260 Journal of Applied Microscopy. 



Second Stain — Anilin Safranin, Gentian Violet. 



This makes a good double stain for many purposes. Stain first in the aniUn 

 safranin from two to four hours ; then wash in 25 per cent, alcohol ; next in 

 water ; and then stain from one to four minutes in the gentian violet. After 

 washing in water, pass through the grades of alcohol, clear in xylol or clove oil, 

 and mount in balsam. 



Third Stain — Heidenhain's Iron-Alum-H/Ematoxylin Stain. 



Run the slides down to water, and from this transfer to the iron-alum. 

 Keep the sections in this from two to four hours, and after washing well in tap 

 water, stain for twelve hours (or over night) in the haematoxylin. After this 

 wash the slides again in water and wipe them clean, and as close to the sections 

 as is safe. The sections are now black and must be cleared. To do this they 

 are placed again in the iron-alum, which gradually takes out the excess of stain. 

 They must be closely watched and examined from time to time under the low 

 power of the microscope. When of a light greyish-blue color they are washed 

 again very thoroughly in tap water so that all iron salt is removed, and are then 

 carried through the grades of alcohol, cleared in xylol, and mounted in balsam. 



Fourth Stain — -Anilin Safran,in, Iron-Alum-Haematoxylin. 



After one has become accustomed to use the foregoing combinations success- 

 fully, the following is well worth trying. Stain first in anilin safranin or in 

 anilin safranin and gentian violet, as described above ; wash in water ; and then 

 stain in the iron-alum-hi-ematoxylin according to the directions given, just as 

 though the sections had not been stained at all. After staining, removing excess 

 of stain, and washing in tap water, pass through the grades of alcohol, clear in 

 xvlol, and mount in balsam. This is one of the clearest stains I know of. 



There are many other excellent combinations and stains, all of which should 

 be learned gradually and employed. The person who uses but one method of 

 killing and staining without trying others and comparing with them, may get 

 certain results, but such results are always to be taken with some reserve. The 

 stains mentioned above, it is believed, will give quite uniform and satisfactory 

 results for general purposes, and they may be taken as the starting point for 

 acquiring a technique which is both elaborate and extensive. 



John H. Schaffner. 

 Ohio State University. 



Carbolic Acid as a Clearing Agent. 



This is particularly useful in mounting marine diatoms /// situ on seaweeds. 

 Algee covered with the frustules of Rhabdoncma arcuatum and similar diatoms may 

 often be found tossed up by the storms on the British coasts. To remove air 

 from the interior of the frustules, drying and subsequent soaking in benzine have 

 been recommended, but are only partially successful, many of the diatoms remain- 

 ing filled with air. Soaking in water and passing successively through alcohol 

 and oil of cloves will of course remove all air, but will often bleach the algae on 



