i6 THE CHEMICAL STRUCTURE OF BACTERIA 



kinds of bacterial proteins, for the methods are not sensitive enough and do not give 

 unconditionally reliable results; nor do they permit generalization. At least worthy of 

 mention, however, is the hydraulic-press method which produces from moist, living 

 bacterial cultures, without deep-seated changes in the cell substance, protein-rich 

 fluids. For example, in order to obtain in this way the protein material of the cholera 

 vibrio, colonies of this organism are grown on nutrient agar; the thick, growing layer 

 of vibrios is carefully lifted off by means of a platinum spatula; the colonies thus ob- 

 tained are finely pulverized with diatomaceous earth and quartz sand in a mortar and 

 through the addition of salt solution worked into a pulpy mass. This is thrown into a 

 strong filter cloth and laid under the hydraulic press. By a pressure which is gradually 

 increased to 400 or 500 atmospheres a press fluid is obtained which comes through the 

 thick filter at first as a light, clear, protein-rich liquid, later changing its color in the 

 air to yellow and brownish— the so-called "vibrio plasma." As the chemical investiga- 

 tion shows, the plasma is largely precipitable with acetic acid in the cold; it does not 

 dissolve with excess of acetic acid, i.e., it acts like a nucleoprotein. In other respects 

 the expressed fluid gives the usual protein reactions. 



With the help of a number of color reactions the protein chemical differentiation 

 of many kinds of bacteria can be carried out. The best known in this connection is the 

 usual color method of Gram, which depends on the fact that salts of pararosanilin 

 (for example, gentian violet and methyl violet) unite firmly with iodine and that 

 certain kinds of bacteria take the color compounds thus formed very firmly, i.e., when 

 treated with alcohol later, they do not so readily give up the color substance they have 

 taken as do certain other bacteria. In consequence, bacteria may be separated into 

 those "colored by Gram's method" (gram positive) and "not colored by Gram's 

 method" (gram negative). The differential behavior of bacteria in the color test of 

 Gram is due to an unexplained difference in the physico-chemical structure of the 

 protoplasm.^ 



In analogous ways certain protein or protein-like cell constituents can be detected 

 in the bacteria. Among these may be included the reserve material (volutin) stored 

 up in many bacteria in the form of colorless and strongly light-refracting spheres, vis- 

 ible under the microscope, the presence of which, for example, is utilized practically 

 in the usual bacteriological tests for the diagnosis of diphtheria. Volutin is dissolved 

 by warm water (30°) within two or three days. In water at 80° C. volutin is dissolved in 

 five minutes. Volutin fixed with heat or alcohol (or formol) is insoluble in boiling 

 water. A 5 per cent or saturated aqueous solution of sodium carbonate dissolves volu- 

 tin in five minutes, as does also caustic potash. A freshly prepared solution of Javelle 

 water dissolves volutin in five minutes. A 5 per cent sulphuric or hydrochloric acid 

 solution dissolves volutin in from five to ten minutes; a 25 per cent nitric acid solution 

 dissolves it at once; and a i per cent acetic or osmic acid as well as a 5 per cent car- 

 bolic acid solution dissolves it slowly. On the other hand, alcohol, ether, chloroform, 

 and carbon tetrachloride do not dissolve volutin. Potassium iodide-iodine solution, 

 Millon's reagent, vanillin hydrochloride, and zinc chloride-iodine give no reaction 

 with volutin; and trypsin and pepsin are also without effect. Moreover, dyestuffs also 

 give a characteristic behavior with volutin. Methylene blue and carbol-fuchsin in 10 



' See chap, iii (by Dr. Churchman) in this volume. 



