100 BIOLOGY AND TECHNIQUE 



It is important that the formalin be fresh and the exposure to 

 water momentary. When decolorizing in the Gram method, strong 

 alcohol only should be used. Wadsworth also found that encapsulated 

 pneumococci could be demonstrated in celloidin sections of pneumonic 

 lesions hardened in strong formalin. The lungs should be distended with 

 the formalin or the lesions cut in very thin bits, hardened, dehydrated, 

 embedded, and cut in the usual way. The celloidin sections may be fixed 

 on the slides by partially dissolving the celloidin in alcohol and ether 

 and setting the celloidin quickly in water before staining. Failure to 

 obtain pneumococci encapsulated in such sections is usually due to 

 improper or inadequate fixation in the formalin. 



The differential method employed by Wadsworth for tissue staining 

 is as follows: 



1. Fix in formalin forty per cent, two to five minutes. 



2. Wash in water. 



3. Anilin gentian-violet, two minutes. 



4. lodin solution, two minutes. 



5. Alcohol, ninety-five per cent, decolorize. 



6. Eosin alcohol, counterstain. 



7. Clear in oil of origanum. 



8. Mount in balsam. 



Flagella Stains. All flagella stains, in order to be successful, neces- 

 sitate particularly clean cover-slip preparations, best made from young 

 agar cultures emulsified in sterile salt solution. Scrupulous care should 

 be exercised in cleaning the glassware used. 



LOEFFLER'S METHOD. 1 The preparation is dried- in the air and fixed 

 by heat. It is then treated with the following mordant solution : 



Twenty per cent aqueous tannic acid . . 10 parts. 



Ferrous sulphate aq. sol. saturated at room temperature . 5 parts. 

 Saturated alcoholic f uchsin solution 1 part. 



This solution, which should be freshly filtered before using, is 

 poured over the cover-glass and allowed to remain there for on^-half 

 to one minute, during which time it should be gently heated, but not 

 allowed to boil. 



Wash thoroughly in water. 



Stain with five per cent anilin water fuchsin or anilin water gen- 



Loeffler, Cent. f. Bakt., I, vi, 1889. 



