Journal of 



Applied Microscopy 



and 



Laboratory Methods. 



VOLUME IV. JULY, 1901. Number 7 



The Value of Methylen Blue as an Intravitam Stain in 



the Tunicata. 



While working for special results on the tunicate nervous system, with 

 methylen blue, I found that this anilin could be made of much value as a gene- 

 ral stain where living material was obtainable. Small species, such as Amorxcium, 

 Botryllus, and Pcrophora, as well as young Molguh^, left in sea water containing just 

 enough methylen blue to color the water a lively blue (about 1 part to 500U) for half 

 an hour, will give almost diagrammatically the branchial basket and its organs, as 

 well as the free mesenchyme cells of the body cavity, leucocytes and phagocytes. 

 This method is especially favorable for cilia; the demonstration of cilia in 

 motion, the arrangement of cilia in rows on the surface of the cell, and the pecu- 

 liar thickened basal portion of the tunicate cilium can all be well shown. Long, 

 whip-like flagellae, which are found in the endostyle and ciliated funnel, also take 

 the blue and stand out with wonderful distinctness. The above named cells are 

 usually the first to stain. The sensory or peripheral portion of the nervous sys- 

 tem stains relatively early (from 1 to 1^ hours after immersion), while the 

 deeper lying nerve cells and motor fibers stain later. Cells of the central nervous 

 system are sometimes found colored blue as much as five hours after immersion. 

 But all of this so-called staining of the different tissues is transitory, sometimes 

 lasting only a few minutes — as in the case of the very delicate neurofibrils — or 

 for several hours, or even days, in the case of the mesenchyme cells. 



Special Methods. — The best results for the staining of the nervous system 

 were obtained by the two following methods : Molgulse were placed in a weak 

 solution (1-5000) of Meyer's BX methylen blue in sea water, and allowed to remain 

 from one to five hours, according to the size of the animal and the tissue to be 

 stained. For staining by immersion small specimens were used. It was found 

 necessary to have the animals absolutely fresh, or satisfactory results could not 

 be depended upon. Molgulae which had remained in aquaria for so short a 

 period as two to three days, frequently refuse to take the stain ; or give a diffuse 

 staining. The exact intensity of the blue in solution does not seem to be as 

 important a factor as the length of immersion. Just before taking out the animals 



(1357) 



