Journal of Applied Microscopy. 



tate. Only a few species require a nu- 

 trient solution to coax tlie growth, all 

 the remaining forms growing readily in 

 ordinary spring, or other, water. My 

 best results have uniformly been ob- 

 tained by sowing in pasteboard cells and 

 the success of this cell is apparently due 

 to the retention of moisture within the 

 cell. Another excellent feature of such 

 a cell is the rapidity with which water 

 is carried from the outside to the cham- 

 ber inside, thus removing the necessity 

 for disturbing the culture glass on ac- 

 count of drying. These cells are made 

 as follows: A small sized gun wad-cut- 

 ter is used to make holes in a sheet of 

 pasteboard (which must be not too thick 

 since it will swell when wet to perhaps 

 three times its thickness when dry) and 

 then a larger cutter is used to remove 

 a ring of pasteboard not to exceed an 

 eighth of an inch in width. These rings 

 are cemented to the slide by a thin layer 

 of balsam and when thoroughly dry are 

 ready for use. The cover glass, which 

 should overlap the ring all around, is 

 thoroughly cleaned and a drop of nu- 

 trient solution or water, as the case 

 may be, is placed in the center, the 

 spores sown in it with a needle, and it is 

 then inverted over the pasteboard ring, 

 which has, in the meantime, been soaked 

 full of water. A second slide placed on 

 top of the ring during the soaking pro- 

 cess will prevent uneven swelling of the 

 ring. The culture is now ready for ob- 

 servation, and the myxamoebae may be 

 observed with both low and high powers 

 if care is taken in regulating the size of 

 the drop. Such a cell may be arranged 

 for the study of the effects of any of the 

 normal stimuli as well as of reagents. 

 Cultures made in this way serve admir- 

 ably for the study of the transition of 

 protoplasmic into ciliary motion and 

 vice versa, and they are unequaled for 

 the investigation of the streaming of 

 protoplasm and the contents of the vital 

 substance. In all cases the cells must 

 be carefully protected from contamina- 

 tion with fungi and bacteria, both in 

 the sowing and the later stages of the 

 culture, otherwise their period of useful- 

 ness will be short indeed. Loss from 

 evaporation is easily prevented by keep- 

 ing in a moist chamber when not under 

 observation and by the addition of drops 

 of water to the outside of the pasteboard 

 ring when the cell is exposed to dry air. 

 If the purpose of the germination of the 

 spores is not the study of the myxamoe- 

 bae, but the production of Plasmodia, 

 this end may best be reached by sowing 

 on decayed bark or rotten wood under 

 bell jars where the natural conditions 

 are fully reproduced. Plasmodia may 

 thus be raised at any season of the 

 year and in any quantity. A super- 



abundance of excellent material may 

 thus be prepared for the use of the in- 

 vestigator or for demonstrations to 

 large classes. It is essential to keep 

 even temperatures of from 85 to 100 

 degrees F. in order to secure rapid 

 growth of the cultures. 



TO TRAN.SFER PLASMODIA FROM ANY SUB- 

 STRATUM TO GLASS SLIDES FOR 

 MICROSCOPIC STUDY. 



Plasmodia may readily be transferred 

 to glass slides in a short time in either 

 of the following ways: A clean glass 

 slide is placed against a piece of the de- 

 cayed wood under a bell jar where it is 

 being kept as described in a previous 

 paragraph and supported in such a man- 

 ner that its lower end is in contact with 

 the edge of the Plasmodium. A gentle 

 current of water is now allowed to flow 

 down the slide, either from wash bot- 

 tle as shown in Fig. 1, or by dropping 

 water from a pipette, when the proto- 

 plasm will begin to flow upwards on the 

 slide and soon cover it sufflciently for 

 the purpose of the study. This reaction 

 to a current of water is known as Rheo- 

 tropism and is as remarkable as it is evi- 

 dent. Another, and in many respects 

 similar, method is to place as many 

 slides as desired on a glass plate and on 

 these small pieces of the plasmodium- 

 bearing wood, covering the whole with 

 a bell jar. In the course of a few hours, 

 the time depending on the temperature 

 and other conditions of the environments, 

 the Plasmodia will be found beautifully 

 expanded and in full migration upon the 

 surface of the slides. These may be 

 taken up and immersed in the killing re- 

 agents or used for study by classes oi 

 in original investigations, without fur- 

 ther preparation. This is the easiest and 

 surest way of securing those huge mul- 

 tinucleate amaeboids for the use of 

 classes, and since the supply at any 

 time is limited only by the collections (if 

 in summer) or by the sowings (if in win- 

 ter) there need be no lack of either 

 myxamoebae or Plasmodia in any 

 laboratory. 



THE STUDY OF VITAL PHENOMENA. INTRA 

 VITAM STAINING. 



To stain the plasma in living Plas- 

 modia and myxamoebae. — If the former, 

 get the Plasmodium on the slide in the 

 manner already described and allow it 

 to be spread out and form slender radia- 

 tions. Cover some of these with a suit- 

 able sized cover glass supported on wax 

 feet, after which the stains may be run 

 under the cover. The action is prompt 

 and the colored solution may be readily 

 washed out or replaced with rain or tap 

 water when the preparation is ready for 

 study. 



