January 10, 1913] 



SCIENCE 



59 



eentriole, karyosome and sub-membranous 

 granules, take the stain in diflferent degrees, 

 yet it is the same chemical basophilic sub- 

 stance that becomes stained. 



HEematoxylin tells us nothing about the 

 acidophilic substance which seems to play an 

 important part in the physiology of the 

 nucleus. 



During the past year, working with 

 Entamceha tetragena, I have been impressed 

 by the lack of information in literature on the 

 subject of the acidophilic substance in the 

 nucleus of protozoa; and in descriptions of 

 protozoa, I have noticed the frequency with 

 which acidophilic substance has been confused 

 with true chromatin (basichromatin). This 

 appears to be due to the use of polychrome 

 stains which have not been thoroughly dif- 

 ferentiated, and to the absence of a satisfac- 

 tory technique for demonstrating acidophilic 

 substance in wet fixed films. 



Those who have used the Eomonowsky 

 modifications have usually been content with 

 over-toned or blurred pictures. In attempting 

 to identify " E. histolytica" in this region 

 and differentiate it from E. tetragena, our 

 common pathogenic entamoeba, I have used 

 Eomonowsky stains on films which have been 

 so differentiated that excessive amounts of 

 the stain have been washed out as one would 

 differentiate preparations stained with hsema- 

 toxylin. Inasmuch as Eomonowsky stains 

 have almost as much tendency to overstain as 

 haematoxylin, the necessity for extraction of 

 superfluous stain is manifest. 



I have usually selected cover-slip prepara- 

 tions that contained a sufficient number of 

 entamcebae to warrant further study, and 

 stained both cover-slip and object slide, 

 thereby obtaining three pictures from one 

 film, fresh, hsematoxylin and polychrome. 

 After staining, the excess of polychrome stain 

 has been removed by means of 95 per cent, 

 ethyl alcohol and ammoniated 60 per cent, 

 alcohol, and I have found that when properly 

 differentiated, the polychrome stain after dry 

 fixation gives a picture entirely different from 

 that of hematoxylin after wet fixation. It is 

 different in two respects. It not only shows 

 that there is an acidophilic substance — oxy- 



chromatin — within the nucleus quite different 

 from anything yet described for E. tetragena, 

 but the remainder and larger portion of the 

 nucleus has a different structure and staining 

 characters from that described from hsema- 

 toxylin preparations of this entamoBba. 



The nucleus of E. tetragena, when stained 

 with Hasting's stain followed by Giemsa's 

 stain, and carefully differentiated with 60 per 

 cent, ethyl alcohol, to which a few drops of 

 aqua ammonia have been added (1 per cent, 

 aqua ammoniae in 60 per cent, alcohol) is 

 seen to be made up of a clearly defined red 

 substance which takes the form of a ring 

 about the size of the karyosome or smaller. 

 Oftener, it takes the form of a delicate 

 reticulum or of discrete granules lying within 

 the nuclear ruembrane. This red substance 

 does not correspond in location with true 

 chromatin (basichromatin) which stains with 

 hsematoxylin, and it should not be confused 

 with basichromatin as some writers have 

 done. The red substance, or oxychromatin, 

 is imbedded in an ill-defined nuclear struc- 

 ture, staining faintly blue which sometimes 

 is made up of slightly refractile achromatic 

 granules of uniform size, imbedded in 

 faintly staining blue substance and sur- 

 rounded by an achromatic or faintly staining 

 blue ring, corresponding with the nuclear 

 membrane. The cytoplasm stains various 

 shades of blue. 



Attention is drawn to this subject with the 

 suggestion that those interested in the cytol- 

 ogy of protozoa pay more attention to the 

 acidophilic substance of the nucleus — oxy- 

 chromatin, for the purpose of learning what 

 part it plays in relation to synchronous 

 changes in the basichromatin of the nucleus 

 and in the physiology of the cell. 



It is extremely likely that so clearly defined 

 a substance as the oxychromatin of the nucleus 

 of E. tetragena Is has an important physiolog- 

 ical function, and it would seem that other 

 protozoa might yield interesting and no doubt 

 important information if stu.died from prepa- 

 rations designed to satisfactorily show baso- 

 philic and acidophilic nuclear substance. 



Samoel T. Darling 



