Mutagen isolation methods

These possibilities could be due to a parameter inherent in a given method. Among these three isolation methods, the differences in parameters include pH, temperature, polarity of extraction solvents, moisture, and particle size. Any single parameter or combination of parameters could be affecting the yield in mutagen levels recovered from the sludge samples. Therefore, a series of experiments was conducted to examine possible effects of these parameters. 

In our laboratory we have been using M2 seed families and mutagenized haploid protoplasts of Nicotianaplumbaginifolia in a combined approach to search both for mutants resistant to toxic concentrations of auxins and cytokinins, and for temperature-sensitive auxin auxotrophs. The hope has been that variation isolated using these rather unspecific selection procedures would include mutations in hormone uptake, biosynthesis, catabolism and reception. The isolation methods, particularly for the cloning of auxotrophs, are laborious, and during the last five years we have slowly assembled a small collection of mutants which we feel will allow us to test several current theories about auxin and cytokinin synthesis and mode of action. This paper briefly reviews our work on the mutants and their characterization. 

Epler, J. L., The Use of Short-Term Tests in the Isolation and Identification of Chemical Mutagens in Complex Mixtures, in Chemical Mutagens Principles and Methods for Their Detection (F. J. DeSerres and A. Hollander, Eds.), Vol. 6, pp. 239-270, Plenum, New York, 1980. 

The liquid-liquid, acid-base fractionation method was preferred over HPLC by the liquid waste panel members because it is a better validated technique for the isolation of components that are toxic or that might otherwise interfere with the assay. Also, the distribution of activity in the acid, base, and neutral fractions provides a preliminary estimate of the types of chemicals responsible for the mutagenicity. 

New Methods for the Isolation of Mutagenic Components of Organic Residuals in Sludges... 

Effects of pH on the Recovery of Mutagenic Activity. The effect of pH, characteristic of the modified EMSL method, was examined in the following experiment. Residue organics extracted from a sample of primary sludge first were isolated via the milling procedure and then recombined in proportions representative of the original sample. An aliquot (ALIQ-I) of this reconstituted sample was bioassayed. A second aliquot (ALIQ-II) was spiked into aqueous base of pH 11. Then, the... 

Table II. Effects of Method of Moist Pellet Processing on the Isolation of Mutagenic Residue Organics from Secondary Sludge...

---