ZOOLOGY AND BOTANY, MIOROSOOPY, ETC. 149 



In order to obtain a deep black tbe whole process must be repeated 

 three or four times. 



The advantages of this black stain are that the negatives of bacteria 

 are extraordinarily sharp and vs^ell defined both with sun and artificial 

 light. The details in the bacteria, spores, &c., appear with the greatest 

 clearness. The flagella, too, unstainable with anilin dyes, are stained 

 quite black. 



Lastly the preparations do not lose colour. 



Nucina as a Staining Agent.* — Prof. N. Leon calls attention to the 

 value of the black substance of " nuts " (Nucina) as a staining agent. 

 Though chemists are, as it seems, still ignorant of its chemical formula, 

 solutions are easily obtained. Nucina has the property of actively dif- 

 ferentiating the parts of which cells are composed; it blackens nuclei, 

 bacteria, and the leucites of vegetable cells and easily differentiates the 

 constituent parts of spermatozoa. The aqueous solution is obtained by 

 putting nuts into alcohol ; as soon as the spirit becomes green, owing to 

 the solution of chlorophyll, the nuts are carefully washed with water 

 so as to extract the alcohol. 25 nuts were then placed in a 

 porcelain vessel with 500 grams of distilled water, which was 

 boiled till half the water had evaporated. The liquid, after being 

 filtered several times, was boiled afresh with 10 per cent, of alum ; the 

 solution has a blood-red colour with direct light. The alcoholic solu- 

 tion is made by boiling nuts in water, removing them, and allowing the 

 water to deposit the black nucina ; 100 grams of alcohol at 80° 

 were added for every three grams of nucina. This solution has a black 

 colour ; after its use a few drops of hydrochloric acid should be applied 

 to the section. 



Baumgarten's Triple Staining Method.f — This method as given by 

 M. A. Lewin consists in the following series of operations : — 



(1) After having washed the sections in absolute alcohol, they are 

 placed for 5 minutes in borax-picrocarmine ; excess of stain is then 

 removed with blotting-paper. (Borax-picrocarmine is prepared by 

 adding powdered picric acid to a solution of Grenacher's borax-carmine 

 until the fluid assumes a blood-red colour.) 



(2) The sections are then plunged for 2 minutes into absolute alcohol 

 to which crystals of picric acid have been added until the spirit re- 

 sembles hock. This operation is to be performed twice. 



(3) The sections are next immersed for 1 minute in Ehrlich's gentian- 

 violet solution. This solution should be freshly made. Excess of stain 

 should be removed with filter-paper. (The gentian-violet solution is 

 prepared by adding 11 volumes of a saturated alcoholic solution of the 

 pigment to 100 volumes of a 5 per cent, solution of anilin oil in water 

 and then filtering. 



(4) The sections are next immersed for one minute in a solution of 

 iodine (iodine, 1 ; iodide of potassium, 2 ; water, 300) ; from this they 

 are transferred to absolute alcohol, wherein they remain for 30 seconds. 



(5) Excess of gentian-violet is then removed with hydrochloric acid 

 and alcohol (HCl 3 ; CaHgO 97). In performing this step it is neces- 

 sary to watch the decoloration carefully, as the reaction is very delicate. 



* Zool. Anzeig., xi. (1888) pp. 624-5. 



t Journ. de Microgr., xii. (1888) pp. 415-6. Cf. Bull. Soc. Belg. Micr., 1887, 



No. 7. 



