{"id":1277,"date":"2013-04-03T13:55:51","date_gmt":"2013-04-03T17:55:51","guid":{"rendered":"http:\/\/www.ambrosiasymbiosis.org\/labprotocols\/testing-fungi-for-cellulose\/"},"modified":"2015-10-19T08:47:11","modified_gmt":"2015-10-19T12:47:11","slug":"testing-fungi-for-cellulose","status":"publish","type":"post","link":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/testing-fungi-for-cellulose\/","title":{"rendered":"Testing Fungi For Cellulose"},"content":{"rendered":"

Assays<\/span><\/p>\n

Filter paper assay<\/h2>\n

Testing for utilization of pure unaltered cellulose<\/b><\/p>\n

\n
    \n
  • control-noble (neg. control)<\/li>\n
  • filter paper<\/li>\n
  • PDA (pos. control)<\/li>\n<\/ul>\n<\/blockquote>\n

    CMC & Congo Red assay<\/h2>\n

    Testing for cellulose-degrading enzymes<\/b><\/p>\n

    \n
      \n
    • control-salts (neg. control)<\/li>\n
    • CMC-salts<\/li>\n
    • CMC-PDB_0.25<\/li>\n
    • dextrose-salts<\/li>\n
    • PDA (pos. control)<\/li>\n<\/ul>\n<\/blockquote>\n

      MCC assay<\/h2>\n

      Testing for cellulose carbon utilization (i.e., growth)<\/b><\/p>\n

      \n
        \n
      • control-noble (neg. control)<\/li>\n
      • MCC-salts<\/li>\n
      • MCC-PDB_0.25<\/li>\n
      • PDA (pos. control)<\/li>\n<\/ul>\n<\/blockquote>\n

        Media needed (all media per 1L, all with 20ml\/L Penicillin&Streptomycin):<\/p>\n

        \n
          \n
        • control-noble: 15g noble agar, salts* 0NS<\/li>\n
        • control-salts: 15g normal agar, salts* 0S<\/li>\n
        • MCC-salts: 5g MCC, 15g noble agar, salts* MNS<\/li>\n
        • MCC-PDB_0.25: 5g MCC, 0.25g PDB, 15g regular agar M-green<\/li>\n
        • CMC-salts: 5g CMC, 15g normal agar, salts* CS<\/li>\n
        • dextrose-salts: 5g dextrose, 15g normal agar, salts*<\/li>\n
        • CMC-PDB_0.25: 5g CMC, 0.25g PDB, 15g regular agar C-green<\/li>\n
        • filter paper: piece of autoclaved Whatman filter paper 15g regular agar, salts* 0S<\/li>\n<\/ul>\n

          salts*<\/h3>\n

          (prepare 10x solution ahead= 10X of the following per 1L, autoclave):<\/b><\/p>\n\n\n\n\n\n\n\n\n\n\n
          Salts<\/th>\nAmount<\/th>\n<\/tr>\n
          KH2PO4<\/td>\n0.2 g<\/td>\n<\/td>\n<\/tr>\n
          NH4Cl<\/td>\n0.25 g<\/td>\n<\/td>\n<\/tr>\n
          KCl –<\/td>\n0.5 g<\/td>\n<\/td>\n<\/tr>\n
          CaCl2<\/td>\n0.15 g<\/td>\n<\/td>\n<\/tr>\n
          NaCl<\/td>\n1 g<\/td>\n<\/td>\n<\/tr>\n
          MgCl<\/td>\n0.6 g (or 1.2g if hydrated)<\/td>\n<\/td>\n<\/tr>\n
          K2SO4<\/td>\n2.84 g<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

          include in media through filter after autoclaving: 1ml of 1000x trace minerals solution 10ml of 100x trace vitamins solution<\/p><\/blockquote>\n

          Media based on Czapek-Dox<\/h2>\n

          Czapek-Dox-CMC (cellulose degradation test)<\/b><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
          Ingredient<\/th>\nAmount<\/th>\n<\/tr>\n
          penicillin & streptomycin<\/td>\n20 ml of 100x stock\/L<\/td>\n<\/td>\n<\/tr>\n
          KH2PO4<\/td>\n1g<\/td>\n<\/td>\n<\/tr>\n
          MgSO4*7H2O<\/td>\n0.5g<\/td>\n<\/td>\n<\/tr>\n
          KCl<\/td>\n0.5g<\/td>\n<\/td>\n<\/tr>\n
          FeSO4*7H2O<\/td>\n0.01g<\/td>\n<\/td>\n<\/tr>\n
          cellulose<\/td>\n30g<\/td>\n<\/td>\n<\/tr>\n
          NaNO3<\/td>\n2g<\/td>\n<\/td>\n<\/tr>\n
          noble agar<\/td>\n20g<\/td>\n<\/td>\n<\/tr>\n
          water<\/td>\n1L<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

          Czapek-Dox neg. control (no carbon source)<\/b><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
          Ingredient<\/th>\nAmount<\/th>\n<\/tr>\n
          penicillin & streptomycin<\/td>\n20 ml of 100x stock\/L<\/td>\n<\/td>\n<\/tr>\n
          KH2PO4<\/td>\n1g<\/td>\n<\/td>\n<\/tr>\n
          MgSO4*7H2O<\/td>\n0.5g<\/td>\n<\/td>\n<\/tr>\n
          KCl<\/td>\n0.5g<\/td>\n<\/td>\n<\/tr>\n
          FeSO4*7H2O<\/td>\n0.01g<\/td>\n<\/td>\n<\/tr>\n
          cellulose<\/td>\n30g<\/td>\n<\/td>\n<\/tr>\n
          NaNO3<\/td>\n2g<\/td>\n<\/td>\n<\/tr>\n
          noble agar<\/td>\n20g<\/td>\n<\/td>\n<\/tr>\n
          water<\/td>\n1L<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

          Czapek-Dox-dextrose (positive control)<\/b>\u00a0Czapek-Dox neg. control (no carbon source)<\/b><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
          Ingredient<\/th>\nAmount<\/th>\n<\/tr>\n
          penicillin & streptomycin<\/td>\n20 ml of 100x stock\/L<\/td>\n<\/td>\n<\/tr>\n
          KH2PO4<\/td>\n1g<\/td>\n<\/td>\n<\/tr>\n
          MgSO4*7H2O<\/td>\n0.5g<\/td>\n<\/td>\n<\/tr>\n
          KCl<\/td>\n0.5g<\/td>\n<\/td>\n<\/tr>\n
          FeSO4*7H2O<\/td>\n0.01g<\/td>\n<\/td>\n<\/tr>\n
          dextrose<\/td>\n30g<\/td>\n<\/td>\n<\/tr>\n
          NaNO3<\/td>\n2g<\/td>\n<\/td>\n<\/tr>\n
          noble agar<\/td>\n20g<\/td>\n<\/td>\n<\/tr>\n
          water<\/td>\n1L<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

          The Czapek-Dox doesn\u2019t work for Raffaelea \u2013 doesn\u2019t grow on positive control. Try different media \u2013 Czapek-Dox with proteins, or with little bit of yeast extract, or dextrose+salts+NaNO3.<\/p>\n

           <\/p>\n

          Yeast Nitrogen Base media – Liquid<\/h3>\n

          Tested media:<\/p>\n

          1) Prepare YNB&AA& antibacterials 10x stock: 6.7g YNB&AA and 20mL Streptomycin\/Penicillin concentrate, in 100mL water. Use warm water, but do NOT autoclave! Store in dark and cold place.<\/p>\n

          2)YNB- CMC-liq<\/b>\u00a0(do not use MCC \u2013 not soluble, not usable in absorbance measurements)<\/p>\n

          \n
            \n
          • water 90mL<\/li>\n
          • CMC 3g<\/li>\n
          • after autoclaving: 10ml YNB&AA&antibacterials 10x stock through filter. pH: 5.7<\/li>\n<\/ul>\n<\/blockquote>\n

            YNB- dex-glu-liq<\/b><\/p>\n

            \n
              \n
            • water 90ml<\/li>\n
            • dextrose 1.5g<\/li>\n
            • glucose 1.5g<\/li>\n
            • after autoclaving: 10ml YNB&AA&antibacterials 10x stock through filter<\/li>\n<\/ul>\n<\/blockquote>\n

              YNB- blank-liq<\/b><\/p>\n

              \n
                \n
              • water 90mL<\/li>\n
              • after autoclaving: 10ml YNB&AA&antibacterials 10x stock through filter<\/li>\n<\/ul>\n<\/blockquote>\n

                 <\/p>\n

                Yeast Nitrogen Base media<\/h2>\n

                1) prepare YNB&AA 20x stock: 26.8 g in 200mL warm water, do NOT autoclave! Store in dark and cold place.<\/p>\n

                2) autoclave 50 pieces of filter paper (the same size as last time) as you are autoclaving the media<\/p>\n

                3) media:<\/p>\n

                YNB-CMC<\/b><\/p>\n

                \n
                  \n
                • water 950 mL<\/li>\n
                • agar 15g<\/li>\n
                • CMC 30g<\/li>\n
                • after autoclaving: 50ml YNB&AA 20x stock through filter<\/li>\n<\/ul>\n<\/blockquote>\n

                  YNB-dex-glu<\/b><\/p>\n

                  \n
                    \n
                  • water 950ml<\/li>\n
                  • agar 15g<\/li>\n
                  • dextrose 15g<\/li>\n
                  • glucose 15g<\/li>\n
                  • after autoclaving: 50ml YNB&AA 20x stock through filter<\/li>\n<\/ul>\n<\/blockquote>\n

                    YNB-filter_paper<\/b><\/p>\n

                    water 950 mL<\/p>\n

                      \n
                    • agar 15g<\/li>\n
                    • one piece of filter paper,<\/li>\n
                    • after autoclaving: 50ml YNB&AA 20x stock through filter<\/li>\n<\/ul>\n<\/blockquote>\n

                      YNB-blank<\/b><\/p>\n

                      \n
                        \n
                      • water 950mL<\/li>\n
                      • agar 15g<\/li>\n
                      • after autoclaving: 50ml YNB&AA 20x stock through filter<\/li>\n<\/ul>\n<\/blockquote>\n

                        Congo Red assay<\/h2>\n

                        Prepare Congo Red solution (1g\/L) and 1M solution of NaCl (58g\/L). Overlay plate with Congo Red for 15 minutes, pour off, overlay with NaCl for 15 minutes, pour off, look for zone of clearing<\/p>\n","protected":false},"excerpt":{"rendered":"

                        Assays Filter paper assay Testing for utilization of pure unaltered cellulose control-noble (neg. control) filter paper PDA (pos. control) CMC & Congo Red assay Testing for cellulose-degrading enzymes control-salts (neg. control) CMC-salts CMC-PDB_0.25 dextrose-salts PDA (pos. control) MCC assay Testing for cellulose carbon utilization (i.e., growth) control-noble (neg. control) MCC-salts MCC-PDB_0.25 PDA (pos. control) Media […]<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_s2mail":"yes","footnotes":""},"categories":[5],"tags":[],"class_list":["post-1277","post","type-post","status-publish","format-standard","hentry","category-fungi"],"_links":{"self":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1277","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/comments?post=1277"}],"version-history":[{"count":2,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1277\/revisions"}],"predecessor-version":[{"id":1848,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1277\/revisions\/1848"}],"wp:attachment":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/media?parent=1277"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/categories?post=1277"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/tags?post=1277"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}