EXPERIMENT STATION REPORTS. 181 



From the foregoing tests it is reasonable to conclude that Ever Fresh Milk" 

 while verj'- low in germ content cannot be said to be sterile. 



IV. ■Examination of Spoiled Beet Sugar for Microorganisms. 



Just before going on vay vacation last summer a sample of beet sugar was 

 submitted to me for analysis for microorganisms by a sugar company. The 

 sugar was sticky or waxy in appearance. It was stated by the company's 

 chemist that the sugar contained invert sugar and the question was 

 whether there were organisms present which could invert sucrose. 



Some of the sugar was dissolved in sterile water and tfhen centrifuged. 

 After centrifuging, the sediment was examined microscopically. This ex- 

 amination revealed some bacteria and much debris of a mold-like character, 

 i. e., mold spores and fragments of mold hyphae. The sugar was then plated 

 out by the loop dilution method and the plates turned over to Dr. Snyder, 

 who also made some further cultural examinations of the sugar. He isolated 

 four species of molds and three species of bacteria from the sugar, the most 

 predominant species found being a Penicillium species capable of producing 

 acid from sucrose. He was of the opinion that the acid caused the inversion 

 (jf the sugar. Of course, the presence of molds, in itself, could account for 

 the inversion since many species of molds contain invertase. Kopeloff has 

 shown that even mold spores, without the presence of hyphae, are capable of 

 inverting sucrose. 



It developed later that this sugar was from a shipment which was con- 

 signed to a warehouse for stornge and which became sticky and then caked. 

 The warehouse people submitted a sample of the sugar in a sugar sack. This 

 f;ontained some hard lumps and some apparently unchanged sugar. Starting 

 with a 10 gram sample of the caked sugar, it was plated out in dilutions of 

 1/10 to 1/10,000. A count of 60 bacterial colonies per gram was obtained. Only 

 one Penicillium colony and one Aspergillus colony developed on the 1/10 dil. 

 plates, the jDlates of the other dilutions being free of mold growths and 

 practically free of bacterial colonies. 



Some of the sticky sugar, which Ijad been held over from last summer in 

 a sterile petri plate was also plated at the same time. A count of 2,000 

 molds per gram and 77,000,000 bacterial colonies per gram was obtained. 

 The molds were identified as Penicillium species and Aspergillus species. 

 The bacterial colonies consisted of apparently only one species. This formed 

 a small round, white, glistening surface colony and a small, yellowish, lens- 

 shaped, sub-surface colony. The colonies consisted of small rods, many of 

 which contained oval spores placed near one end of the sporangia, the spores 

 being about twice the width and taking up most of the length of the sporangia. 



Experiments were also conducted with some of the sugar which had not 

 caked to determine how soon caking would take place after wetting and also 

 if heavily inoculating ^vith the microorganisms isolated above affected the 

 results appreciably. For this purpose some of the uncaked sugar was placed 

 in small sacks made of cheese cloth, and the sugar dipped into distilled water 

 until thoroughly wet when they were hung up to dry, some at room tem- 

 perature and some at 37°C. Another set was also prepared in the same way, 

 but instead of being dipped in distilled water, they were dipped in distilled 

 water containing the growths — mold and bacterial— from the plates from 

 which the Aspergillus, Penicillium and the peculiar spore forming bacillus 

 were isolated. The following scheme will illustrate the test: 



