Permafluor scintillation cocktail

Five weeks after shoot emergence, the above-ground tissues of plants were enclosed within airtight labelling chambers (Polybags Ltd, London, UK). At the beginning of the photoperiod, ¹⁴CO₂ was liberated into the chamber by adding 2 ml 10% lactic acid to 28 µl ¹⁴C-sodium bicarbonate (1 MBq). One ml of labelled chamber headspace gas was sampled immediately using a hypodermic syringe and every 1.5 h to monitor the drawdown of ¹⁴CO₂ by plants. Gas samples were injected into separate gas-evacuated 20 ml scintillation vials containing equal volumes (i.e., 10 ml) of the liquid scintillants Carbo-Sorb and Permafluor scintillation cocktail (PerkinElmer, Beaconsfield, UK) and quantified through liquid scintillation counting (Packard Tri-carbon 3100 TR, Isotech, Chesterfield, UK).

Two weeks after ³³P and ¹⁵N tracer additions, plants were prepared for ¹⁴CO₂ labelling, to allow movement of carbon from plant to fungus to be quantified. A 110 µl solution of NaH¹⁴CO₃ (Perkin Elmer) containing 1.0175 MBq ¹⁴C (specific activity = 1.621 GBq/mmol) was added to a cuvette in each pot. The tops of all mesh‐windowed cores were sealed using gas‐tight rubber septa (SubaSeal) to minimize diffusion of ¹⁴CO₂ into the cores. ¹⁴CO₂ gas was liberated from the NaH¹⁴CO₃ by addition of 10% lactic acid, generating a 1.0175 MBq pulse of ¹⁴CO₂. Samples of 1 ml above‐ground gas and 1 ml below‐ground gas (via the glass wool‐filled core) were taken 1 hr after release of ¹⁴CO₂ and every 4 hr thereafter to monitor the drawdown, respiration and flux of ¹⁴C through the plant–AMF network. Gas samples were injected into gas‐evacuated scintillation vials containing 10 ml Carbosorb^® (Perkin Elmer), a carbon‐trapping compound. To this, 10 ml Permafluor scintillation cocktail (Perkin Elmer) was added, and ¹⁴C content of each sample was quantified by liquid scintillation counting (Tricarb 3100TR scintillation counter; Perkin Elmer).
Pots were maintained under cabinet conditions until detection of maximum below‐ground ¹⁴C flux (20–22 hr after ¹⁴CO₂ liberation) at which point 3 ml 2 M KOH was added to cuvettes within each microcosm to capture remaining gaseous ¹⁴CO₂.