DS 23.20 



DYE STAINS OF SPECIAL APPLICATION 



471 



attempt to stain flagella for demonstra- 

 tion purposes in any other than a culture 

 which has been specially prepared for the 

 purpose. It is desirable to take an actively 

 growing culture and to check, under a 

 dark field illuminator, that the organisms 

 are motile. This culture is then subcul- 

 tured, in the evening, into the same me- 

 dium, and checked again the next morn- 

 ing. The temperature of the culture is then 

 raised about ten degrees above that at 

 which it has been maintained overnight, 

 and left at this temperature for about an 

 hour. A final check should be made on the 

 motility of the organisms. 



The chief difficulty in staining these or- 

 ganisms lies in the fact that the stain is not 

 in any sense of the term a specific stain 

 for flagella. It is only designed to make 

 certain that any minute particle present 

 on the shde will be colored so densely as to 

 become apparent when examined under a 

 microscope. The stain is deposited just as 

 enthusiastically on the body of the bac- 

 teria as it is on the flagella, and, if any- 

 thing, more enthusiastically on every min- 

 ute speck of organic detritus which may 

 remain on the shde. Little or nothing can 

 be done about this organic detritus which 

 may be present in the culture medium, 

 and the reason for subculturing the se- 

 lected culture is to make sure that only 

 young organisms shall be present, and 

 that numerous decomposing bodies of bac- 

 teria shall be absent. 



The method employed is a mordant 

 process, using an alum-tannic acid solu- 

 tion which must be prepared with some 

 care. Take 100 milhUters of water and add 

 to it at the same time 3.5 grams of potas- 

 sium alum and 3 grams of tannic acid. 

 These should be placed in a 250-milhliter 

 Ehrlenmeyer flask, and warmed over a 

 flame with constant agitation until they 

 are boihng. The flask is now plugged with 

 cotton, autoclaved at 20 pounds pressure 

 for 30 minutes, and allowed to cool until 

 it can be handled. It is filtered, and the 

 filtrate is placed in a refrigerator until 

 chilled. While the filtrate is cooling, pre- 

 pare a 1 % solution of crystal \'iolet. The 

 staining solution, which consists of about 

 10 parts of the crystal violet to 100 parts 

 to the tannic acid solution, should be pre- 



pared immediately before use and, after 

 mixing, should be passed through a bac- 

 terial filter to remove from it any frag- 

 ments which might possibly remain and 

 which, adhering to the dried film, would 

 stain just as readily as would the flagella 

 themselves. 



Having secured an appropriate culture 

 and having made up the staining solution, 

 make a smear in the manner described in 

 the first example of bacterial technique, 

 dry it, flame it, and pour over it a good 

 supply of the mixed solution. Then hold 

 the shde over a very low bunsen flame and 

 heat until bubbles appear. The heating 

 should be continued for about 20 seconds 

 beyond the time when the bubbles first 

 appear. The sUde is then washed off with 

 water, until no more color comes away, 

 and dried before being examined under an 

 oil immersion objective. If everything has 

 gone correctly the bacteria and their flag- 

 ella will be stained a dense black against a 

 background absolutely free from debris. 

 If, however, there is a granular deposit on 

 the background, which has not been de- 

 rived from the culture, a fresh solution 

 should be made, using, say, 7 parts of 

 violet to 100 of the mordant. If the bac- 

 teria, but not the flagella, are stained, 

 take 15 parts of violet to 100 of the 

 mordant, and repeat the })rocess again. 

 No success is possible save by trial and 

 error. 



Demonstration of diplococci in the 



Uver of the rabbit using the phloxine- 



methylene blue-azur II stain of 



Mallory 1938 



This method of Mallory is the best of 

 all the eosin-methylene blue methods 

 which have from time to time been sug- 

 gested for staining bacteria in sections. It 

 has the advantage of giving a first-class 

 histological stain, in addition to differ- 

 entiating bacteria, and it might well be 

 used as a standard procedure in place of 

 the more customary hematoxyUn-eosin, at 

 least in pathological investigations. The 

 liver of a rabbit is so frequently infected 

 with diplococci that it has been selected as 

 a type demonstration, for such infected 

 animals will be found in ordinary labora- 



