Methods and Techniques 



33 



trifuge is a tool which can translocate sub- 

 stances from a particular part whose behavior 

 is to be studied in their absence, but who 

 knows what effects it may have had on the 

 invisible components of the zone? 



Nor are the mechanical methods the only 

 ones open to suspicion. It is unthinkable that 

 in anything kinetically as complex as the 

 simplest protoplasm, alteration of its equili- 

 bria by chemical or thermal changes could 

 be limited in its effects to a single system 

 alone. Temperatme changes probably affect 

 all its constituent systems in some measure, 

 and indeed the laws of thermodynamics are 

 hardly such as to permit such effects to re- 

 main local. Methods involving the use of 

 radiations and electricity, in spite of the ad- 

 vantage that the amounts of energy applied 

 may be measured, cannot be construed in 

 most cases as affecting single localizable or 

 identifiable systems within the cell. Excep- 

 tion, however, may be made in the cases 

 where radiations or other agents affect the 

 known gene; the most reliable defect method, 

 perhaps the ideal, is that which excludes 

 identified genes (Poiilson, '40, '45). Manipu- 

 lation of the gene, by man or nature, is 

 probably the most satisfactorily controlled 

 experimental method available to the em- 

 bryologist to date, and it will become the 

 more useful the more exhaustively the inter- 

 mediate steps between the primary action of 

 the gene and the end-effects of its activity, as 

 expressed in differentiation, become known. 



Despite the reservations enumerated, how- 

 ever, excellent contributions to embryology 

 have been made by the use of all of the de- 

 fect methods. These methods were the first 

 to demonstrate the high degree of regulabil- 

 ity of which the embryo is capable, and thus 

 have led directly to the primary embryolog- 

 ical problem of ultimate organization, and 

 the data which they have provided will be 

 indispensible for the final solution of it. 



A fact to be kept in mind in this connection 

 is the impossibility of consideration of the 

 defect experiment as separate from the iso- 

 lation or explantation experiment, which is 

 its corollary as well as its complement. In 

 the ideal situation the embryologist wishes 

 to consider both experiments. In one, the in- 

 vestigator studies what remains after some- 

 thing has been taken away; in the other, 

 he studies the behavior of what he has re- 

 moved. In this sense, the study of a single 

 blastomere isolated from a two-celled egg 

 may be regarded either as a defect experi- 

 ment or as an isolation experiment. Roux, as 

 a preformationist, considered it as an isola- 



tion experiment. He knew that in the egg one 

 blastomere forms half an embryo, and in- 

 terpreted the blastomere in his experiment 

 as duplicating its normal action. He con- 

 cluded that its processes of development were 

 identical in normal and experimental mate- 

 rial, and the experiment seemed to him 

 essentially a way to confirm what he had 

 postulated the behavior of the normal part 

 to be. 



The questions that are put to the isolated 

 part are now framed by more open minds, 

 but they still deal in the main with the de- 

 gree to which a cell is dependent for its 

 differentiation on factors impinging upon it 

 from its surroundings. To what degree does 

 a part begin or continue differentiation when 

 isolated from its usual cellular surroundings? 

 Does it differentiate the same structures it 

 was destined to form in the normal embryo? 

 If not, what is the direction of its differenti- 

 ation and what factors determine this direc- 

 tion? Attempts to isolate these factors involve 

 not only the negative phase of the experi- 

 ment and determining what factors usually 

 present are lacking when the part is isolated, 

 but also the more positive one of demonstrat- 

 ing the new or different ones to which it is 

 subjected. This was a side of the problem on 

 which Roux did not concentrate, though it 

 is clear that he recognized its importance; 

 and it is this aspect of it which raises some 

 of the most immediate issues facing the pres- 

 ent interpreters of isolation or explantation 

 experiments. 



Ideally the investigator may express a 

 wish to culture his isolate in a neutral or 

 indifferent medium (cf. Needham, '42, p. 

 175) which will permit it to continue its 

 own development in its own way. This is 

 essentially what Harrison ('07) did when he 

 isolated the neuroblast in clotted lymph, a 

 medium which did not inhibit the produc- 

 tion of the axon yet which excluded the 

 presence of the cells which had been thought 

 by some to manufacture it. If the behavior 

 of a cell is to be studied in the absence of 

 influence from surrounding cells, it is es- 

 sential that the medium to which it is re- 

 moved cannot itself alter the chain of reac- 

 tions to be studied. To what degree can this 

 ideal be achieved? 



First and foremost, the emancipation of a 

 cell from influences emanating from its 

 neighbors may now be recognized as more 

 difficult to achieve than formerly was antici- 

 pated, in view of Holtfreter's ('44) recent 

 demonstration that some of the cells constitut- 

 ing the very cultures being studied may at- 



