78 LABORATORY MANUAL OF MICROBIOLOGY 



HUMUS DETERMINATION 1 



Place six 50-gram samples of soil, previously well mixed and 

 sieved through a 1-millimeter sieve, into six 500-cubic centimeter 

 beakers. Add to each 50-cubic centimeters of 2.5 per cent NaOH 

 solution. Place beakers in autoclave and heat for 30 minutes 

 at 15 pounds pressure. Add 50-cubic centimeter portions of 

 cold distilled water to each beaker, and filter the dark-colored 

 solution through folded filter paper. After draining off all the 

 dark solution from the soil, add again fresh portions of 50-cubic 

 centimeters of 2.5 per cent NaOH solution to the same soils 

 previously extracted, and heat again as before. Add water and 

 filter upon the same papers. Wash soil with 50-cubic centimeter 

 portions of hot distilled water. 



The combined filtrates are collected in 500-cubic centimeter 

 Erlenmeyer flasks and treated with a hot solution of hydrochloric 

 acid 1:1, until a heavy precipitate is formed; add half as much 

 more acid as was required to form the precipitate; warm and 

 shake well. Filter off precipitates (a-fraction of soil organic 

 matter or ''humic acid") through filter papers, previously 

 weighed and dried, or through Gooch crucibles. Wash with 

 warm 10 per cent hydrochloric acid, then a number of times with 

 distilled water. Dry the precipitates at 65 to 70°C. for 24 hours 

 and weigh. Three portions are used for ash determination and 

 three for total nitrogen determinations. If the precipitation 

 and washing with acid have been thorough, the ash content should 

 not exceed 1 to 2 per cent. 



The filtrate from the acid precipitate is now treated with a 5 

 per cent solution of NaOH, until just neutral to litmus or until 

 solution has reached a pH of 4.8 to 5.0. A heavy precipitate 

 will be formed in case of mineral soils. The precipitate (j8- 

 fraction) is filtered off through a series of fresh papers, previously 

 dried and weighed, or through Gooch crucibles. These precipi- 

 tates are washed thoroughly with distilled water, dried and 

 weighed. Three portions are used again for ash and three for 

 nitrogen determinations. The nitrogen content of an aliquot 

 portion of the filtrate from the second precipitate is also 

 determined. 



Tabulate results. 



1 Waksman, S. a., Soil Sci., 22: 221-232, 1926; Springer, U., Zischr. 

 Pflanzenern, Dung. Bodenk., IIA: 313-359, 1928. 



