Staining Bacteria 149 



it making an ordinary cover-slip preparation. Place the slide and block in a 

 37°C. incubator for five to ten minutes to dry slightly. Then lay a clean sterile 

 cover-slip on the inoculated surface of the block in close contact with it, carefully 

 avoiding air-bubbles. Remove the slide from the lower surface of the block and 

 invert 3ie cover-slip so that the agar block is uppermost. With a platinum 

 loop run a drop or two of melted agar along each side of the agar block, to fill 

 the angles between the sides of the block and the cover-slip. This seal hardens 

 at once, preventing slipping of the block. Place the preparation in the incubator 

 again for five or ten minutes to dry the agar-agar seal. Invert this preparation 

 over a moist chamber and seal the cover-slip in place with white wax or paraffin. 

 Vaselin softens too readily at 37°C., allowing shifting of the cover-slip. The 

 preparation may then be examined at leisure." 



With this means of examining the growing cultures, Hill has ac- 

 quired interesting knowledge of the fission and budding of Bacillus 

 diphtherise. 



If the specimens to be examined must be kept for some time at 

 an elevated temperature, some such apparatus as that of Nuttall 

 will be found useful. . 



II. STAINING BACTERU 



In the early days of bacteriology efforts were made to facilitate 

 the observation of bacteria by the use of nuclear dyes. Both carmin 

 and hematoxylin tinge the nuclei of the bacteria a little, but so un- 

 satisfactorily that since Weigert introduced the anilin dyes for the 

 purpose, aU other stains have been abandoned. The affinity be- 

 tween the bacteria and the anihn dyes is peculiar, and in certain 

 cases can be used for the differentiation of species. 



Readers interested in the biochemistry of the subject will do well 

 to refer to the excellent papers by Arnold Grimme,* upon "The 

 Important Methods of Staining Bacteria, etc.," and Marx,t upon 

 "The Metachromatic and Babes-Ernst Granules." 



In this work special methods for staining such bacteria as have 

 pecuUar reactions will be given together with the description of the 

 particular organisms, general methods only being discussed in this 

 chapter. 



Rreparations for General Examination. — For bacteriologic pur- 

 poses thin covers (No. i) are required, because thicker glasses may 

 interfere with the focussing of the oil-immersion lenses. Where 

 cover-glasses are not employed, and the staining is done upon the 

 slide, only the best quaUty of thin glass slides free from bubbles 

 should be used. The cover-glasses must be perfectly clean. It is 

 therefore best to dean a large quantity in advance of use by immers- 

 ing them first in a strong mineral acid, then washing them in water, 

 then in alcohol, then in ether, and finally keeping them in ether until 

 they are to be used. Except that it sometimes cracks, bends, or 

 fuses the edges, a convenient method of preparing cover-glasses is to 

 wipe them as clean as possible with a soft cotton cloth, seize them 

 with fine-pointed forceps, and pass them repeatedly through a small 



* "Centralbl. f. Bakt.," etc., 1902, Bd. xxxii, Nos. 2, 3, 4, and 5. 

 flbid., 1902, XXXII, Nos. 10 and 11, p. 108. 



