BS ISO 23734:2021 pdf download – Marine technology — Marine environment impact assessment (MEIA) — On-board bioassay to monitor seawater quality using delayed fluorescence of microalga 7.2 Preparation of the algal stock culture Either a living stock culture or a cryopreserved stock culture of strain NIES-981 shall be prepared at a land-based laboratory. The stock shall then be used to prepare an inoculum culture on-board before testing. As soon as possible after the living stock culture arrives on-board, it should be placed under culture condition. This step is important, to shorten the preparation time of the alga inoculum on-board. The density of the living stock culture should be 10 8 algal cells per millilitre in the exponential growth phase. This corresponds to between 1 000 counts and 1 500 counts of photons at 0,1 s after the start of the measurements. The living stock culture should be pre-cultured to obtain the inoculum culture for the bioassay. First, determine the cell density of original stock culture using a luminometer (the same device as that for DF measurements, see 6.2). Add a sufficient amount of cells from the algal stock culture to 100 ml of the growth medium (7.1) for the cell density to be 2 × 10 6 algal cells per millilitre, and pre-culture for 3 days. After pre-culturing, the cell density in 10 ml of the living stock culture in the exponential growth phase shall be determined and adjusted to 10 8 algal cells per millilitre. If cell density at that stage is much lower than 10 8 algal cells per millilitre, the culture is not at the exponential growth phase. In that case, repeat the 3-day pre-culturing step until cell density exceeds 10 8 cells per millilitre. The living stock culture shall be incubated under the same conditions as those for the test (8.4). Determine the cell density of the living stock culture immediately before use, to calculate the required culture volume. If the stock culture is in the stationary phase, at least two or three pre-culturing rounds are necessary to obtain culture in the exponential phase of growth. When older stock cultures are used (more than 3 weeks old), more time may be required for recovery. For the preparation of the cryopreserved stock culture, see Annex D. All steps should be performed on a clean bench (6.6). 8 Test procedure on-board 8.1 Preparation of the algal inoculum culture...
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