meth_2.19 PAM Fluorometry in situ measurements of seagrass Marine Microbes Coastal Project

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AM Project ID: Legacy

PAM fluorometry measurements in situ: After bringing the algal tissue to the surface and swabbing ~20 cm² to sample surface-associated microorganisms, use a ‘leaf clip’ provided with a diving Pulse Amplitude Modulated (diving-PAM) fluorometer (Walz, Germany) to first ‘dark-adapt’ for 15 minutes an area of algal thallus adjacent to where the swabbing was done (i.e., not swabbed) that is not covered by visible epiphytes (note: the leaf clip must be in the ‘closed’ position). A 0.8 s ‘saturating pulse’ of light (> 4500 µmol photons m^-2s^-1) is delivered to the algal surface via a 1.5 mm diameter optical fibre. The same fibre also delivers the ‘measuring light’ (which should be set < 0.15 µmol photons m^-2s^-1, below the level required to initiate photosynthesis. Chlorophyll fluorescence (wavelength > 710 nm) will be measured on the Diving-PAM (gain = 4). Minimum dark-adapted fluorescence (F) is determined prior to the saturating flash, while the maximum dark-adapted fluorescence (FMʹ) will be determined as the fluorescence value during the saturating flash. The difference between FMʹ and F is the variable fluorescence, ΔF. Effective quantum yield (EQY, ΔF/FMʹ) is a measure of photosystem II photochemical efficiency in the dark-adapted algae. Area of thallus appropriate for sampling and PAM measurements: Because E. radiata has a basal meristem, the tissues at the top of the thallus (primary lamina) and the ends of each lateral ‘branch’ are typically older, may be more poorly chemically defended and are thus more likely to show evidence of epibiosis or natural senescence. We wish to sample young, healthy tissue, so avoid those areas. Select a lateral ‘branch’ that is in the lower half of the alga and sample that lateral ‘branch’ on the half closest to the primary lamina.