170 PLASMA STAINS 



aqueous solution of orange G. After at most a few minutes, whilst 

 pale violet clouds are still being given off from the sections on agitation, 

 bring them into absolute alcohol until hardly any more colour comes 

 away, clear in clove or bergamot oil, and mount in damar or balsam 

 before the last pale clouds of colour have ceased to come away. The 

 orange must be orange G. 



Winiwarter and Sainmont (Zeit. iviss. Mik., xxv, 1908, p. 157, 

 and Arch. Biol., xxiv, 1909, p. 15) stain for twenty-four hours in the 

 gentian, wash out after the orange for two to three hours in 100 c.c. 

 absolute alcohol with 3 to 4 drops of HCl, and differentiate finally with 

 oil of cloves. 



This is not a triple stain in the sense of giving three different colours 

 in the result ; it is a nuclear and plasmatic stain in mixed tones ; the 

 orange, apparently, combines with the gentian to form a " neutral " 

 dye, soluble in excess of the orange, which thus differentiates the stain. 



See also Flemming in Arch. Ancit. Phys. Anat. Ahth., 1897, p. 175. 



328. Reinke's Orange Method {Arch. mik. Anat., xliv, 2, 1894, p. 262). 

 To a concentrated aqueous solution of gentian violet are added '" a few 

 drops " of a like solution of orange G. The solution precipitates in part, 

 owing to the formation of an imperfectly soluble " neutral " colour, but 

 becomes almost clear again if an excess of water be added. The 

 solution is not to be filtered, but the sections are to be stained in the 

 mixture made almost clear by addition of water. It is said that the 

 " neutral " solution may be preserved for future use by adding to it 

 one-third of alcohol. After staining (sections previously stained with 

 safranin), you differentiate rapidly with alcohol and clear with clove oil. 



Lee has tried this process and obtained exactly the same results as 

 with Flemming's process, and so have other workers. 



Arnold's Orange Method {Arch. Zellforsch., iii, 1909, p. 434). Sections 

 (of chrome material) are treated for five minutes with solution of 

 equal parts of iodine and iodide of potassium in alcohol of 40 per cent., 

 then washed and stained for four hours in a saturated solution of safranin 

 in alcohol of 75 per cent. : then washed and put for five to fifteen 

 minutes into solution of 7 parts of methylen blue, 0-5 of carbonate of 

 soda and 100 of water, washed, dehydrated, and treated until pale blue 

 with solution of orange G in oil of cloves. Cytoplasmic reticulum blue 

 on orange ground, nucleoli and centrosomes red. Instead of the 

 safranin, basic fuchsin may be taken. 



329. Bonney's Triple Stain {Virchozv's Arch., cxciii, 1908, 

 p. 547). Stain sections (of acetic alcohol or sublimate material, 

 not chrome or formol material) for two minutes in a solution 

 of 0-25 parts methyl violet and 1 part pyronin in 100 of 

 water. Wipe slide dry, and flood twice with the following : 

 2 per cent, aqueous solution of orange G, boiled and filtered, 

 is added drop by drop to 100 c.c. of acetone, with agitation, 

 until there is formed a floccident precipitate, which redissolves 

 on further addition of the orange. Wash rapidly in pure 

 acetone, and pass through xylol into balsam. Chromatin 

 violet, cytoplasm red, connective-tissue yellow, keratin violet. 

 Not adapted for blood films. 



330. Bordeaux R. An " acid '" dye, giving a general stain 

 taking effect both on chromatin and cytoplasm, and a very 

 good plasma stain. Lee used for chrome-osmium material a 



