APPENDIX. 



General Technic. 

 I. PROCURING AND HANDLING MATERIAL.* 



Methods of procuring and treating material for the study of maturation, fertilization 

 and segmentation are given under "Practical Suggestions" at the ends of the chapters treating 

 of those subjects (pp. 33, 41 and 53, respectively). 



AMPHIBIAN EMBRYOS. The eggs of the common wood frog (Rana sylvatica) can be 

 found in ponds during the first few warm days of spring (usually the latter part of March in 

 the vicinity of New York City). When deposited, the eggs are embedded in a compact mass 

 of transparent gelatinous substance. After lying in the water for a few hours, this substance 

 swells so that each egg appears to be surrounded by its own little spherical mass; all the masses, 

 however, still clinging together to form a cluster. 



The eggs are fertilized soon after being deposited, the spermatozoa floating free in the 

 water, and within a few hours begin to segment. The stage of segmentation can usually 

 be discerned with the naked eye, but it is always well to be provided with a hand lens. 



If the eggs are to be used for gross study, the clusters should be put into a large quantity 

 of 4 per cent, formalin. Stronger formalin will render the gelatinous substance opaque. 

 The eggs can be preserved in this fluid indefinitely. 



The later stages of cleavage and the young embryos can also be obtained in ponds, but 

 it is usually more convenient to take a number of the clusters to the laboratory in water. 

 If the water is kept fresh, development goes on almost as well as in the natural surroundings. 

 In this way it is possible to obtain whatever stages are desired. 



If the eggs or young embryos are to be used for the study of finer structures in sections, 

 Flemming's fluid is one of the best fixatives. The spherules of gelatinous substance should 

 be cut apart, however, before fixing. After the embryos have grown to a length of 3 or 4 mm., 

 the gelatinous substance is easily removed before fixing. During the cleavage stages, how- 

 ever, the removal is a difficult but necessary procedure, either before or after fixation. One 

 of the best methods is to dissolve the capsules with some fluid which does not injure the eggs. 

 Put the fixed eggs in a 10 per cent, solution of sodium hypochlorite diluted with 5 to 6 volumes 

 of water and leave them until they can be shaken free (several minutes or longer). The 

 following method is one of the most successful. Cut the fresh spherules from the general 

 mass and put for several hours in 



Saturated solution picric acid in 35 per cent, alcohol . . 100 volumes. 



Sulphuric acid 2 volumes. 



Wash for several hours in graded alcohols up to 70 per cent. If left for three or four days 

 in the 70 per cent, alcohol, the capsule swells and can be picked off with needles. The 



* Embryolo?ical material can also be purchased at Johns Hopkins University, at the South 

 Harpswell Laboratory (Prof. J. S. Kingsley, Tufts College, Mass.), and at the Marine Biological 

 Laboratory (Mr. George M. Gray, Woods Hole, Mass.). Models of embryos and developing parts 

 are always of great assistance to the student and teacher. These can be obtained from firms deal- 

 ing in scientific apparatus. 



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