The Microscope. 69 



NEW METHODS IN DOUBLE STAINING. 



.T. D. BECK. 



UNDOUBTEDLY many microscopists as well as myselt have 

 met with difficulties in double-staining animal and vegetable 

 sections or specimens, viz: That when sections were beautifully 

 double-stained with iodine or cerrulin green and carmine, and hardly 

 dehydrated, the green stain would be nearly or totally obliterated or 

 washed out by the absolute alcohol. The ammonia or acid car- 

 mine stains having too much ammonia or acid, also remove a certain 

 portion of the green stain, unless the section has a strong affinity 

 for the stain. I have also known the essential oils to remove or 

 bleach out what little was left of the green stain in the specimen. 

 Thus the green stained section goes through three, special processes, 

 each one inclined to remove the green stain, viz: first, staining in 

 carmine; second, dehydrating sections; third, clarifying in essential 

 oils. Tannic acid was recommended as a mordant to fix the stain. 

 I tried it moderately strong. It fixed the stain and also fixed the 

 sections as useless. After diluting it with distilled water, alcohol, 

 etc., separately, until it lost all its mordant properties, it continued 

 to precipitate the carmine (and thus ruin sections) until its strength 

 was reduced to 300 or 400 times with distilled water or alcohol. I 

 found it the greatest nuisance in the laboratory. 



I experimented with a large variety of chemicals, metallic salts, 

 acids, etc., without reaching satisfactory results. Finally I prepared 

 an acid green dye (Echein green), but this also precipitated the car- 

 mine. Then I prepared some more stains, (chrokallus,) pink, crim- 

 son, orange and blue. 



I found those stains to work admirably in mauy specimens, but 

 I found that some plant sections (nasturtiums, etc.,) did not hold the 

 pink stain permanently, and that the crimson would mix too much 

 with the blue or Echein green in some sections, and did not differ- 

 entiate so admirably as the pink, but was much more permanent — as 

 much so as carmine. For some purposes I prefer the crimson to 

 the other red stains, when it differentiates beautifully. The blue 

 and red stains, blue and orange stains work well on some sections. 

 I considered carmine a stain too valuable to abandon entirely, and 

 so I repeated my experiments with Echein green and carmine, and 

 met with splendid results. 



THE PROCESS. 



The process is simply as follows: First, stain the sections well 

 in Echein green, which takes from five seconds to ten minutes.* 



*The time varies very mucli in various plants or tissues, depending on the affinity 



