PROTOZOA 585 



volume of a saturated solution of picric acid, and staining for five 

 minutes, after magenta 1 per cent, (aqueous), one hour. 



Borrel fixes in OSO4, 2 grm., platinum chloride 2 grm., chromic acid 

 3 grm., glacial acetic 20 c.c, water 350 c.c, for twenty-four hours. Then 

 running water for some hours. Stained slides are differentiated in 

 absolute alcohol and then oil of cloves. 



DoBELL (Amcebce in Man, 1919, p. 7) says he obtained excellent 

 results by using Borrel's method after safranin with sections containing 

 E. histolytica. 



HoARE (Parasit., xv, 1923, p. 357), uses, for sections of ked's gut 

 containing trypanosomes, Heidenhain's iron ha-matoxylin and counter- 

 stains by Ramon y Cajal's magenta and picro-indigo-carmine). 



DOUBLE AND TRIPLE STAINING METHODS 



1152. Mann's methyl-blue-eosin mixture (§ 356). This mixture 

 keeps indefinitely and can be used repeatedly. 



Transfer films or sections from distilled water (on no account 

 from alkaline liquids, such as tap-water, since methyl blue is 

 insoluble in alkalies as well as in alcohol, and eosin is soluble 

 in both) to the mixture for five to ten minutes, then wash in water. 

 In distilled water both dyes wash out slightly, but in tap-water 

 only the eosin. Dehydrate rapidly and mount. 



If used for fresh material, it is best to dilute the solution about 

 ten times. It may be used after most fixatives, especially those 

 containing sublimate or chromic acid ; also as a counterstain 

 after mueh-washed-out iron ha?matoxylin. 



Mann's long method comprises staining for twelve to twenty- 

 four hours, rinsing in distilled water for half a minute, thoroughly 

 dehydrating in caustic alcohol (see Mann's " Histology," 1902, 

 p. 217). 



Chatton's modification of Mann's stain (Arch. Zool. Exper.,\\Ti.,\^2Q, 

 p. 22), recommended for sections after fixation with picric acid mixture 

 is to use distilled water saturated with methyl blue and eosin W.G., a 

 mixture of equal volumes of the two saturated solutions appears to be 

 satisfactory. The mixture keeps indefinitely. 



Stain sections for fifteen minutes — they will be violet — pass them 

 quickly into water, 95 per cent, alcohol, and absolute alcohol, the latter 

 containing 1 drop of ammonia per 10 c.c. They should now be rose. 

 Differentiate in clove oil while examining under the microscope and, 

 if necessary, return to the akaline alcohol. 



Chitin, connective tissue, nuclear chromatin, not associated with 

 plastin should be blue, while muscles, nucleoli, caryosomes remain 

 brilliant rose. 



Dobell's modification of Mann's method is to stain for four to 

 twelve hours and, after washing in distilled water, to differentiate in 

 70 per cent, alcohol containing a little orange G. (A few drops of a 

 saturated solution added to 100 c.c. of 70 per cent, alcohol.) The 

 differentiation is controlled under a microscope and very pretty pre- 

 parations of amcEbfc and cysts obtained. Of course the alcoholic solu- 

 tion must not be allowed to act long enough to wash all eosin out of the 

 nuclei. 



