FUNGUS OCCURRING IN PULVINARIA INNUMERABILIS. 307 



CHANGES IN THE SYMBIONTS AFTER HIBERNATION. 



Our observations on these are very fragmentary as our available 

 time at this season was occupied with the cultural experiments 

 described below. It appears quite certain, however, that the oval 

 symbionts of the early spring Pnhnnaria nymphs undergo further 

 multiplication and morphological changes in late April, and dur- 

 ing May. A cursory examination of specimens about May 10 

 showed the symbionts in groups sometimes forming short strings, 

 somewhat similar to the typical mycelium which was obtained in 

 cultures. The picture at this time indicates cmite clearly the 

 fungous nature of the yeast-like organisms just described, found 

 before spring growth begins. 



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ISOLATION OF THE FUNGUS FROM PULVINARIA. 



Our first attempts to cultivate in artificial media the yeast-like 

 cells present in Puh'inaria were made in early April. The over- 

 wintered, partly grown scales were brought into the laboratory 

 still attached to the maple twigs on which they occur. The scales 

 can readily be detached from the twig by means of a sterile 

 needle and allowed to fall upon a sterile microscope slide. The 

 scales thus removed, were treated in several ways as experience 

 had taught us that material of this sort is very apt to be contami- 

 nated on the surface by various microorganisms. Some were 

 treated with 85 per cent, alcohol for a few minutes, others rapidly 

 passed through the flame of a Bunsen burner, and others were 

 used without treatment, except to avoid contact with any un- 

 sterile object. Our first media were potato agar and " sugar 

 agar " which consisted of potato agar to which varying amounts 

 of maple sugar (2 l / 2 per cent.- 20 per cent.) had been added. 

 These tubes are readily inoculated by crushing the scale insect 

 between two microscope slides and streaking the agar with a loop 

 dipped in the body-juices thus extracted. It is also easy to drop 

 the whole insects into culture tubes and then crush them on the 

 agar by means of a dissecting needle. From a large series of 

 tubes inoculated according to these methods, nearly one half 

 showed after three days a good growth, white in color, spread- 

 ing on the surface of the media. These colonies appeared to de- 



