REMARKS ON TECHNIQUE 11 



them, so that in individual cases a variety of methods may be 

 available. 



1. Nicolle's Thionin. Saturated solution of thionin in 50 per cent, alcohol, 

 10 parts ; 1 per cent, carbolic acid in water, 100 parts. Stain for about one minute ; 

 wash with water, alcohol, xylol, balsam. 



2. Pappenheim's Stain. 1 Fix sections or films in alcohol or sublimate ; embed 

 in paraffin, etc. ; stain in a solution of methylene green, 2 pinches (amount on the 

 point of a knife) ; pyronin, 1 pinch; aq. dist., 5 c.cm. Mix and leave for fourteen 

 days, then filter, and stain for five minutes; differentiate in a dish of absolute 

 alcohol containing 3 pinches of resorcin, then pass through absolute alcohol 

 and xylol. 



3. Polychrome Methylene Blue (Unna, 2 after E. Frankel). After staining the 

 sections for several houi-s in the polychrome methylene blue, they are differentiated 

 in Unna's tannin-orange mixture, washed in water, and then taken through alcohol 

 and oil into balsam. If the staining has been successful, the nuclei and bacteria 

 are blue, the red blood-corpuscles orange, and the collagen tissues unstained. 



4. Zieler's Stain. Fix in Mtiller's formol ; embed in paraffin, or remove celloidin 

 before staining. 3 Stain over-night (eight to twenty-four hours) in Tranter's weak 

 orcein solution (orcein D [Grtibler], 1 part ; official nitric acid, 2 parts ; 70 per cent, 

 alcohol, 100 parts) ; rapidly wash in 70 per cent, alcohol, then in water. Stain in 

 polychrome methylene blue for ten minutes to two hours ; wash in water. 

 Thoroughly differentiate in Griibler's glycerine-ether mixture; wash in distilled 

 water. Take through 70 per cent, alcohol into abs. alcohol, then xylol to balsam. 

 The bacteria are dark blue or blue-black. 



5. E. S. Thomson's 4 Carbol Toluidin. Equal parts of 1 per cent, toluidin and 

 1 to 2 per cent, formol in water ; stain for fifteen to thirty minutes ; decolorize in 

 95 per cent, alcohol ; if this be difficult, add a drop or two of glacial acetic acid , 

 and again pass through 95 per cent, alcohol. 



Clear in carbol xylol, origanum oil, and balsam. Gcod results were often 

 obtained by this method by the introducer. 



Both paraffin and celloidin sections often stain well with warm Loffler's 

 methylene blue. 5 The dye is carefully filtered and placed with the section in 

 a closed watch-glass for one to twenty-four hours in the brood oven; it is 

 then poured out into a larger dish with water, and then taken through alcohol. 

 The degree of decolorization, and also the intensity of staining, can only be decided 

 in any particular case by trial. In many cases the sections may be placed in water 

 acidulated with acetic acid, or some of the acid can be added to the alcohol. In 

 other cases this will cause too much loss of colour. A little methylene blue can be 

 sometimes added to the alcohol to prevent too thorough a decolorization. Then 

 carbol xylol and balsam (oil must be avoided, as the sections bleach in it, or if used, 

 some methylene blue should be added to the oil). 



The following sources of error must be avoided in all cases where 

 Bacteria are stained in sections, or in fluids from the interior of the eye : 



1 According to Mayon, better than polychrome methylene blue. 



2 Prepared by Grubler (Leipzig). 



3 Spread section carefully on the slide, dry with filter-paper ; drop on a few drops of 

 absolute alcohol and then alcohol and ether, and when section begins to dry wash quickly 

 with 80 per cent, alcohol. Some celloidin must remain, or the section will fall to pieces. 



4 ' The Staining and Examination of the Bacteria of the Eye by Simple Practical 

 Methods ' (Jour. Amer. Med. Assn., 1906). 



5 A few drops of heematoxylin can be added (Wagemnann). Hjematoxylin alone often 

 shows organisms when overstained, especially Staphylococci and Streptococci in masses. 



