Micro-organisms of Maple Sap 493 



increased in amount for one or two days, but began to diminish 

 on the fourth day and was entirely gone on the sixth daw Mean- 

 time the milky appearance of the sap remained unchanged. A 

 slimy consistency developed and the material sometimes became 

 stringy, but the pronounced ropy character described under 

 cultures in unsterilized sap did not appear. 



Unsterilized sap was employed in a large number of the 

 inoculation experiments. Care was taken to procure this fresh 

 from the trees and as slightly contaminated as possible by bits of 

 dust, bark, and other extraneous matter, in order that the natural 

 inoculations should be minimized. In the first series a young 

 culture in beef broth was employed for the inoculations, about 5 

 cc. of culture being added to a liter of sap ; in other series vary- 

 ing small amounts of broth culture were employed, and in still 

 others pure cultures upon sterilized sap were used to produce the 

 inoculations. The cultural characters were invariably the same, 

 except that the heavier inoculations produced the results more 

 quickly. The sap took on a deep milky appearance but developed 

 no pellicle. A slime was deposited upon the walls of the con- 

 taining vessel but the gummy consistency of the deposit described 

 under sterilized sap was not observed. Moreover the liquid 

 developed a ropy character which was very pronounced after 24 

 to 48 hours and persisted for the two weeks that the cultures were 

 held under observation. The character of the culture produced 

 by inoculation with the organism after it had been cultivated in 

 the laboratory for one year was exactly similar to that observed 

 in the original material from which the organism was isolated. 

 The maximum reaction observed in unsterilized sap in which 

 this organism was cultivated for three days was h : 7 Fuller's 

 scale. Before inoculation the reaction was +.3. Associated or 

 ganisms, when introduced in maple sap in such quantities as (<> 

 cause an overgrowth, produce a reaction of -f-.6 to -|-i. 



Artificial maple sap was prepared from maple sirup as fol- 

 lows : 25 cc. of sirup, 475 cc. of water and 25 cc. of nutrient 

 bouillon were mixed, heated to boiling, filtered through filter 



