76 LABORATORY MANUAL OF MICROBIOLOGY 



and waxes. After the ether has been removed, the residue is 

 placed in a glass-stoppered flask or bottle and covered with 10 

 cubic centimeters of 18 per cent hydrochloric acid solution and 

 50 cubic centimeters of 72 per cent sulphuric acid solution. 

 The flask is stoppered, shaken, and immediately immersed in cold 

 water. The reacting mixture is allowed to stand for 2 to 3 

 hours, then transferred with 300 cubic centimeters of water to 

 500-cubic centimeter flasks and boiled for 30 minutes. The 

 residue is filtered off upon dried and weighed paper or Gooch 

 crucible, and washed with an excess of water. The residues 

 are then dried at 70°C. to constant weight. Two portions are 

 used for ashing and two for determination of total nitrogen by 

 the Kjeldahl method. (The weight of the dry residue) — (the 

 weight of ash + weight of nitrogen X 6.25) = amount of lignin. 



COMPLETE ANALYSIS OF NATURAL OR DECOMPOSED PLANT 



MATERIAL! 



An approximately complete analysis of a natural organic 

 material is carried out as follows: 



1. Moisture is determined on two 5-gram portions of material. 



2. Total nitrogen is determined on two 2-gram portions of 

 material. 



3. Ash on two 2-gram portions. 



4. Pentosans on two 2-gram portions. 



5. Two 5-gram portions are analyzed as follows: 



(a) Treat for 12 hours with ether in Soxhlets. The ether 

 extract is evaporated to a small volume, then transferred to 

 weighing bottles and dried to constant weight, giving ether 

 soluble fraction. 



(6) Residue from ether treatment is extracted for 24 hours 

 with cold water. Extract is made up to volume and divided into 

 four portions, one to be used for total nitrogen determination, 

 one for determination of total soluble organic matter by evaporat- 

 ing in silica dishes, one for determination of reducing sugar, 

 and one for the determination of ammonia or nitrate. 



(c) Residue from cold water extraction is treated with 100 cubic 

 centimeters hot water, boiling to be continued for 30 to 60 

 minutes. The solution is analyzed as in (b). 



1 Waksman, S. A., and F. G. Tenney, Soil Sci., 24: 317-341 (1927) 26: 

 113-137 (1928). 



