STAINS. 



189 



Wash the sections in distilled water and then 

 in tap-water. Stain (until very black) in water 

 containing I per cent haematoxylin. Differen- 

 tiate in 30 per cent acetic-acid water with care- 

 ful watching, or in more dilute acid, or in very 

 dilute (i : 20) liquor ferri, if it is to be followed 

 by acid fuchsin as a contrast stain. (Verhandl. 

 d. Anat. Gessellsch., 1893, Jena, Gustav Fischer.) 



Heidenhain's Iron Haematoxylin. 

 Mordant the sections from one-half hour to 

 12 hours in a 2.5 per cent watery solution of iron 

 alum (ammonio-ferric sulphate (NH 4 )i Fe 2 

 (SO<)i dissolved cold). This salt comes in 

 violet crystals. Yellow or green crystals should 

 be rejected. Rinse well in water. Stain in 

 water containing 0.5 per cent of haematoxylin. 

 Rinse, expose again to the iron-alum solution, 

 watching the differentiation under the micro- 

 scope, the examination being made in tap-water. 

 When properly differentiated wash 15 to 45 min- 

 utes in running water. Dehydrate, clear only 

 with xylol, and mount in xylol balsam. The 

 mordant does not keep indefinitely, but is said 

 to retain its properties for some time (Dodge). 



Schaffner's Safraiiin Picro-nigrosin. 



The slides are stained for a few minutes in 

 anilin-safranin made as follows : 



1 i ) Anilin water 50 



Saturated alcoholic (95 per 



cent) solution of safranin 50 



Rinse quickly in water. They are then stained 

 in (2). 



(2) Distilled water 100 



Picric acid thoroughly dissolved 



in the above i 



Then add nigrosin i 



Rinse in water, wash rapidly in 95 per cent al- 

 cohol, dehydrate, and mount in balsam. 



Malachite Green. 



For plant tissues, as well as animal tissues, 

 this may be used as a contrast stain, follow- 

 ing carbol-fuchsin. It is dissolved in anilin 

 (i : 1000) and used fresh; generally the ex- 

 posure to it should be for only a very brief 

 period, '. e., i to 3 minutes. If not fresh, much 

 longer exposures are required. 



CLEANING COVER-GLASSES FOR FLAGELLA STAINS. 



Van Ermengem recommends boiling in a mixture of water 100 cc., concen- 

 trated sulphuric acid 60 cc., potassium bichromate 60 grams. The covers are 

 afterward thoroughly washed, first in water and then in absolute alcohol. They 

 are set on edge and dried under a bell-jar. 



Loeffler recommends heating covers in concentrated sulphuric acid. They are 

 then washed in distilled water and put into alcohol-ammonia, from which they are 

 wiped with a very clean linen cloth. 



Covers cleaned in the ordinary way may be freed from fat by passing them 

 through a Bunsen flame immediately before using. 



FLAGELLA STAINING. 



There is no easy road to success. Some of the common stones of stumbling 

 are (i) oily or otherwise dirty covers ; (2) cultures unsuited either by age or by 

 composition of the medium ; (3) the casting off of flagella on dilution or during 

 the slow drying of the fluid on the cover ; (4) an uneven or too copious distribution 

 of the organisms on the cover ; (5) imperfect mordanting ; (6) excessive mor- 

 danting ; (7) understating ; (8) overstating ; (9) precipitates on the cover-glass 

 during some stage in the process. 



If clean covers are used, if the bacteria are derived from young moist agar cult- 

 ures, if a very small quantity of such culture is put into a large drop of well aerated 

 water, or, better, into a test-tube or watch-glass containing 5 or 10 cc. of water, and a 

 tiny quantity of this dilution is taken on a platinum needle and deftly swept over 

 the whole cover ; or if the needle is touched to the bacterial fluid and then touched 



