APPENDIX 7
PREPARATION OF SEEDLINGAGAR SLANTS FOR CULTIVATING SMALL SEEDED LEGUMES
Small seeded legumes can be cultured enclosed in tubes if these plants are to be used for the authentication of rhizobia or for enumerating rhizobia by the plant-infection technique. One of the limitations of strain evaluation in enclosed tubes is that a tube environment restricts growth conditions and proper differentiation of the plant. A nitrogenfree nutrient solution is solidified with agar for slant preparation or without agar for NifTAL-tubes.
(a) Tubes 250 mm x 25 mm (Figure A.9) are required. Tubes are stoppered with cotton plugs sufficiently loose to allow good air exchange and simultaneously filter off contaminants.
(b) A total of 1.62 l of the Nfree nutrient solution is needed for 54 tubes at the rate of 30 ml per tube. For convenience, divide the nutrient solution into manageable volumes in beakers or Erlenmeyer flasks prior to the addition of the agar powder. (Example: It is convenient to have 500 ml of the N-free nutrient solution in a 1 l container as this will greatly facilitate stirring when the agar is being melted or dispensed). Add 1.5% (w/v) agar to the N-free nutrient solution (24.3 g of agar powder will be needed for 1.62 l of N-free nutrient solution). Melt the agar either by steaming in an autoclave or by direct heating over a bunsenflame. If direct heating is used, the mixture must be constantly stirred over gentle heat to prevent charring of the agar on the bottom on the container.
(c) Dispense the melted agar in 30 ml portions into the tubes and plug. To facilitate dispensing of the agar, a simple setup is illustrated in Figure A.9 which is adequate for approximate volumes. Arrange tubes in suitable metal baskets and autoclave at 121°C for 30 min. To make slants, support the tubes at an angle as illustrated.
Figure A.9. Simple set up for dispensing seedling agar into tubes and forming slants.