546 POWDERED DRUGS 



be transferred to the cover-glass. More organisms should be transferred from the 

 pure culture to the test-tube, or more nutrient liquid should be added to that al- 

 ready in the test-tube, until it is found that a single individual of the organism to be 

 studied occurs in each drop taken from the test-tube. If, in inverting the cover- 

 glass over the ring, the drop runs to one edge or spreads out over the cover-glass 

 so that it comes in contact with the ring, the cover-glass is to be washed off and the 

 process repeated until the drop hangs free near the center of the cover-glass. The 

 drop must not be so large that if the organism should sink to the lower surface of 

 the drop it could not be brought into focus with the highest power objective to be 

 used in studying it. The circulation in the plasmodia of myxomycetes may be 

 conveniently studied if a bit of the substratum containing the plasmodium is 

 moistened and placed on the cover-glass, which is then inverted over the ring as 

 before. The preparation is then set aside in a warm, dark place until the 

 plasmodium has grown out over the cover-glass. 



It is sometimes of advantage to place a drop of water on the glass slip within 

 the ring, so that the atmosphere within the cell will be kept quite humid. Instead 

 of the glass or vulcanite ring, thick cardboard may be used. A piece is cut i inch 

 square, if the glass slip is 3 x I inch, and a round or square hole is cut in the center 

 of the cardboard somewhat smaller than the cover-glass to be used. The card- 

 board is sterilized in boiling water, and pressed into position on the slide. The 

 hanging-drop culture is prepared as above described, but no medium is needed to 

 fasten the cover-glass to the cardboard, other than the water with which the card- 

 board is soaked. Thereafter the cardboard should be moistened from time to time 

 as needed. Bits of the plasmodia of myxomycetes, when suspended on the cover- 

 glass of such a cardboard cell or of the glass or vulcanite cell, as above described, 

 will grow out over the cover-glass, and may be studied throughout a protracted 

 period without being disturbed. If a solid nutrient medium is required, a drop of 

 nutrient gelatine may be placed on the cover-glass instead of the drop from the 

 fluid nutrient medium. Under certain circumstances it is of advantage to flatten 

 out the drop of nutrient substance, after the organisms have been planted in it, 

 by placing over it a smaller cover-glass, which, of course, should be so small that 

 when the larger cover-glass is inverted over the ring or piece of cardboard, the 

 border of the smaller cover-glass will not come in contact with the inner edges of 

 the cell. 



Hardening Processes. The hardening of tissues is accomplished by the with- 

 drawal of water from them. This is, in most cases, best accomplished by means 

 of successively higher grades of alcohol, as described elsewhere. 



A quick method of hardening fresh tissues, and at the same time preparing them 

 for immediate sectioning, is to freeze them by the evaporation of ether or the ex-< 

 pansion of liquid carbonic-acid gas. This process requires the use of special appa- 

 ratus, easily obtainable. For an imbedding mass, either a drop of the white of 

 egg, or a thick solution of dextrin in a solution of carbolic acid, I part, water, 40 

 parts, may be placed about the object before freezing. If the dextrine solution 

 is used, it would be better to pump the air from the object while immersed in the 

 solution; then place on the object-holder, pour a small amount of the solution about 

 it, and freeze. This method will answer very well in some cases, when it is desired 

 to prepare a large number of sections quickly for class use, but it can by no means 

 take the place of fixing the material in an appropriate fixative, hardening slowly 

 in alcohol, and imbedding in paraffin or collodion. 



The mucilaginous layer of certain seed coats may be hardened with a 10 per 

 cent, solution of neutral acetate of lead. The sections are cut from dry seeds, hard- 

 ened in the lead acetate, and stained with methyl blue. They are then washed 

 in water and mounted in a 2 per cent, solution of boracic acid. 



