Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions

Simplified protocols for ingredients of Aspergillus minimal medium (MM) are presented. These either do not change the final composition of MM or at most involve only minor modifications which, in extensive comparative tests, have not shown any effects on growth patterns of all strains/cultures tested. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol48/iss1/8 20 Fungal Genetics Newsletter Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions Terry W. Hill and Etta Kafer. Department of Biology, Rhodes College, Memphis, TN 38112 USA. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., V5A 1S6, Canada. Simplified protocols for ingredients of Aspergillus minimal medium (MM) are presented. These either do not change the final composition of MM or at most involve only minor modifications which, in extensive comparative tests, have not shown any effects on growth patterns of all strains/cultures tested. ______________________________________________________________________________ I. Improved preparation of Hutner =s trace element (TE) solution, three versions: A. Procedure for Hutner=s TE with full solubility at room temperature and during storage. By first dissolving the Fe-salt and adjusting its pH separately, as follows, there is no need for heating, and occasional problems with precipitation are avoided. To prepare 200 ml of the 1000x TE solution (i.e., 1 ml required for 1 liter of MM) the Astandard@ ingredients and proportions are used, as listed below [from Scott and Kafer 1982 de Serres and Hollaender (eds.) Chemical Mutagens vol. 7 (Plenum) 447-479]. 1. Dissolve the listed salts in 80 ml of distilled water in the order indicated: FeSO4 •7H2O (Ferrous sulphate) 1.0 g EDTA 10.0 g Adjust pH upwards with KOH pellets. A golden yellow solution results above around pH 5.5, and this is sufficient to proceed. 2. Dissolve the listed salts in 80 ml of distilled water in the order indicated: ZnSO4 •7H2O (Zinc sulphate) 4.4 g H3BO3 (Boric acid) 2.2 g MnCl2 •4H2O (Manganous chloride) 1.0 g CoCl2 •6H2O (Cobaltous chloride) 0.32 g CuSO4 •5H2O (Cupric sulphate) 0.32 g (NH4)6Mo7O24 •4H2O (Ammonium molybdate) 0.22 g Combine Solutions (1) and (2), and readjust the pH to 6.5 using first KOH pellets, then KOH solutions of decreasing concentration. Bring the final volume to 200 ml with distilled water, and store at 4-8°C. As with traditionally prepared Hutner=s TE, this solution is initially bright green, turning purple upon storage. Precipitates are never formed. a In general, precipitation, if it happens to occur in Hutner=s TE, will not affect growth, provided noticeable mismeasurement is avoided. In fact, at least for Chlamydomonas, it is rumoured to work better if a precipitate has formed b Alternate protocols for trace element solutions and MM, especially types more suitable for tests of N-metabolism mutants, are presented in this reference (e.g., Scott and Alderson, 1972, Radiat. Bot. 12:45-50) c In all protocols, distilled water can be replaced by nanopure water. B. Modified Hutner=s TE solution: pH established with tetra-sodium-EDTA. This method is simple and easy, and TE will contain the standard concentrations of all critical nutrients. However, there are two differences compared to standard Hutner=s TE: namely, the Na/K balance is changed in favor of Na, and the molarity of EDTA is reduced. (The latter can be corrected, however; see protocol C below.) Published by New Prairie Press, 2017