336 



METHODS AND FORMULAS 



DS 12.30-DS 12.31 



rolled into position on the slide to avoid 

 the almost inevitable curling which will 

 result from the absorption of water by 

 sections of this very large area and their 

 subsequent swelling within the bounds of 

 the paraffin which retains them. 



The sections are then dewaxed in the 

 ordinary manner and passed to water to 

 await staining. 



The stains which are required in the 

 present instance are the acid alum hema- 

 toxylin of Masson (DS 11.123 Masson 

 1934) and the three solutions specified by 

 Patay 1934 for his triple staining method 

 (DS 12.32 Patay 1934). A description has 

 already been given (DS 11.10 typical 

 preparations) of the method by which an 

 acid alum hematoxylin of this kind can be 

 utilized to secure a sharp and clear differ- 

 ential staining of nuclei. In the present 

 instance, however, a diffuse blue stain, 

 particularly of cartilaginous areas, is pre- 

 ferred to a sharp definition of nuclei. One 

 need, therefore, have no hesitation in 

 placing the sections directly into this stain 

 where they may remain overnight or for 

 such period of time as is convenient to the 

 operator. They will usualh' be satis- 

 factorily stained within an hour and some- 

 times in less time ; though this depends so 

 much upon the time which they have 

 spent in previous solutions, that it cannot 

 be forecast with accuracy. When the sec- 

 tions are removed from hematoxyhn they 

 should be differentiated in a 0.1 % solution 



of hydrochloric acid in 70% alcohol until 

 the nuclei and the matrix of the cartilage, 

 wliich will be clearly visible in large 

 areas, alone remain blue. It is more 

 damaging to over-differentiate than to 

 under-diff erentiate . 



When differentiation is complete, the 

 sections should be accumulated in a jar 

 of alkaline tap water (rendered alkaline 

 when necessary by the addition of sodium 

 bicarbonate) until they have turned from 

 dull purple to clear blue. The sections are 

 now taken from the water and j^assed to 

 the 1% solution of ponceau 2R for a 

 period of two minutes, briefly rinsed in 

 water, passed into 1% phosphomolybdic 

 acid for approximately two minutes, or 

 until such time as the cartilage is freed 

 from the red stain, rinsed again briefly 

 in water, and then dipped two or three 

 times in 90*^0 alcohol to remove the exces- 

 sive water before being placed for about 

 30 seconds in the ^2% solution of fight 

 green in 90 ';o alcohol, which constitutes 

 the final stain of the series. The section 

 should then be passed into absolute alco- 

 hol until such time as no further green 

 color comes away before being passed to 

 xylene and mounted in balsam. 



This method of triple staining is one of 

 the most foolproof and satisfactory of any 

 known to the author, and it is particularly 

 applicable to those structures which con- 

 tain bone as well as cartilage. 



12.31 TECHNIQUES EMPLOYING THE PHOSPHOTUNGSTIC (-MOLYBDIC-) REACTION 



WITH ACID FUCHSIN 



12.31 Brillmeyer 1929 acid fuchsin-anilin blue-orange G 



11571b, 12:122 

 REAGENTS required: .4. 0.2% acid fuchsin; B. water 100, phosphomolybdic acid 1, 



anilin blue 0.5, orange G 2.0 

 method: [blue nuclei (original specifies DS 11.122 Delafield 1885)] -♦.4,1 min. — > drain 



— > B, 2-3 hrs. — » water, quick wash -^ balsam, via usual reagents 

 note: Weiss 1932 (20540b, 7:131) differs only in the dilution of A to 0.04% and in the 

 substitution of 4 minutes for 3 hours immersion in B. DS 11.122 Mayer 1901 is 

 recommended for prior staining of sections from picric fixed or mordanted material. 



12.31 Crossmon 1937 acid fuchsin-orange G-light green {or -anHin blue) 



763, 69 :33 

 reagents required: A. water 100, acetic acid 1, acid fuchsin 0.3, orange G 0.13, thymol 

 0.06; B. 1% phosphomolybdic acid; C. either water 100, acetic acid 1, light green 1 or 

 water 100, acetic acid 2, aniline bhxe 2; D. 1% acetic acid 

 method: [sections, nuclei hematoxyUn-stained] — > water —> A, 1 min. — > rinse -^ B, till 

 collagen decolorized — > quick rinse -^ C, 5 mins. — > rinse -^ D, till differentiated —* 

 rinse -> abs. ale. — > balsam, via xylene 



