154 



Methods of Observing Micro-organisms 



III. Glycerin-gelatin. — ^As the penetration of the tissue by cel- 

 loidin is attended with deterioration in the staining qualities of 

 the tubercle bacillus, it has been recommended by KoUe* that the 

 tissue be saturated with a mixture of glycerin, i part; gelatin, 2 

 parts; and water, 3 parts; cemented to a cork or block of wood, 

 hardened in absolute alcohol, and cut as usual for celloidin with a 

 knife wet with alcohol. 



Staining. — ^Simple Method. — For ordinary work the following 

 simple method can be recommended: After the sections are cut 

 and cemented to the slide, the paraflSn and celloidin should be re- 

 moved by appropriate solvents. The sections are immersed in the 

 ordinary aqueous solution of the aniUn stain and allowed to remain 



about five minutes, next washed 

 in water for several minutes, then 

 decolorized in 0.5 to i per cent 

 acetic acid solution. The acid re- 

 moves the stain from the tissues, 

 but ultimately from the bacteria 

 as well, so that one must watch 

 carefully, and so soon as the color 

 has almost disappeared from the 

 sections, they must be removed and 

 transferred to absolute alcohol. At 

 this point the process may be in- 

 terrupted to allow the tissue ele- 

 ments to be countercolored with 

 alum-carmin or any stain not re- 

 quiring acid for differentiation, 



CROSS-SECTION 

 SHOWING SHOES 

 IN POSITION. 



Fig. 28. — Coplin's staining jar. 



after which the sections are dehydrated in absolute alcohol, cleared 

 in xylol, and mounted in Canada balsam. 



The greater number of appUcations can be made by simply 

 dropping the reagents upon the shde while held in the fingers. 

 Where exposure to the reagents is to be prolonged, the Coplin jar 

 or some more capacious device must be employed. 



Pfeiffer's Method. — The sections are stained for one-half hour in 

 diluted Ziehl's carbol-fuchsin (?.!».), then transferred to absolute 

 alcohol made feebly acid with acetic acid. The sections must 

 be carefully watched, and so soon as the original, almost black-red 

 color gives place to a red-violet color they are removed to xylol, 

 to be cleared preparatory to mounting in balsam. 



LofHer's Method. — Certain bacteria that do not permit ready 

 penetration by the dye require some more intense stain. One of 

 the best of these is LofBer's alkaline methylene-blue: 



Saturated alcoholic solution of methylene-blue 30 



I : 10,000 aqueous solution of caustic potash ,. . loc 



* Fliigge's "Die Mikroorganismen," vol. i, page S34. 



