APPENDIX. 411 



the groove), the cover glass, after being thoroughly heated in the flame, is 

 allowed to cool ; a ring of vaseline is painted on the slide around the 

 groove ; on to this the cover glass is lowered, where it compresses the drop 

 of culture fluid, which thus presents a flat surface and allows of the 

 development of the bacteria being exceedingly well followed. The groove 

 around the disc forms a kind of air chamber. If it is required that the 

 oxygen should be absorbed this groove may be partially filled with the 

 alkaline pyrogallic acid solution. 



Various modifications of the hanging drop culture method are described. 

 Buchner dries the spores upon a sterilized cover glass, then places on this 

 a drop of his culture fluid and inverts the cover glass ; fragments of cover 

 glass are then arranged around the drop so as to support the cover glass on 

 which it is hanging and the whole is cemented down to form a perfectly 

 air-tight cell in which the development of the organisms may be readily 

 watched. 



Salomonsen describes a moist chamber constructed of a thin sheet of card- 

 board which is sterilized by boiling, and is rendered so soft that it fits 

 accurately to the slide and allows of the cover glass with its hanging drop 

 of inoculation fluid being pressed down so as to form an air-tight chamber ; 

 the moisture from the cardboard serving to prevent the evaporation of the 

 fluid. It is difficult to keep these cardboard supports sufficiently moist and 

 sterile without the use of somewhat complicated moist sterilized chambers. 

 The best way of keeping them, however, is under bell jars, the air of which 

 is saturated with moisture. These hanging drop cultures may be dried, and 

 the organisms are then stained as in the case of an ordinary cover glass pre- 

 paration. Watson Cheyne has utilized this method in a most ingenious 

 manner for obtaining a permanent record of the various phases of develop- 

 ment of micro-organisms. He makes a series of hanging drop cultures of 

 any organism that is to be studied, and then dries and stains them, 

 taking them at stated intervals, say, five, ten, fifteen, twenty minutes, and 

 so on through a whole series. In this manner the whole cycle of develop- 

 ment may be watched and referred to again and again, especially if different 

 series of these cultures be grown at different temperatures. 



METHODS OF STAINING BACTERIA. 



Methylene Blue. 



Methylene blue may be kept as a saturated alcoholic solution ; a few 

 drops of this filtered into water will give a very beautiful stain in cover 

 glass preparations. The sputum, &c., on the cover glass, after being dried 

 and passed three times rapidly through a spirit lamp or Bunsen flame (see 

 page 206), is floated on the surface of the methylene blue, it is allowed to 

 stain for five or ten minutes, washed in water (sometimes first in alcohol), 

 tilted on edge and allowed to dry. It is then mounted in xylol balsam. 



Kiihne's Methylene Blue Method. 



One of the best general stains for bacteriological work is Kiihne's methylene 

 blue solution. 1.5 grammes of methyl blue is dissolved in 10 cc. of absolute 

 alcohol and 100 cc. of a I to 20 watery solution of carbolic acid. Specimens 

 are stained in this for from five minutes to two hours, although sections 

 may be left in it for a whole day without becoming overstained. They 

 are then carefully washed in water, then with acidulated water made by 

 adding a couple of drops of hydrochloric acid to 100 cc. of water. As soon 



