Dec. i6, 191S Bacteriological Studies on Alfalfa Silage 573 



glucose agar, to which was added i cc. of a i per cent sterile acetic-acid 

 solution. The acid solution was added directly to the plates and the 

 glucose agar was mixed with it when the plates were poured. Lactose 

 agar was used in place of glucose agar in the preliminary work, but from 

 subsequent work it was observed that the glucose medium favored this type 

 of organisms more than the lactose agar. The small amount of acid 

 added was sufficient to check practically all types of organisms except the 

 Bulgarian group and the yeasts. After a little experience there was no 

 difficulty in distinguishing between these two types, on account of their 

 characteristic colonies. 



Litmus-lactose agar was used to determine total numbers, as well as the 

 acid and neutral types, of microorganisms. 



Bile lactose fermentation tubes were employed in determining colon 

 organisms. The Dunham fermentation tubes were inoculated with 

 dififerent dilutions of the silage extract. The tube with the highest 

 dilution showing gas production was used as an estimate of the total num- 

 ber of organisms of the colon group present. To substantiate further this 

 presumptive test, different dilutions were plated out from time to time, 

 and the organisms of the colon type isolated and identified. 



Glucose fermentation tubes were used in determining the total number 

 of yeasts present by noting gas production in the different dilution tubes. 

 To be certain that the gas was due to yeast fermentation, stained prep- 

 arations were made from each dilution and examined for the presence of 

 yeast cells. If gas was present and no yeast could be dernonstrated, it 

 was taken for granted that the gas formation was not due to yeasts, but 

 to other causes. The yeast count from the acid agar was used to check 

 this method, and they compared very favorably. The general rule, how- 

 ever, was that higher numbers were obtained from the fermentation 

 tubes than from the plates. 



All media used were made from Liebig's beef extract, and a reaction 

 of 4-1.0 to phenolphthalein required. The period of incubation, unless 

 otherwise stated, was always four days at 37.5° C. The long period of 

 incubation was used to favor the complete development of the Bulgarian 

 type. The enumeration of all plates was done by the aid of a hand lens. 



The principal and predominating types of microorganisms as they ap- 

 peared on the dift'erent media from time to time were isolated. A mor- 

 phological, cultural, and biochemical study was made from the organ- 

 isms thus isolated. , 



Stained preparations were made directly from the silage infusion in 

 order to check the results of the cultural analysis. It seemed unneces- 

 sary to employ the use of synthetic media after comparing the micro- 

 scopic appearance of the silage infusion with the results obtained from 

 the culture media, as the media gave a very good estimate of the true 

 microbial content of the silage. 



