Staining 171 



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 at- 

 tempted. 



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 employed 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 c.c. 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 stir- 

 ring, 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 c.c. 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 fuchsin (rosanilinchlorhydrate) 2.5 grams 



Absolute alcohol 100.0 c.c. 



(C) Potassium hydrate i .o gram 



Distilled water 100.0 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 15 to 20 c.c. of the A B mixture into a glass-stop- 

 pered test-tube and adds 2 or 3 drops of solution C. A precipitate 

 forms, but quickly dissolves on shaking. More of solution C is added, 

 and the tube shaken until the solution becomes brown and clouded and 

 one can see a fine precipitate in a thin layer of the fluid. The fluid is 

 next filtered several times through the same filter and caught in the 

 same glass until it w r ill remain clear for several minutes. Then it is 

 poured on the filter a last time and 4 or 5 drops allowed to fall upon each 

 of the prepared cover-glasses. In a short time a sheen is observed upon 

 the surface of the fluid on the cover-glasses, showing that a fine precipi- 

 tate has formed. When this has occurred, a little experience will show 

 when the proper moment arrives to throw off the fluid and wash the 



* "Centralbl. f. Bakt. u. Parasitenk.," xxxm, Orig., 1903, p. 572. 



