6l6 LABORATORY EXERCISES 



Germination on Solid Media. — Observing precisely similar technique a few drops 

 of liquefied gelatine or agar may be run over the surface of the cover-slip and a 

 hanging-drop plate cultivation thereby prepared. After sealing down the prepara- 

 tion it may be set aside and the growth watched at definite intervals under the 

 microscope. 



Dilution Method to Obtain Material for Inoculating Hanging-drop Media. — In 

 the case of yeast this problem was solved by Hansen, who developed the method to 

 such a degree of perfection as to create, in fact, an exact method (1881). He 

 employed dilution with water. The yeast developed in the flask is diluted with an 

 arbitrary amount of sterilized water, and after vigorous shaking, the number of 

 cells in a small drop of liquid is determined. The counting, in this case, is effected 

 in a very simple manner by transferring a drop to a cover-glass, in the center of which 

 some small squares are engraved and this is then connected with a moist chamber; 

 the drop must not be allowed to extend beyond the limits of the square. The cells 

 present in the drop are then counted. Suppose, for instance, that ten cells are 

 found: a drop of similar size is transferred from the liquid, which must first be 

 shaken vigorously, to a flask containing a known volume of water, e.g. 20 c.c. of 

 sterilized water. This flask, then, will in all likelihood contain about ten cells. If 

 it is then vigorously shaken for some time until the cells are equally distributed in 

 the water, and then i c.c. of the liquid introduced into each of twenty flasks contain- 

 ing nutritive liquid, it is probable that half of these twenty flasks have received one 

 cell each. But, here again, as in Lister's experiments, it is entirely a calculation 

 of probabilities. If the flasks are allowed to stand for further development of micro- 

 organisms, there will be a chance of getting a pure culture in some of them. Hansen 

 succeeded, however, in adding a new factor, which first gave certainty to this experi- 

 ment. Thus, if the freshly inoculated flasks are vigorously shaken, and then left in 

 repose, the individual cells will sink to the bottom and be deposited on the walls of 

 the flask. It is self-evident that if a flask contains, for instance, three cells, these 

 cells will always, or at least in the great majority of cases, be deposited in three 

 distinct places on the bottom. After some days, if the flask is raised carefully, it 

 will be observed that one or more white specks have formed on the bottom of the 

 flask. If only one such speck be found, we have a pure culture by the dilution 

 method. 



Method of Preparing Squared Cover-glasses.- — Since such cover-glasses are some- 

 what expensive and can be easily etched, the method of their preparation is de- 

 scribed below. A little paraffin or wax is melted in a saucer and the cover-glass 

 dipped into it, being held at one corner by a forceps; it is taken out quickly and as 

 much as possible of the melted parafiin is allowed to run off, leaving on either side a 

 thin cover of parafiin which is allowed to harden. By a very fine needle and a small 

 ruler the required lines are then scratched on the wax, and the cover-glass immersed 

 for a moment in hydrofluoric acid which should be poured into a platinum crucible 

 or dish. The paraffin can now be dissolved off in xylol, leaving the surface etched 

 with the squares used in making bacterial, or fungous spore counts (Fig. 217). 



These squared covers may be raised above the slide, while the count is being made, 

 either on four pillars of paraffin, or in a moist chamber. 



