DEVELOPMENT AND LIFE-HISTOEIES OF TELEOSTEAN FISHES. 805 



hatched embryos, and it is certainly one of the best that can be used for killing and hardening 

 them. 



Perenyi's Fluid (Chrom-nitric Solution). — This fluid kills instantaneously, and preserves 

 Teleostean ova better than any other medium tried ; but in the processes subsequent to hardening, 

 its action proves defective. The staining fluid is best added during the fixing process, borax- 

 carmine being mingled with the solution. Pelagic eggs preserved their form admirably, and 

 fixation was apparently most satisfactory, but staining was not very successful, and in the clearing 

 and imbedding processes the ova shrunk, and good sections were found to be impossible. With 

 some change in the mode of imbedding, this fluid would be most efficient. Whitman, however, 

 states that he obtained good sections " more instructive than any obtained from eggs hardened in 

 other fluids " {op. cit., p. 154). 



Chromic Acid. — The merits of this fluid for killing and fixing Teleostean eggs need not be 

 insisted on. It acts perfectly ; but the long washing and difficulty of subsequent staining are 

 objections. 



Osmic Acid. — Alone and in various combinations osmic acid is much recommended. The fatty 

 elements in Teleostean eggs, however, render it a doubtful medium, and no good results were ob- 

 tained. Marshall used it for the embryos of Scyllium, which were placed in \ per cent. sol. chromic 

 acid and a few drops of 1 per cent, osmic acid for twenty-four hours — thence into alcohol. 



Chrom-Platinicm. — This mixture is said to be admirable for fixing, but Whitman found that 

 embryos are often rendered brittle, and contours are indistinct. It is very slow in action, but after 

 washing in alcohol, staining is said to be easy and successful. 



Alcohol-Method. — A great number of ova and embryos were not subjected to special treatment, 

 but were simply transferred from the tanks (sea-water) to 60 per cent, alcohol. In this they were 

 killed and hardened, as ordinary museum-specimens are. Much distortion often resulted, yet some 

 good sections were made of blastoderms thus simply prepared. The capsule of the egg was 

 usually pierced with a fine needle to ensure entrance of the alcohol, stain, &c. 



The graduated series of alcohols was tried, and, producing less distortion, gave fair results. 

 The objects were transferred from the sea-water into dilute alcohol, " Dritteralcohol," i.e., 33'3 per 

 cent. ; thence in 40, 50, and 60 per cent. On account of the small size of the ova, five or six hours 

 in each sufficed, extended in the stronger alcohols to ten or twelve hours. 



III. Staining. — Only alcoholic stains were used, and Beale's solution, if not too newly made, 

 gave very satisfactory results. It requires long immersion, rarely less than twenty to thirty hours, 

 and is apt to be diffuse, but acidulated alcohol in a short time makes it markedly nuclear. Diluted 

 with alcohol, the penetrative power of this stain is increased. 



Borax Carmine (Naples formula) is one of the most successful stains — penetrating and nuclear, 

 and sections are additionally valuable if, after overstaining, the eggs are placed in acidulated alcohol 

 until the surplus is removed. 



Hccwiatoxylin (Kleinenberg's). — This proved less useful than might have been supposed ; no 

 good sections of early blastoderms were obtained after the employment of this stain, but more mature 

 tissues were very satisfactorily treated, the stain being of the most pronounced nuclear character. 



On the whole, the carmine stains are found to be the best. 



IV. Imbedding. — Prior to imbedding, the ova were finally dehydrated by an immersion for two 

 or three hours in absolute alcohol, and transferred thence either into benzine, oil of bergamot, 

 or chloroform — clove-oil, creosote, &c, not being found to act well. The transference was made 

 gradual by the method of Giesbeecht. Turpentine succeeded the bergamot, in other cases 

 a mixture of the clearing agent and paraffin followed, fragments of paraffin being added until 

 finally the objects were transferred to pure melted paraffin in the usual way. Mixtures of the 

 hard and soft paraffin, supplied by the Cambridge Instrument Company, were used — the proportions 

 varying according to the temperature of the laboratory. Before transferring from the final absolute 



