BACTERIA WITH MYXOMYCETES. 171 



The presence of aerobic bacteria seems to be necessary to the life of the myxomyeete outside 

 of the host cells. Thus among the great number of tubes of agar sowed with the spores, the greater 

 part of those containing bacteria gave at once a development of the fungus (formation of the amoeboid 

 individuals which soon perished) while in two containing no bacteria, the spores, though perfectly- 

 preserved did not germinate. 



Pinoy thinks, therefore, that the bacteria introduced with the parasite contribute to the decay 

 of the tumor of the cabbage when the conditions are favorable to their multiplication. 



Pinoy continued his interesting studies, publishing a monograph in 1907 wherein he 

 considered at length the relations of various bacteria to several species of Myxomycetes 

 in the Group Acrasieae ; then to several species of the endospore-bearing forms, and finally 

 to Plasmodia phor a brassicae. I summarize as follows: 



Pinoy, like Nadson, found Bacterium fluoresceins liquefaciens associated with Dictyostelium 

 mucoroides. 



On agar poured plates the spores of this slime mold germinate only in the presence of bacterial 

 colonies. Elsewhere the spores do not germinate. But those spores which do not germinate can 

 not for that reason be assumed to be bacteria-free. They also bear bacteria which can be resusci- 

 tated by putting them into bouillon, which clouds after 6 to 8 days. Nadson, Potts, and Vuillemin 

 did not take into account these tardily developing bacteria. 



B. fluoresceins is killed by exposure to 50 C. for 1 hour. On the contrary, 80 per cent of the 

 spores of Dictyostelium mucoroides are still able to germinate after such an exposure. It is possible 

 also to purify the spores by heating them for 2 minutes at 56 C. Spores about 8 days old are most 

 resistant. Spores thus purified will never germinate on any culture medium whatsoever, unless 

 suitable bacteria are added. 



Potts' statement that he was able to grow Dictyostelium mucoroides on the dead bodies of his 

 Bacterium fimbriatum is regarded as illusory, i. e., the purity of his spores is questioned. Pinoy 

 showed the need of living bacteria quite clearly as follows: Into a flask of culture medium he plunged 

 a collodion sack attached to a projecting tube. After proper sterilization, the medium inside the 

 collodion sack was inoculated with purified spores of D. mucoroides, that outside with Bact. fluoresceins. 

 The spores of the Myxomyeete germinated but the amoebae soon rounded off and died. The dead 

 bodies of Bad. fluoresceins (killed by heat, ether and chloroform) were also placed in the collodion 

 sack without result. 



" En resume, le D. mucoroides ne peut vivre qu'en association avec une Bacterie vivante. Toutes 

 les Bacteries ne conviennent pas egalement." 



The culture medium exerts a marked influence, e. g., on potato sowed with mixtures of Diet v. 

 mucoroides and Bad. fluorescens the slime mold does not develop. The same is true on this medium 

 whatever bacterium isused. With most bacteria this is also true on peptone agar, or meat broth agar. 

 The best culture medium found was flax-seed agar. On this the bacteria grow abundantly and the 

 harvest of Dictyostelium mucoroides reaches its maximum. 



Bacteria that stain by Gram are not suitable for such cultures. In general, bacteria which do 

 not stain by Gram allow the Dictyostelium mucoroides to grow. The Dicty. mucoroides will not grow 

 in the presence of pure cultures of Bacillus megaterium, but if this organism is added to Dictyostelium 

 mucoroides with Bad. fluorescens growth may be had on beef agar. 



The growth of the Dictyostelium mucoroides may be regarded as a parasitism on the bacteria. 

 They are absorbed into the vacuoles and digested. Pinoy confirms Metchnikoff's observation that 

 the liquid in the vacuoles is acid. 



Neutral red is recommended as a stain for the bacteria in process of digestion. It does not 

 stain the living bacteria nor kill the Myxomyeete unless too strong. Vesuvin may also be used. 

 For details see paper. Potts did not find bacteria in the interior of the vacuoles of Dicty. mucoroides 

 because his technic of fixation and staining was insufficient, he therefore formed the erroneous 

 hypothesis of an extra-cellular diastase. 



Grown with Bacillus coli, the enzyme isolated at the end of 40 hours liquefied gelatin. It acts 

 in neutral or feebly alkaline liquids. The acidity of methyl orange inhibits. It is therefore related 

 to trypsin rather than to pepsin. It is destroyed at 55 C. Its maximum of activity is about 38 C. 



It has scarcely any action on fibrin or on albumen coagulated by heat. 



" Acradiastase " does not act on bacteria killed by heat, but readily dissolves those killed by 

 ether or chloroform. 



"The best bacterial test is also B. coli which is not self-autolytic, and a chloroformed emulsion 

 of which remains cloudy. 



