APPENDIX 7
PREPARATION OF SEEDLING-AGAR 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 nitrogen-free 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 N-free 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 bunsen-flame.
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
set-up 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.