Variations in Bacteria Caused by Change of Medium 7 



ration. Part of this solution was weighed to ascertain the approximate 

 protein content. 



This solution heated to 85 to 90 C., with enough acetic acid to 

 concentration of o.oi per cent, produces a coagulum, which when 

 washed and dried on a weighed filter, is estimated as coagulable 

 protein. The filtrate from the previous operation, after being diluted 

 to o.i per cent NaCl and divided into two parts; one part having 

 been precipitated by HC1 o.i per cent, and the other part by o.i 

 per cent KOH. The precipitates were washed, dried, and weighed 

 as acid-precipitable and alkali- precipitable protein. 



The filtrates from the acid- and alkali-precipitations still gave 

 evidence of containing protein not easily precipitated by mineral 

 acids, alkalis, and neutral reagents. After neutralizing, the protein 

 content of these filtrates was estimated by Esbach's method and listed 

 as soluble proteins. 



The insoluble residue containing a protein which resisted extrac- 

 tion, was subjected to tryptic digestion; the difference in weight before 

 and after digestion was recorded as insoluble protein. 



The substance remaining after the last operation, having given no 

 protein reaction, nor showing the presence of nitrogen, was reserved 

 for other examinations as residue. 



FATS 



Preliminary. Determinations of fat could not be done with accuracy 

 until the known methods were examined. Experiments showed fat to 

 be at least partly intracellular, and consequently total disintegration 

 of the cell necessary. The liberation of fat without its deterioration 

 after cell disintegration was effected by means of alkaline hypochlorite, 

 commercially known as 'antiformin'. 



Method. Weighed quantities of wet bacteria were treated with 

 20. per cent antiformin for 24 hours at 30 C., then warmed to 60 C., 

 for one hour. After cooling to 15 C., and neutralizing with N/io 

 sulphuric acid, it was evaporated to dryness at 80 C., ground, and 

 extracted with ethyl- or petrol-ether. 



This residue was slowly boiled in 10. per cent NaOH, and after 

 boiling was rendered acid with dilute sulphuric. It was then diluted 

 to three times the original volume with distilled water and cooled 

 to 7 to 10 C., after which it was filtered. The precipitate was washed, 

 dried, and extracted for five hours with petrol-ether. 



