METHODS OF OBSERVING BACTERIA. 8 1 



two to three minutes ; 5. Wash thoroughly in water ; 

 6. Dry ; 7. Mount in Canada balsam. 



This simple process suffices to stain most bacteria. 



Staining Bacteria in Sections of Tissue. It not 

 infrequently happens that the bacteria to be examined 

 are scattered among or enclosed in the cells of tissues. 

 Their demonstration is then a matter of some difficulty, 

 and the method employed is one which must be modified 

 according to the kind of organism to be stained. Very 

 much, too, depends upon the preservation of the tissue 

 to be studied. As bacteria disintegrate rapidly in dead 

 tissue, the specimen for examination should be secured 

 as fresh as possible, cut into small fragments, and im- 

 mersed in absolute alcohol from six to twenty-four hours 

 to kill the cells and bacteria. Afterward they are re- 

 moved from the absolute alcohol and kept in 80-90 

 per cent., which does not shrink the tissue. Bichlorid 

 of mercury may also be used, but absolute alcohol seems 

 to answer every purpose. 



For ordinary work the following simple method is 

 recommended : After the sections are cut, the paraffin 

 must be, and the celloidin would better be, removed. 

 From water the sections are placed in the same watery 

 stain used for cover-glasses and allowed to remain five 

 to eight minutes. They are next washed in water for 

 several minutes, then decolorized in 0.5-1 per cent, 

 acetic-acid solution. The acid removes the stain from 

 the tissues, and ultimately from the bacteria as well, 

 so that one must watch carefully, and as soon as the 

 color almost disappears from the sections remove them 

 to absolute alcohol. At this point the process may be 

 interrupted to allow the tissue-elements to be counter- 

 stained with alum carmin or any stain not requiring 

 acid for differentiation, after which the sections are 

 dehydrated in absolute alcohol, cleared in xylol, and 

 mounted in Canada balsam. 



As will be mentioned hereafter, certain of the bacteria 

 which occur in tissue do not allow of the ready penetra- 



