CYTOCHEMICAL TESTS ON EMBRYOS 555 



B. THE PLASMAL BEACTION : 



The following procedure haa been used successfully with the amphlhian ovary, 

 particularly the iimnature and post-ovulation ovary. It is haeed on Voss (1922 and 

 1951) and Lison (I956). The reaction is given by a special type of phospholipid 

 in which an aldehyde group appears upon treatment with the sublimate. The pro- 

 cedure follows : 



1. Fix the ovary in saturated corrosive sublimate - 5 minutes. 



2. Wash in distilled water. 



5. Dehydrate, clear, embed, section (10 ^), and then hydrate. 

 k. Place directly In Feulgen reagent for I5 minutes. 



5. Wash with 5 changes of distilled water saturated with SO^. 



6. Einae in distilled water. 



7. Mount in pure glycerine and observe under the microscope. 

 Plasmalogen is a component of the cytoplasm which gives a positive Feulgen 



test. Being a phospholipid, the control for this test consists of fixing the 

 ovary with Carnoy's fluid and washing twice with alcohol for 15 minutes each. 

 This extracts most of the lipids. 



C. THE NUCLEAL BEACTION : 



This reaction depends upon the combination of the N-sulphinlc acid in 

 fuchsin- sulphurous acid with the aldehyde component released from a molecule by 

 mild and partial hydrolysis. However, it nnist be remembered that the failure of 

 any tested tissue to give this reaction may be due to any of four following causes 

 (Gardiner, 1955). 



1. The substance may not contain the aldehyde component. 



2. The hydrolysis may be insufficient to release the aldehyde group from the 

 molecule . 



5. The hydrolysis may result not only in splitting off of the aldehyde group 

 but also in its disintegration, so that it cannot react with the sulphinlc 

 acid to form the new compound. 

 k. The aldehyde-containing substance, and consequently the aldehyde set free 

 on hydrolysis, may be so small in quantity that, although the reaction 

 actually occiu^s, the compound la too minute in amount to be visible even 

 with high magnification. 

 Gardiner (1955) saya further: "lypically chromatin gives the reaction, but 

 it does not invariably do so, nor la it the only cellular substance capable of it." 

 Stowell (19^6) says; "The preponderance of evidence indicates that with the 

 proper precautions the Feulgen technlc for thymonucleic acid la one of the most 

 specific histochemlcal reactions." 



THE PROCEDURE 



The procedure given here is baaed on the paper by Eafalko (19^+6): hydro- 

 chloric acid is neceaaary only to release the sulphur dioxide from the sulphites 

 used in the formation of the leuco-baalc fuchsin and in the sulphurous acid bath 

 following atainlng. Both the acid and the sulphite were eliminated and direct 

 charging of both the basic fuchsin and the bath water with sulphur dioxide gas 

 was substituted. 



Bubble sulphur dioxide gas from a small aperture In glass tubing into 100 cc. 

 of 0.5^ basic fuchsin, beneath a hood. Decolorlzatlon takes place in 1 hour and 

 the reagent is ready for use. Distilled water la aimilarly aaturated, and may be 

 stored In tightly corked bottlea for weeka. To get SOg, use simple flask-and- 

 funnel generator and sodium bisulphite and dilute aulphurlc acid. 



1. Fix tiaaues in Zenkera (or Bouln) for 2-20 mlnutea. 



2. Wash not more than 20 mlnutea, embed and aection in the usual manner. 

 5. Place in distilled water - 2 minutes. 



k. Normal HCl at room temperature - 2 minutes. 

 5. Normal HCl at 60OC, for 8 to 10 mlnutea. 



