52 CULTURE METHODS AND TECHNIQUE 



The method of procedure with the gentian will depend on whether 

 a chromatic or a kinoplasmic stain is desired. In the first case a 

 short immersion in a strong stain will give best results, and in the 

 latter case it is often well to use only a few drops of the gentian 

 to a tumbler of water. The orange acts rapidly upon well-fixed 

 structures, and often an immersion of a few seconds will suffice. 

 Clove oil washes out the gentian somewhat in clearing, but berga- 

 mot oil does not have this effect, and serves rather to fix the stain, 

 so that it may sometimes be necessary to dash the slide with berga- 

 mot oil before differentiating with clove oil. In every case, how- 

 ever, considerable experimentation is necessary for the proper 

 handling of this stain. 



Iron hematoxylin. Where the safranin-gentian-orange is in- 

 effective, iron haematoxylin will often give excellent results. With 

 this process the sections are immersed from one to several hours 

 in about a 3 per cent solution of iron alum (ammonia-sulphate of 

 iron). They are next washed well in water and then stained in a 

 0.5 per cent aqueous solution of haematoxylin. The latter is allowed 

 to act until a considerable overstating has resulted. The slide is 

 then washed and again put into the iron solution until the desired 

 differentiation shall have resulted. It is then dehydrated, etc., as 

 usual. Iron haematoxylin will give some brilliant results when the 

 Flemming combination is ineffective. It is usually necessary to 

 considerably overstain the preparations and then to wash out 

 strongly in the alum solution if chromatin differentiation is 

 desired. It is sometimes well to follow this treatment with a 

 slight ground stain of orange G. 



After Merkel's solution good results have been obtained by 

 Harper with a double stain of acid fuchsin and iodine green. The 

 same stain has also been found useful after corrosive sublimate by 

 Wager in his studies upon the cytology of the yeasts. 



Bacteria. Only a few general directions may be given dealing 

 with some of the ordinary methods now employed for the staining 

 of the bacteria. The concentrated alcoholic solutions mentioned 

 for the fungi are used, and, in addition, a similar solution of 

 gentian violet. These solutions are sometimes made of definite pro- 

 portion, standard strengths being 1 gram of the stain to 10 cc. of 

 95 per cent alcohol. These solutions are diluted for use, just as with 



