EREMASCACEAE 171 



succeeded easily when pns was used. In 1898 Barucliello apparently also 

 cultivated the same or<>anisni as iMarcone and Tokishi<>'e. In 3 906, 8an Felice 

 succeeded in g-rowing mierocolonies, but was unable to keep them alive very 

 long, although he was able to reproduce the disease. In spite of these seem- 

 ing successes, the next few years saw several hypotheses that the organism 

 was a protozoon, the authors explaining the presence of hyphae in the cultures 

 of earlier workers as contaminations in spite of the observations of San Felice 

 who controlled his studies by microscopic examination. In 1916, Lindner and 

 Knutli renamed the organism Monilia co.psidata. During- the decade of the 

 World War, Boquet and Negre and their coworkers made a very thorough 

 study of the problem and developed methods for its cultivation. The last 

 decade has seen their work confirmed by several German workers. 



In the tissues, cells are spherical or ovoid, or acuminate at the two poles, 

 sprouting, 3-5 x 2.5-3.5(U in diameter, membrane of variable thickness, granular. 

 Occasionally elongate cells seen or larger cells, 5-7/t in diameter. 



In cultures, hyphae 2/i, in diameter, septa about 10-20/u apart, finely granu- 

 lar, no oil globules, branched. Yeast cells pyriform, thin-walled, at first, 

 becoming thick-walled after the cell separates from the parent cell, containing 

 oil globules which may be large as the cell increases in size up to 8-12^ or 

 even 15-16//,. Thick-walled hyphae formed from these yeast cells, 3-4// in 

 diameter, with septa 10-18// apart and oil globules present. Chlamj^dospores 

 from thick-walled mycelium, usually terminal, slightly polyhedral, 10-18//, 

 protoplasm granular without oil globules. In old cultures the mycelium breaks 

 up into somewhat irregular, thick-walled arthrospores. Asci 4-spored. [The 

 ascospores figured by Tokishige were undoubtedly drops of oil.] Everbeck 

 (1926) reports ascospores, thick-walled, ovoid, 3 x 2//, regularly produced 

 after 14 weeks. 



In tissues from animal inoculations, yeast cells 3-4//, or, when actively 

 sprouting, 5-6//, in groups of 10-15, and some thick-walled hyiDhae. The anti- 

 bodies appear about the twentieth day of the disease and remain a long time 

 after cure, so that it is very difficult to inoculate a horse which has had the 

 disease. 



For isolation and early subcultures Boquet & Negre report best results 

 with the following medium : 



Macerate 400 gm. horse dung in 2,000 c.c. of water for 24 hours in a cool 

 place ; strain through cheesecloth, squeeze, filter, and add 10 gm. peptone and 

 18 gm. agar for each 1,000 c.c. of filtrate. Sterilize for 30 minutes at 120° C., 

 add 40 gm. glucose per 1,000 c.c, tube and sterilize for 20 minutes at 115° C. 

 For isolation add 20 drops of the following solution to the tubes after inocu- 

 lation : chop 100 gm. lymphatic ganglions of the horse, macerate for 24 hours 

 in 500 c.c. water, strain through cheesecloth, squeeze, filter, add 20 gm. 

 glucose, tube and sterilize for 30 minutes at 115° C. Moisten cultures with 

 this liquid as they begin to dry out. Incubate at 25-30° C. 



On dung agar, after 4-6 weeks, colonies appear on surface, as small, round, 

 elevated, grayish white, slightly velvety, the size of a pinhead. The colonies 



