330 



METHODS AND FORMULAS 



DS 12.30 



them only the techniques of Margolena in 

 1933 and de Winwarter and Saimonte 

 1908 can be regarded as having any wide 

 application. 



12.30 TYPICAL EXAMPLES 



Preparation of a transverse section of 

 an earthworm, using the iron-hema- 

 toxyhn stain of R^gaud 1910, fol- 

 lowed by the acid fuchsin- 

 anihn blue of Masson 1912 



Considering that almost every student 

 of biology studies a transverse section of 

 the earthworm, it is really surprising that 

 sections commonly available from biologi- 

 cal supply houses should have been pre- 

 pared with little thought for their ultimate 

 use. Quite apart from any considerations 

 of staining, it may be pointed out that the 

 average field of view of the 3.5X objec- 

 tive, commonly used in elementary bio- 

 logical laboratories, is about 4 mm., so 

 that a worm larger than 4 mm. in diameter 

 cannot be got into the field of view of this 

 low power at one observation. It is un- 

 doubtedly a technical feat of some skill to 

 cut a transverse section of a large earth- 

 worm, but it is also extraordinarily diffi- 

 cult for a beginning student to envisage 

 the relationships of the whole by suc- 

 cessive examinations of a variety of fields. 

 It may be taken, therefore, as the first 

 prerequisite to a useful preparation that 

 one should select an earthworm of approxi- 

 mately the right size. Though tliis may 

 prove an arduous labor, it may be pointed 

 out that at least 100,000 sections may be 

 obtained from a single worm. Further, 

 the majority of sections which one sees 

 in elementary classes have been stained 

 by a standard hematoxylin-eosin tech- 

 nique which gives students very little 

 chance of envisaging the relationships of 

 the muscles; whereas if one employs a 

 technique of the kind here recommended, 

 the muscles will be stained a brilliant 

 scarlet and the connective tissues to w^hich 

 they are attached will be stained a bright 

 blue. There is also a much better differ- 

 entiation of the various layers of the intes- 

 tine and of the nervous tissue. 



Once the earthworm has been collected 

 and selected, it then becomes necessary 

 to remove from it the grit with winch the 

 intestine is filled. The classic method of 

 doing this, to which the writer strongly 

 adheres, is to place the earthworm in an 

 environment of old coffee grounds. It is 

 not, however, sufficient, to take coffee 

 grounds from which a single drink has 

 been extracted. Grounds left over from 

 the preparation of coffee should be taken, 

 boiled for a considerable period in water, 

 filtered, and again boiled, until fittle or no 

 coloring matter remains to be extracted 

 from them. The earthworm may be ec- 

 lectic in its diet, but its constitution does 

 not enable it to survive the glucosides and 

 alkaloids of a partially extracted coffee 

 bean. These depleted grounds should then 

 be placed in a layer an inch or two thick 

 in a clean glass jar, the selected earth- 

 worms placed on the surface, hghtly 

 sprinkled with coffee grounds, and per- 

 mitted to live undisturbed for a period of 

 about a week. The only critical factor is 

 the degree of humidity of the grounds. 

 This should be such that water cannot be 

 pressed from them when they are squeezed 

 in the hand, yet each grain should present 

 the appearance of being moist. 



After a week on a diet of coffee grounds 

 the worms will have voided most of their 

 grit content and may now be narcotized 

 prior to fixation. Though it is not as 

 necessary to narcotize them for section 

 cutting as it is for dissection, it is never- 

 theless desirable, since it appears that 

 muscles fixed after narcotization (at least 

 those of the Annelida) are not nearly as 

 liable to become brittle as are those fixed 

 in the violent state of contraction which 

 results from dropping the worm directly 

 into the fixative. Earthworms may be 

 satisfactorily narcotized under the surface 

 of water containing almost any known 

 narcotic agent, and the choice between 

 chloroform, chloral hydrate, or cocaine 

 and its substitutes must rest largely upon 

 the availability of the reagents. The worm 

 may be considered adequately narcotized 

 when it does not react rapidly when placed 

 on a flat surface of filter paper and slowly 

 stretched with the fingers. If it does not 

 contract at all, and particularly if its outer 



