Frog embryos 



PARAFFIN SECTIOKS 



133 



Preparation of. Serial Sections of an Amphibian Embryo 



Heavily yolked embryos are among the 

 most difficult objects from which to pre- 

 pare satisfactory serial sections, as may 

 be witnessed by the photographs which 

 illustrate the work of many experimental 

 embrj'ologists. Though the example spe- 

 cificall}' taken is that of an amphibian 

 embryo, the methods to be discussed may 

 l)e used for any heavily 3'olked material 

 such as fish, or even in the preparation of 

 sections of the early stages (segmentation 

 and tlie like) of bird embryos. 



The crux of the entire matter lies in the 

 selection of a fixative and it is doubtful 

 whether a worse fixative for the purpose 

 could be found than the picro-acetic- 

 formaldehj'de of Bouin, so generally em- 

 ployed. Two fixatives have been devel- 

 oped specifically for heavily yolked mate- 

 rial: that of Gregg and Puckett (Chapter 

 18 F 3000.1010 Gregg and Puckett 1943) 

 which is designed specifically for the eggs 

 of frogs, and that of Smith (Chapter IS 

 F 7000.1010 Smith 1912) which was 

 originally develoiied for the eggs of Crypto- 

 hranchus, but which the author has used 

 successfull}^ for a large variety of heavily 

 yolked material. The author has used 

 Smith for so many years that he is prej- 

 udiced in favor of this formula, and the 

 fact that he has not been so successful 

 with the formula of Gregg and Puckett 

 may be due to the fact that he is less ex- 

 perienced with it and not that it is in- 

 herently incapable of giving equally good 

 results. As the techniciues of fixation in- 

 volved are altogether different they will 

 be discussed separately. 



Let us assume first of all that we are 

 using the fluid of Gregg and Puckett, 

 which has been made up according to 

 the formula given in Chapter 18. The 

 mass of embryos and eggs, together with 

 their gelatinous surrounding envelopes, 

 are taken and placed in at least 50 times 

 their own volume of the solution. The jar 

 centaining them should be turned upside 

 down at intervals to insui'e that the fluid 

 around them does not become diluted and 

 they are then left for 24 hours. If the eggs 

 are to be embedded and sectioned at once, 



they may then be removed and waslied in 

 running water for 24 hours or, if they are 

 to be stored for long periods, they may be 

 moved to 2% formaldehyde directly from 

 the fixative and may remain in this fluid, 

 changed possibly after the first 48 hours, 

 until they are required for use. The mass is 

 then removed and broken into small clus- 

 ters which are placed in a test tul)e or 

 small flask about one-third filled with 

 water. The flask is then shaken vigorously, 

 which will remove a portion of the albu- 

 minous envelope, the dirty water poured 

 off, fresh added, and the flask again 

 shaken. After half a dozen treatments of 

 this type the greater part of the albumen 

 will have been removed. When as much 

 of the jelly as possible has been removed 

 by this mechanical treatment the eggs are 

 transferred to a flask of 1 % sodium hyi)0- 

 chlorite and shaken gently and carefully. 

 At intervals eggs will be found to detach 

 themselves. These should be removed 

 either with a section lifter or a small pipet 

 to another flask containing water; b\^ tliis 

 means the whole of the remaining jelly 

 will be removed. The eggs, which are now 

 in water, should be carefully examined to 

 see whether or not the vitelhne membrane 

 is still adherent. If it is still adherent, those 

 which have it should be transferred back 

 to fresh 1% sodium hypochlorite and 

 stirred very gently, being examined at 

 intervals under a binocular microscope, 

 until the membrane has been removed. 

 The eggs are then washed thoroughly to 

 remove all traces of hypochlorite. It is 

 better to do this with half a dozen changes 

 of water rather than in running water, be- 

 cause the eggs at this stage are lirittle and 

 portions may be flaked off the outside if 

 they are subjected to the ))umping which 

 seems to be an inevital)le part of washing 

 with running water. 



Let us now examine Smith's method. 

 The fixative must be made up immediately 

 before use and large volumes are required 

 in relation to the size of the objects. 

 The author once fixed the entire yolk of a 

 hen's egg, using two gallons of solution; 

 and at least 500 milliliters should be em- 



