i6o Methods of Observing Micro-organisms 



2. Bain sensibilisateur: 



5 per cent, solution of nitrate of silver in distilled water. ■ 



The films are allowed to remain in this for a few seconds, and are 

 then immediately transferred to the third bath. 

 3. Bain reducteur et reinforqateur: 



Gallic acid 5 grams 



Tannin 3 " 



Fused potassium acetate 10 " 



Distilled water 350 cc. 



The preparations are kept in this solution for a few seconds, then 

 returned to the nitrate of silver solution until they begin to turn 

 black. They are then washed, dried, and mounted. 



Mervyn Gorden modifies the method by allowing the preparations 

 to remain in the second bath for two minutes, transferring to the 

 third bath for one and a half to two minutes, and then washing, 

 drying, and mounting without returning to the second bath. 



Muir and Ritchie find it advantageous to use a fresh supply of 

 the third solution for each specimen. 



Rossi* gives the following directions for staining flagella: 



The culture to be examined should be a young culture, not more than ten, 

 eighteen, or twenty-four hours old. It should be made upon freshly prepared 

 agar-agar, or upon the reagent after it has been melted and then congealed, 

 as it is of the utmost importance that the surface be moist. The culture 

 should be examined by the hanging-drop method to see that the organisms 

 are actively motile before the staining is attempted. 



The staining should be done only after the greatest care has been taken to 

 see that all the conditions are favorable. For this reason the cover-glasses em- 

 ployed in making the spreads must be carefully cleaned with alcohol, then 

 immersed in steaming sulphuric acid for ten to fifteen minutes. They are then 

 washed in water, then placed in a mixture of alcohol and benzine (equal parts), 

 wiped with a clean soft cloth, and passed through the colorless Bunsen flame 

 forty to fifty times, and then that side of the glass utilized for the "spread" that 

 has been in direct contact with the flame. 



A platinum loopful of the appropriate culture is placed in a drop of distilled 

 water upon a clean slide and slightly stirred. If conditions are favorable, it forms 

 a homogeneous emulsion. If clumps appear, the cultural conditions are not 

 favorable. 



If favorable, a loopful of this dilution is added to i cc. of distilled water in a 

 clean cover-glass and thoroughly stirred. From the center of the surface of this 

 fluid a platinum loopful is next taken and placed upon each of the prepared 

 cover-glasses and, without spreading or stirring, allowed to dry in the air or in an 

 exsiccator. 



The staining solutions are made as follows: 



(A) A solution of 50 grams of pure crystalline carbolic acid in 1000 cc. of 



distilled water, to which 40 grams of pure tannin are added, the whole 

 being warmed on a water-bath until solution is complete. 



(B) Basic f uchsin (rosanilinchlorhydrate) 2.5 grams 



Absolute alcohol 100. o cc. 



(C) Potassium hydrate i . o gram 



Distilled water 100 . o grams 



Mix solutions A and B and preserve in a well-closed bottle. Place solution C 

 in a bottle with a pipette stopper. When the staining is to be done, one pours 

 IS to 20 cc. of the A B mixture into a glass-stoppered test-tube and adds 2 or 3 



* "Centralbl. f. Bakt. u. Parasitenk.," Orig., 1903, xxxiii, p. 372 



