Water-autoclave

Sterile 0.1 M EGTA, pH 7.9—Dissolve 1.9 g of EGTA (ethylene glycol-bis(/3-aminoethyl ether) N, N, N, N -tetraacetic acid) in 35 ml of distilled water, titrate to pH 7.9 with 1 N NaOH, and dilute to 50 ml final volume with distilled water. Autoclave in a capped test tube (20 min at 105-110°C) and cool. 

GTE lysis buffer—Dissolve 1.8 g of D-glucose, 0.606 g of Tris free base, and 0.744 g of disodium EDTA 2H20 in 150 ml of distilled water. Adjust the pH to 8.0 with dilute HC1. Bring the final volume of the solution to 200 ml with distilled water. Autoclave the solution and store at 4°C. If you wish, you may add lysozyme to this solution to a final concentration of 10 pg/ml. We find that this is not necessary. 

M potassium acetate solution—Dissolve 29.4 g of potassium acetate in distilled water to a final volume of 60 ml. Add 11.5 ml of glacial acetic acid and 28.5 ml of distilled water. Autoclave the solution and store at 4°C. 

Bicarbonate 44 g sodium hydrogen carbonate, 8 g sodium chloride/L in high purity water, autoclaved. 

X SSC stock solution 100.2 g trisodium citrate, 175.0 g sodium chloride, 1.0 L double-distilled water, autoclave. 

Phosphate buffered saUne (PBS) 200 g NaCl, 5 g KCl, 5 g KH2PO4,37 g Na2HP04-12 H20,1.0 L double distilled water. Autoclave. 

Low-gelling-temperature agarose Add 0.4 g of low-gelling-temperature agarose to 50 ml water. Autoclave and keep at approximately 50°C. 

Low-gelling-temperature agarose Dissolve 1 g low-gelling-temperature agarose in water. Autoclave for 20 min at 121°C. 

Hot water sterilisers are also called hot water (immersion) autoclaves. In these sterilisers, hot distilled water, freshly filled in the stainless steel steriliser bath and heated in a sanitary heat exchanger is pumped around inside the autoclave and is continuously and with high flow sprayed top down over the load to be sterilised. The hot water autoclave contains a mixture of water and air before and during the sterilisation process no vacuum phase is used (see Fig. 30.5). 

Heating in closed containers in a steam or hot water autoclave is the method of choice for aqueous pharmaceutical products. 

To extract phenolic acids in plant tissues/residues ground plant tissues/residues were extracted with water, EDTA, citrate plus or minus imidazole, KCl, or dibasic sodium phosphate and the water-autoclave procedure (Blum et al. 1992 Blum 1997). For procedures used for " C-labeled phenolic acids see Section 2.2.10... 

Water-autoclave extractions were also much more efficient than neutral EDTA extractions in recovering sorbed/fixed phenolic acids from plant tissues/residues (Blum et al. 1992). For example HPLC analysis of EDTA extracts for wheat stubble collected after a wheat harvest contained no detectable phenolic acid peaks. Water-autoclave extracts of this wheat stubble had 11 distinct peaks. Concentrations for ferulic acid, vanillic acid, p-coumaric acid, and p-hydroxybenzoic acid were 33, 22, 1034, and 47 p,g/g dry weight, respectively (Fig. 3.8). On the other hand water and... 

One hundred days after the addition of 1,000 p.g/g (approximately 5 timol/g soil) ferulic acid to sterile Cecil soil samples, water, neutral EDTA, and water-autoclave extractions recovered 28% (277 tig/g), 37% (373 tig/g), and 32% (322 p.g/g) of the ferulic acid added, respectively, from Cecil A soil samples (3.7% organic matter) and 17% (167 tig/g), 52% (524 tig/g), and 30% (304 p.g/g) of the ferulic acid added, respectively, from Cecil B soil samples (0.2% organic matter Blum et al. 1992). The recovery of femlic acid from soil by neutral EDTA extraction was thus more effective than the water-autoclave extraction. 

We suspect that both neutral EDTA and water-autoclave extraction procedures readily recovered the free phenolic acid fraction. So how effective were they in extracting reversibly sorbed phenolic acids (i.e., sorbed recovered by neutral EDTA or water-autoclave extraction minus free recovered by water extraction) For the 1,000 p.g/g added 723 p.g/g was sorbed by Cecil A and 833 p,g/g by Cecil B. For Cecil A samples, 13% (96 of 723 tig/g) and 6% (45 of 723 p-g/g) of the sorbed ferulic acid was recovered by the neutral EDTA and the water-autoclave procedures, respectively. Of this 96% (92 pg/g) and 55% (25 pg/g), respectively, was utilized by microorganisms over the 30 days (Blum et al. 1992). For Cecil B samples, 43% (357 of 833 pg/g) and 16% (137 of 833 pg/g) of the sorbed ferulic acid was recovered by the neutral EDTA and the water-autoclave procedures, respectively. Of this 100% (357 pg/g) and 97% (133 pg/g), respectively, was utilized by microorganisms over the 30 days. 

In conclusion, the water-autoclave extraction procedure when compared to the EDTA extraction procedure underestimated the total available ferulic acid in the soil by roughly 5% for Cecil A and 22% for Cecil B. In addition to the quantitative difference there also appeared to be a difference in the types of the sorbed ferulic acid recovered. The water-autoclave-procedure recovered some irreversibly sorbed phenolic acids from Cecil A soil since only 55% of the sorbed phenolic acid recovered was utilized by microbes. This difference should not be surprising since the physical and chemical processes of the two extraction procedures, i.e., chelation vs. 

So what were the concentrations of phenolic acids in the Cecil A soil wheat stubble (Triticum aestivum L. Coker 916 )/soybean (Glycine maxL. Deltapine417 ) systems Subsamples taken from wheat stubble/soybean (no-till), wheat stubble tilled under/soybean (conventional-till), and fallow/soybean soil (conventional-till) cores were extracted by the water-autoclave procedure and analyzed for 7 common phenolic acids (ferulic, caffeic, p-coumaric, p-hydroxybenzoic, sinapic, syringinc, and vanillic) and total phenolic acid (Blum et al. 1991). With minor exception, individual phenolic acids were correlated with each other, with the sum of the 7 phenolic acids identified by HPLC analysis, and total phenolic acid as determined by the Folin Ciocalteu s phenol reagent method. 

All total phenolic acid values in ferulic acid equivalence were determined by water-autoclave extraction and the Folin Cicoltaeu s phenol reagent approximately biweekly during the 1992 and 1993 experimental periods. Mean total phenolic acid for the upper 2.5 cm of the soil in 1992 were as follows plots without cover crops (reference plots) 41 1 rg/g soil (mean standard error), crimson clover plots 55 1 JLg/g soil, subterranean clover plots 58 1 p,g/g soil, rye plots 50 1 irg/g soil, and wheat plots 45 1 rg/g soil (Fig. 3.14 Blum et al. 1997). In 1993 soil total phenolic acid values for the upper 2.5 cm of the soil were higher than in 1992 (Fig. 3.14 Blum et al. 1997). The values for the April glyphosate desiccated plots were plots without cover crops (reference plots) 62 2 t,g/g soil, crimson clover plots 83 4 jtg/g soil, subterranean clover plots 83 3 t,g/g soil. 

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