GAS GLANDS OF SOME TELEOSTEAN FISHES. 237 



The Picric Acid, of course, differentiates the.Borax Carmine, leaving 

 scarlet nuclei, and the Picro-indigo-carmine stains the cytoplasm 

 of the gas gland a dull green, the cytoplasm of red blood corpuscles 

 and seci'etion products like zymogen granules a brilliant emerald- 

 green, and connective tissue blue. Curiously enough, I did not 

 always obtain these brilliant colour-contrasts with Zenker, espe- 

 cially if the material had been preserved in Alcohol and Glycerine 

 for some time after fixation, but fixation with Mann's Fluid always 

 yielded the best results. Objects which did not stain well with 

 the Borax Carmine I stained with Ehrlich's Htematoxylin and 

 Picro-indigo-carmine, and obtained sufliciently good contrasts. The 

 principal object to be attained by using Picro-indigo-carmine is to 

 show up the blood corpuscles and granules. In exceptional cases 

 I employed the Iron-hagmatoxylin method for nuclear details (in 

 Pe7'ca, e.g.), also Kernschwarz for rendering visible cytoplasmic 

 edges and hence the limits of intercellular and intracellular ducts 

 (in Gadus, e. g.). As above mentioned, most of my gas glands were 

 cut both transversely and longitudinally. 



Appendix B. 



On the Artificial Production of Gas Bubbles in Cells of the 

 Gas Gland. 



Of the various methods available for compelling fish to produce 

 gas in their bladder I have employed several, but only with one 

 have I succeeded in detecting the gas bubbles in the act of being- 

 formed by the cells. For the guidance of others I will first men- 

 tion my unsuccessful experiments. I first experimented with 

 Gobius pagaoiellus at Plymouth, subjecting this fish to increased 

 pressure due to increased depth — one of the easiest methods of 

 inducing gas-production. I took tank specimens from the Biolo- 

 gical Laboratory on board the steam launch, and when anchored 

 well out at sea I enclosed three or four specimens in each of five 

 cages. One cage I let down to a depth of 30 feet and kept it 

 there for one hour and a half ; a second cage 1 let down 60 feet 

 for one hour, a thiid 90 feet for one hour, a fourth 180 feet for 

 half an hour and a fifth 180 feet for two and a half hours. On 

 examining sections of the gas glands of these fish I could detect 

 no decisive differences in the gland cells. 1 am unable to explain 

 why this experiment was a failure. Possibly even the maximum 

 time allowed (2| hours) was too short in which to allow these cells 

 to become active ; possibly also the fact that Gobius paganellus is 

 a bottom form may have contributed to this negative result. 

 Another experiment which I may mention was to stimulate the 

 two vagus nerves of an Eel (Angioilla) with a battery for three 

 hours, the two sympathetics having been cut and the bladder 

 previously emptied and ligatured. At the end of the three hours 

 the bladder certainly contained about one-sixth of its usual volume 

 of gas, but I was unable to detect bubbles in the cytoplasm. I did 

 not try the experiment of injecting fish with Pilocarpine nitrate 

 or any similar drug. 



