CHAPTER XXVI. 321 



heat being used as before to aid differentiation ; blot, dip into 

 90 per cent, or absolute alcohol, xylol, balsam. This method only 

 stains granules which can be seen intra vitam ; properly used it 

 never produces artifacts, and Fischer's critique is quite wrong 

 (Fixirung Faerbung u. Bern des Protoplasms). Altmann's original 

 method has been superseded more or less by the following method 

 of Champy-Kull. (Both Dr. J. A. Murray and I find that the 

 20 grms. of acid fuchsin will not dissolve in 100 c.c. of aniline oil- 

 water ; only about 5 to 7 grms. will dissolve, and this quantity will 

 make a perfectly efficient solution.) 



681. CHAMPY-KULL'S Acid Fuchsin, Toluidin Blue and Aurantia 



(KuLL, Anat. Anz., Bd. xlv, 1913). The following method, while 

 being generally useful, will be found very convenient for work on 

 Invertebrata. It gives results intermediate between those of Benda 

 and Altmann, but is shorter and undoubtedly better than the 

 method of Benda. It will be found very useful for embryological 

 research, and probably also for protozoology. Fix in Champy 

 ( 43) (we find Flemming- without-acetic acid will do, too) for twenty- 

 four hours. Pieces to be fixed must be small. After fixation wash 

 half an hour in aq. dest., and then transfer to a mixture of 1 part 

 acid acet. pyrolignosum rect., and 2 parts 1 per cent, chromic acid, 

 for twenty hours. Wash half an hour in aq. dest., and transfer 

 to a 3 per cent, solution of potassium bichromate for three days. 

 Wash under tap for twenty-four hours ; pass through up-graded 

 alcohols to xylol ; embed in paraffin wax (or celloidin method, if 

 desired). Section 4 or 5 /a. Proceed as follows : (1) Stain in 

 Altmann's acid fuchsin aniline oil mixture (5 to 10 grms. of acid 

 fuchsin in 100 c.c. of aniline oil-water), and heat till steaming. 

 (2) Set slide aside to cool for six minutes (this is important), pour 

 off, and wash quickly in aq. dest. (3) Counter-stain in either a 

 0-5 per cent, solution of toluidin blue or a saturated solution of 

 thionin in aq. dest. for one to two minutes. Wash in aq. dest. 

 In some cases the time in the blue stain must be shortened. Transfer 

 to a 0-5 per cent, solution of aurantia in 70 per cent, alcohol for from 

 twenty to forty seconds, watching extraction of fuchsin stain under 

 microscope. Differentiate the blue stain in 96 per cent, alcohol, 

 then absolute, xylol, and balsam. The chromatin is generally blue, 

 mitochondria (and occasionally Golgi apparatus) are red, and the 

 ground cytoplasm is golden-yellowish to green. This modification 

 of Altmann's method is a most brilliant three-colour stain which is 

 highly recommended. We have found that it is useful for histo- 

 logical as well as cytological purposes ; sections of Annelids, or of 



M. 



