Journal of Applied Microscopy. 



97 



over night. The following- day the water 

 is poured off. filtered, diluted with fresh 

 water until it is of a white wine color, 

 and two per cent, of milk is added to the 

 infusion. It is then filtered, put into a 

 flask, and sterilized for future use. The 

 macerated hay is cut and placed in 

 Erlenmeyer flasks; the first portion is 

 cut short enough so as to form a toler- 

 ably compact layer in the bottom of the 

 flask to the depth of one centimeter; 

 the rest is cut sufficiently long to form 

 a very loose layer reaching about two- 

 thirds the way up the sides of the flask, 

 care being taken not to let any of the 

 stems reach the cotton. Sufficient water 

 is placed in the flasks to cover the hay, 

 and they are sterilized for fifteen min- 

 utes. On the following day fresh water 

 is substituted, and they are again steril- 

 ized. The water is once more poured 

 off, and enough of the hay infusion and 

 milk previously prepared is added until 

 it is about one centimeter deep. The 

 flasks are then sterilized in a steam ster- 

 ilizer for ten minutes on three succes- 

 sive days. They are then ready for use. 



After soaking the hay for twenty-four 

 hours in water, and boiling it several 

 times in fresh water, about all of the 

 soluble substance has been extracted, 

 and the diluted hay infusion with two 

 per cent, of milk is added. We thus 

 have a medium of tolerably uniform 

 composition. 



Of the cultures gotten from the air 

 several contained mould fungi, which 

 were eliminated by putting the cultures 

 in the oven at a temperature of 37°C. 



One culture contained Chroococci, and 

 these were eliminated by keeping a 

 series of cultures in a dark closet. It is 

 not possible in every case to eliminate 

 other protozoic forms that may be pres- 

 ent, but one may at times succeed by 

 taking advantage of the fact that the 

 encysted forms withstand drying. In 

 this way one may sometimes succeed in 

 separating mycetozoa from the infusoria, 

 amoebae, and other protozoic forms 

 found in hay infusions. 



The cultures are usually transplanted 

 by means of a sterilized pipette. The 

 Plasmodia form on the hay at the sur- 

 face of the water or on the glass. Pre- 

 vious to forming the sporangia, they 

 crawl up above the surface of the water, 

 sometimes (Physarum) covering the 

 entire inner surface of the flask, but 

 not extending over upon the cotton plug. 

 The sporangia of the various species 

 formed in from twelve hours to thirty 

 days. The sporangia are more numer- 

 ous when many, rather than few, stalks 

 of hay project above the surface of the 

 infusion. "As a rule, but one set of 

 sporangia developed in the same cul- 



ture. Sporangia do not develop in all 

 cultures; at times large Plasmodia form 

 on the hay and degenerate without 

 forming sporangia." The formation of 

 sporangia is favored by keeping the 

 cultures in the light. 



As to the manner in which the large 

 Plasmodia are formed, the author states 

 that while he does not question the 

 "accuracy of the observations of such 

 competent observers as Cienkowski. 

 Strasberger, Lister, and others," who 

 have seen, figured, and described the 

 fusion of zoospores, he himself has seen 

 nothing of the kind, though he has at 

 times watched on the slide the coales- 

 cence of fairly large Plasmodia into 

 still larger ones. Referring to this point 

 he says, "What takes place in the cul- 

 ture seems to be as follows: the bacteria 

 multiply at the expense of a portion of 

 the nutrient material; the zoospores 

 multiply at the expense of the bacteria; 

 and possibly some nutrient material 

 which was not consumed by the bacteria; 

 the majority of the zoospores encyst, 

 small Plasmodia develop from a single 

 zoospore or by the fusion of several 

 zoospores; the Plasmodia take in and 

 digest active and encysted zoospores 

 and bacteria; finally, the small Plasmodia 

 fuse to form the large Plasmodium." 

 The author saw the zoospores capture 

 bacteria and particles of carmine by the 

 stroke of a flagellum, which threw them 

 into a funnel-like depression in the 

 protoplasm, the sides of the depression 

 then closing, thus forming a vacuole. 

 The fission of the zoospores was also 

 seen. For microscopic study Plasmodia 

 were placed on the slide in a few drops 

 of the infusion and covered with a 

 cover glass supported by wax feet. They 

 may be fixed and hardened on the slide 

 by using picric and acetic acids, and 

 stained in picro-carmine. 



Charles Wright Dodge. 



University of Rochester. 



Staining Yeast.* 



Stain for fifteen minutes in alum- 

 haematoxylin, wash in water, and then 

 stain with very dilute phenol fuchsin 

 (one part Ziehl's Solution, twenty parts 

 distilled water) for thirty minutes to 

 twenty-four hours. After this, decolor- 

 ize and dehydrate for fifteen seconds to 

 one minute in alcohol, then ninety-five 

 per cent, followed by absolute alcohol, 

 xylol, and balsam. 



* O. Busse, Central-BI. Bakt. u. Par., 

 XXII., P. 349. 



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