FLAGELLA STAINS. 



193 



The gold method consists of placing on the 

 mordanted cover 4 or 5 drops of an aqueous 

 solution of neutral gold chloride (i :2ooo). 

 This is then heated until steam is given off 

 freely. If the mordanting has been sufficient 

 there will be a deposit of metallic gold on the 

 bacteria. 



Hugh Williams' Method. 



(Copied from Mallory & Wright's Patholog- 

 ical Technique.) 



This is a modification of van Ermengem's 

 method along the lines of the modification of 

 Hinterberger and others. It has been adopted 

 by Dr. Hugh Williams after a large experience 

 with various methods in the laboratory of the 

 Massachusetts General Hospital. 



The method is capable of giving black bac- 

 teria and flagella, with little or no precipitate. 

 The method is as follows : 



(1) Cover the cover-glass with a mordant con- 

 sisting of 



Alumnol,* i per cent solution... I part 

 Osmic acid, 2 per cent solution. . i part 



Tannin, 20 per cent solution 3 parts 



Shake the mixture, add three drops of glacial 

 acetic acid, and again shake. 



(2) Apply the mordant less than one minute 

 without heating. Wash thoroughly in water. 



(3) Cover the preparation, during about i 

 minute, with a i per cent solution of silver ni- 

 trate to which sufficient ammonium hydroxid 

 has been added to keep the silver in solution.-j- 



(4) Wash in water. 



(5) Wash with 0.6 per cent solution of sodium 

 chlorid. 



(6) Flood the preparation with a 30 per cent 

 solution of ammonium hydroxid, and imme- 

 diately wash in water. 



('/) Apply a few drops of Ortol photographic 

 developer. (The directions for making up this 

 developer come with the Ortol.) 



(8) Wash in water. 



(9) Cover with a i per cent solution of gold 

 chlorid during a few seconds. 



(10) Wash in water and apply Ortol developer 

 for a few seconds. 



(u) Wash in water and cover with a i per 

 cent solution of mercuric chlorid for a few sec- 

 onds. 



(12) Wash in water. 



(13) Apply Ortol developer for a few seconds. 



(14) Wash in water and repeat the application 

 of chlorid of gold, the washing, and the applica- 

 tion of the developer two or more times. Be- 

 tween the various applications of the chlorid of 

 gold the preparation should be inspected with a 

 high, dry lens to determine the progress of the 

 staining. This is readily done by placing the 

 cover-glass, charged side upward, on a slide. In 

 this way the process of impregnation with gold 

 may be controlled; for the flagella, if stained, 

 may be easily seen with the high-power dry lens. 



The preparation is very conveniently held dur- 

 ing the process in cover-glass forceps. The 

 washing is best done in a small stream of water 

 from a faucet. The various solutions are con- 

 veniently applied from dropping-bottles. 



It will be seen that the process consists essen- 

 tially in the impregnation of the flagella with 

 silver, followed by intensification, in the photo- 

 graphic sense, with mercury and gold. The ob- 

 ject of the application of the sodium chlorid and 

 ammonia is to remove the excess of silver com- 

 pounds which adhere to the surface of the cover- 

 glass in spite of washing. This excess of silver 

 compounds is chiefly responsible for the precipi- 

 tates which appear on the preparation after the 

 intensification. In spite of the application of 

 the sodium chlorid and ammonia solutions, 

 some precipitate will occur if the intensification 

 is pushed too far. On this acount it is advisable 

 to observe the progress of the intensification 

 under the microscope, as above indicated. 



Although this method may appear compli- 

 cated, in practice it requires but a few minutes 

 to stain a preparation. 



Duckwall's Method. 



Streaks are made on 2 per cent agar in Petri 

 dishes from young growths in bouillon. Sus- 

 pensions are made in water according to nature 

 of organism, the motile bacteria being divided 

 into six groups for staining purposes. Pigments 

 and slime are removed by shaking with chloro- 

 form. Cover-glasses must be absolutely clean. 

 Mordant must be used only when fresh; dye 

 must be fresh and used warm (Loeffler's stain) ; 

 streaks on cover-glass should not be confluent. 

 Fix without injury to flagella; stain without 

 overheating; wash in alcohol and water without 

 breaking film; clear in xylol; mount in xylol 

 balsam without previous examination. For de- 

 tails respecting method of making suspensions 

 see The Canner and Dried Fruit Packer, Vol. 

 XX, Feb. 16, 1905, p. 23. 



*Farhwerke vorm. Meister I.ucius u, Briining, Hochst a. M., Germany. 



fWorkers in the Bureau of Animal Industry U. S. Department of Agriculture state that too great an excess of 

 ammonia In the silver nitrate may interfere with the working of the method. 



