T. S. intestine 



PARAFFIN SECTIONS 



129 



tral nervous system) should not be pre- 

 served in this fluid for subsequent 

 investigation. 



The frog is killed by any convenient 

 method, but it is usually best for histo- 

 logical purposes to sever a large blood 

 vessel and permit as much l)lood as possi- 

 ble to drain out from the heart before 

 opening the abdominal cavity and remov- 

 ing the intestine. One or more lengths of 

 about 3'3 of an inch should then be cut 

 from the intestine and transferred directly 

 to fixative where they may remain from a 

 few hours to several weeks. 



When they are next required the speci- 

 mens should be removed from fixative, 

 washed in running water for a few hours, 

 and then transferred directly to 70 'o alco- 

 hol. The easiest method of washing objects 

 of this size in running water is to take one 

 of the coplin jars previoush' described, to 

 fill it with water, insert the specimen, and 

 then to attach a cover of coarse cheese- 

 cloth with a rubber band. This is then 

 placed in the sink and a narrow stream of 

 water permitted to fall on it from the tap. 

 It will be found that the specimen will 

 swirl round and round in the jar in a most 

 satisfactory manner. This simple device 

 saves all the trouble of rigging up glass 

 tubes and boring corks to make the cum- 

 bersome apparatus sometimes recom- 

 mended for the purpose. 



The specimen is transferred, after 

 twentj'-four hours in 70% alcohol, to 

 95% alcohol. It is better to use a large 

 volume of alcohol and to suspend the 

 object in it than to use relatively small 

 volumes which have to l^e frequently 

 changed. It is recommended that a wide- 

 mouthed stoppered jar of about 500 milli- 

 liters capacity be fitted witli a hook in the 

 center of its stopper, from which the ob- 

 ject can then be suspended. The majority 

 of stoi)pers for wide-mouthed glass jars 

 have a hollowed undersurface which may 

 be filled with plaster of Paris, and a glass 

 hook (which is verj^ easily bent from thin 

 glass rod) may be inserted in the litiuid 

 plaster. This must naturally be done some 

 days beforehand, and the plaster must 

 finally thoroughly be dried out in an oven 

 before the jar is usetl for dehydrating. If 

 the worker does not wish to go to this 



much trouble, it is also easy to screw a 

 small metal "pot hook" into the under 

 surface of a plastic screw cover for a jar of 

 the same size. Alcohol is, however, so 

 hygroscopic that it is better to employ a 

 glass-stoppered jar, the stopper being 

 greased with stoj)C()ck grease, or petro- 

 latum, for a permanent setup. An object 

 as coarse as the one under discussion may 

 be sus])ended in a loop of thread or cotton 

 directly from the liook; or if this is not 

 desirable, it may be enclosed in a small 

 fold of cheese cloth for suspension. After 

 twentj'-four hours in this volume of alco- 

 hol, the ol)ject will be completely pene- 

 trated, and should then be transferred to 

 absolute alcohol using the same volume in 

 a jar of similar construction. It is useful to 

 place about a (luarter-inch layer of an- 

 hydrous copper sulfate at the bottom of 

 the absolute alcohol jar, not only to make 

 sure that the alcohol is absolute, but also 

 to indicate, as it changes to blue, when 

 this jar should be removed from service. 

 Of the mau}^ de-alcoholizing (clearing) 

 agents which may be used, the writer 

 would in the present case select benzene 

 because it is less liable to harden the 

 circular muscles of the intestine than is 

 xylene. As benzene is fighter than an ab- 

 solute alcohol, it is not possible to employ 

 the hanging technique for clearing, and 

 the object should be placed in about 25 

 milliliters of benzene which should be 

 changed when diffusion currents are seen 

 to have ceased to rise from the object. 

 This will take about six hours for an object 

 of the size under discussion and a secoufl 

 bath of at least six houis should also be 

 given. 



It is now necessai-y to select the medium 

 in which em])edding is to be done and the 

 writer would recommend the rubber par- 

 affin of Hance (Chapter 17— E 21.1 Hance 

 1933) which must, of course, have been 

 prepared some time before. The melting 

 l)oint of this medium is about 56°C. so 

 (hat ail oven should be availal)le whi(Oi is 

 lliermostatically controlied at about 5S"('. 

 This oven should contain three stender 

 dishes as well as a 500 cc beaker contain- 

 ing about a pound of the embedding me- 

 dium. The ol)ject is removed from ben- 

 zene, drained briefly on a piece of filter 



