320 



METHODS AND FORMULAS 



DS 12.1 DS 12.14 



are employed, and second, those which 

 give a double contrast from a single solu- 

 tion. As the latter are no more difficult to 

 apply than are the single contrast mate- 

 rials, there seems to be no reason for the 

 retention of the former group save custom. 

 The third division contains those complex 

 stains which are designed to provide a 

 differential staining of the cytoplasmic 

 elements present. 



12.1 Single-contrast Formulas 



The division of solutions designed to 

 produce a single contrast to the nuclear 

 staining has been based entirely on the 

 solvent employed. It is unnecessary, in 

 most cases, to list references, since the 

 majority of them are recommended by so 

 many persons that it would be a waste of 

 time to secure the original recommenda- 

 tion. The difference between the aqueous 

 solutions and the weak alcohol solutions 

 is very slight, since the alcohol in the 

 latter is almost certainly designed as a 

 biostatic agent. Almost any antiseptic 

 can be used for this purpose, alcohol being 



retained as a matter of convenience. The 

 strong alcohol solutions are either of dyes 

 insoluble in water or of materials so 

 readily extracted by alcohol that their 

 use must be left to the very last stage of 

 dehydration if they are not to be lost. 

 Clove oil solutions are widely used both in 

 zoological and botanical techniques, but it 

 is a pity that more attention has not been 

 paid to the formula of Johansen 1940, 

 given under 12.16, which combines all 

 the desirable characters of an alcohol and 

 a clove oil solution. The phenol solu- 

 tions are necessitated either through the 

 desirability of having an acid solution, or 

 by the difficulty of securing a solution of 

 the dye without the special technique em- 

 ployed, or by the impossibility of preserv- 

 ing the solution without a biostatic re- 

 agent being included. They are far more 

 widely employed in bacteriology than in 

 either zoology or botany. The few stains 

 given in the last division, other than 

 Johansen 1940 which has already been 

 noticed, are unusual formulas specifically 

 designed to take part in a more complex 

 technique given later in the work. 



12.11 AQUEOUS SOLUTIONS, compl. scHpt. 



formula: water 100, acid fuchsin 0.2 or instead of acid fuchsin either Bordeaux red 1, or 

 Congo red 0.5, or erythrosin 1, or methyl green 1, or orange G 1, or orange II 1, or 

 ponceau 2R 1 



method: [sections, with desired nuclear staining]—* water—* stain, ^2 to 2 mins. -^ water, 

 wash -^ balsam, via usual reagents 



note: Congo red and acid fuchsin are very alkali-sensitive and not safe after hematoxylin 

 stains which have been "blued" in alkalis. Erythrosin is practically confined to botan- 

 ical techniques, as orange II and ponceau 2R are confined to animal procedures. The 

 writer considers the latter the best general-purpose counterstain to blue. Methyl green 

 tends to be fugitive. 



12.12 WEAK ALCOHOL SOLUTIONS, compl. script . 



formula: water 90, 95% alcohol 10, benzopurpurin 0.5 or instead of benzopurpurin either 

 anilin blue 1, or Biebrich scarlet 1, or eosin B 0.5, or eosin Y 0.2, or methyl blue 1, or 

 phloxin 0.2, or rose bengal 0.5 



method: as 12.11 



note: The use of alcohol i.s either to assist in forming a smooth suspension (as in Biebrich 

 scarlet) or to discourage the growth of molds (as in the eosins). The "Magdala Red" 

 commonly specified in botanical techniques is actually phloxine (Conn 1946, 112). 



12.13 STRONG ALCOHOL SOLUTIONS, compl. script. 



formula: 95% ale. 100, aurantia 0.5 or instead of aurantia, either chromotrope 2R 2.0 or 

 ethyl eosin 0.5 or light green 0.5 or fast green FCF 0.5 or spirit blue 0.5 



method: [sections, with desired nuclear staining] -* 70% ale. -^ stain, }i to 2 mins. — > 95% 

 ale, rinse — > balsam, via usual reagents 



12.14 CLOVE OIL SOLUTIONS, compl. script. 



formula: oil of cloves 100, light green to sat. or, instead of light green, either i&st green FCF, 

 or orange G, or safranin O 



