Staining Bacteria in Tissues 149 



directly upon the smear, and no cover-glass used. If the staining 

 has been done upon a cover-glass, it can be mounted upon a slide 

 with a drop of water between, and then examined, though this is 

 less satisfactory than examination after drying it and mounting it 

 in Canada balsam. 



Sometimes the material to be examined is solid or too thick to 

 spread upon the glass conveniently. Under such circumstances a 

 drop of distilled water or bouillon can be added and a minute portion 

 of the material mixed in it and spread upon the glass. 



When the bacteria are contained in urine or other non-albuminous 

 fluid, so that the heat used for fixing has nothing to coagulate and 

 fix the organisms to the glass, a drop of Meyer's glycerin-albumen 

 can be added with advantage, though the precaution must be taken 

 to see that this mixture contains no bacteria to cause confusion with 

 those in the material to be studied. 



The entire process is, in brief: (i) Spread the material upon the 

 glass; (2) dry do not heat; (3) pass three times through the flame; 

 (4) stain one minute; (5) wash thoroughly in water; (6) dry; (7) 

 mount in Canada balsam. 



Fig. 33. Stewart's cover-glass forceps. 



To Observe Bacteria in Sections of Tissue. Hardening. It 



not infrequently happens that the bacteria to be examined are scat- 

 tered among or inclosed in the cells of tissues. The demonstration 

 then becomes a matter of difficulty, and the method employed must 

 be modified according to the particular kind of organism. The 

 success of the method will depend upon the good 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 immersed in absolute alcohol from six 

 to twenty-four hours, to kill and fix the cells and bacteria. The 

 blocks are then removed from the absolute alcohol and kept in 80 

 to 90 per cent, alcohol, which does not shrink the tissue. Solutions 

 of bichlorid of mercury* may also be used and are particularly useful 

 when the bacteria are to be studied in relation to the cells of the 

 tissues. 



* Zenker's fluid: 



Bichromate of potassium 2.5 grams 



Sulphate of sodium i . o 



Bichlorid of mercury. 5.0 



Water 100.0 



At the time of using add 5 grams of glacial acetic acid. Permit the specimens 

 to remain in the solution for a few hours only, then wash for twenty-four hours in 

 running water and transfer to 80 per cent, alcohol. 



