

 Nicolle, and Morax. 



Recently Bunge has employed a somewhat different method, which 

 has given us good results also, but, like Loffler' s process, it has also 

 sometimes left us capriciously in the lurch: (1) The preparation is 

 made as for Loffler' s ; (2) warming with Bunge's mordant (p. 476) 

 until it steams for one minute ; (3) careful washing with water and 

 drying ; (4) staining with slightly warm carbol-gentian-violet or car- 

 bol-fuchsin ; (5) washing with water, drying, mounting in Canada 

 balsam. 



The silver method of van Ermengem is also much employed for 

 staining flagella. The caver-glass preparations are stained with a mix- 

 ture of 1 part 2% osmic acid and 2 parts 10% to 25 % solution of tan- 

 nic acid, to 100 c.c. of which 4 to 5 drops of glacial acetic acid are 

 added. It is then washed with water, and afterward with absolute 

 alcohol, and the preparation is next moistened for a few seconds with 

 0.5% to 2.5% silver solution. The preparation, without washing, is 

 brought into a solution of tannic acid 3.0, gallic acid 5.0, sodium ace- 

 tate iO.O, and water 350, for a few moments, and again back to the 

 silver solution until it begins to get black. Finally it is again washed 

 in water. 



Most of our preparations have been made with Bunge's mordant 

 when it was several months old. 



5. Staining of Endospores. 1 According to Hauser : 



1. Preparation of the film. (It is recommended to pass it through 

 the flame ten times, instead of three.) 



2. Stain with watery fuchsin or carbol-fuchsin ( Ziehl's solution ), the 

 preparation being covered with abundance of the staining solution and 

 warmed above a flame for one or two minutes until it begins to simmer 

 (not boil). The fluid which evaporates is constantly replaced by fresh 

 staining solution. 



1 Arthrospores possess no undisputed tinctorial reaction. Regarding 

 metachromatic bodies, Ernst's and Bunge's granules, preliminary stages 

 of spores and their demonstration, see page 21. 



