particular experiment were inoculated with small sections, 

 approximately 2 inches in length, of the appropriate species 

 removed from continuously maintained stock cultures. 

 Culture periods of 4-5 weeks were necessary to obtain the 

 yields reported. 



To develop macrophytes deficient in the essential trace 

 elements (Mn, Zn, B, Cu , and Mo) it was necessary to use 

 standard procedures for reducing environmental contamina- 

 tion, that is in acid-washing culture and media containers, 

 double-distilling water used to prepare culture solutions, 

 and in purifying culture media salts (Hewitt, 1966; Stout 

 and Arnon, 19 39) . 



Inorganic analyses were by quantitative procedures in 

 general use for plant analysis. Total nitrogen was 

 determined by a semi-micro Kjeldahl procedure. Phosphorus 

 determinations were by a vanado-molybdate yellow-complex 

 procedure following dry ashing of oven-dried plant material 

 at 550°C (Jackson, 1958). Potassium analysis was by emission 

 flame photometry of 1 N ammonium acetate extracts of tissue 

 samples. Tissues were prepared for iron analysis by a 

 combined wet-dry ashing procedure which permits ashing at 

 low temperatures. Iron then was determined as a complex 

 with o-phenanthroline. Following dry ashing and acid 

 solution of the residues, calcium, magnesium, zinc, manganese 

 and copper were determined by atomic absorption using a 

 Jarrell-Ash instrument. Boron was determined as a curcumin 

 complex (Johnson and Ulrich, 19 59) and molybdenum as a 

 complex with thiocyanate following stannous reduction 

 (Johnson and Arkeley, 1954). Both analyses were on dry- 

 ashed tissues. Analyses for sulfur were by turbidimetric 

 measurements of BaSOi* precipitated in HNO3-HCIO4 digests of 

 plant tissues (Blanchar, Rehm, and Caldwell, 1965). 



To minimize contamination, samples analyzed for trace 

 elements and iron were ground with an agate mortar and 

 pestle. When sample size permitted, analyses for the major 

 essential elements were on plant material ground in a 

 Wiley Mill equipped with a stainless steel screen. Other- 

 wise the samples again were ground with a mortar and pestle. 

 In the initial work on this project, critical concentra- 

 tions were established and evaluated from analyses of 

 entire plants. As in agricultural applications, this proved 

 unsatisfactory, particularly in the collection and analysis 

 of plants from lakes in which nutrient supplies were being 

 evaluated. Large portions of plants too often were 

 unhealthy for reasons other than nutrient deficiency; 

 reduced light for example, and were low in nutrient 



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