80 Papers from the Marine Biological Laboratory at Tortugas. 



It has not been possible to dissect the adult lymphatic system of 

 the loggerhead turtle in time for this preliminary paper. The anatomical 

 description will, therefore, be based on the excellent work of Miiller on 

 the green turtle, supplemented by Fritsch's work on the land turtle, 

 and by such dissections as the writer has made on the mud turtles found 

 in the vicinity of Iowa City, The specimens dissected were mostly 

 Chrysemys marginata, although specimens of Chelydra serpentina and of 

 Amyda mutica were also studied. The turtles were killed with chloral 

 hydrate or with chloroform. The blood-vessels were injected with red 

 and with blue gelatin, while the lymphatics were injected with a thin 

 yellow gelatin mass. The melting-point of the gelatin was lowered by 

 adding potassium iodid to the melted solution. The posterior lymph 

 hearts were studied by exposing them after the animal had been chloro- 

 formed. As they continue to beat for some time after they have been 

 exposed, they are very easily found and studied. Their connections 

 with adjacent veins can readily be demonstrated by injecting India ink 

 into their cavity. 



For the preparation of the embryonic material a large number of 

 fixing agents were used. Corrosive sublimate solutions were, as a rule, 

 not satisfactory, although Zenker's fluid gave fairly good results. Gilson's 

 fluid proved very unsatisfactory, especially with large embryos. The 

 mesenchymal tissues were poorly fixed and the material did not keep 

 well. By far the best results for embryos of all stages were obtained 

 by using mixtures of chromic and acetic acids with pure formaldehyde. 

 These mixtures seem to penetrate the thick shell more readily and to 

 fix the internal organs more thoroughly and with less distortion than 

 any other combination tried. The following mixture for embryos up to 

 about 30 days of development was found satisfactory: 



Chrom-aceto-Formaldehyde:^ Chromic acid, i per cent aq, solution, 64 parts; 

 glacial acetic acid, 4 parts; formaldehyde, pure 40 per cent, 32 

 parts. 



The strength of the chromic-acid solution may be varied somewhat 

 to suit conditions. The solution should be allowed to stand mitil it 

 assumes a greenish or "chrom-alum" color before using. Embryos are 

 fixed from 15 minutes to 6 hours according to size, and washed thor- 

 oughly in running water. Although Lee refuses to recognize chromic- 

 formalin fixing agents in the later editions of "The Microtomist's Vade- 

 mecum," the writer is compelled to confess that, after using the mixture 

 mentioned above for a number of years, none of the standard fixing 

 agents have given such uniformly good results as has this one. If the 

 material is not left in the fixing solution entirely too long, and is thor- 

 oughly washed out, there is no difificulty whatever in obtaining beautiful 

 stains with any of the standard methods. 



Various methods of staining were employed. The most satisfactory 

 results were obtained with Delafield's hematoxylin or iron haematoxylin 

 followed by slightly acidulated orange G. or eosin-aurantia-orange G, 



' Lee "The Microtomist's Vade-mecum." 4th ed., page 55, Norris, Proceed- 

 ings of the Iowa Academy of Science, vol. 8, page 78 (1900). 



