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 For reproducible results, the algal inoculum culture shall be in the exponential growth phase, with the biomass increasing ca. 16-fold over 72 h. First, immediately before use, determine cell density of the living stock culture using a luminometer, and add a sufficient amount of cells from the living stock culture to the growth medium (7.1) for a cell density of 10 6 algal cells per millilitre. A three-day pre- culture is necessary to obtain an algal inoculum culture with a cell density of over 10 8 algal cells per millilitre (i.e. sufficient density for starting the test) in the exponential phase of growth. If cell density after pre-culturing is much lower than 10 8 algal cells per millilitre, the cells are not in the exponential growth phase. In that case, repeat the 3-day pre-culturing step until cell concentration exceeds 10 8 cells per millilitre.
8.2 Choice of the test concentrations Two cyanobacterium cultures (Cyanobium sp. NIES-981) in triplicate shall be established in the control tubes (growth medium with no test seawater) and tubes containing diluted test seawater [test seawater mixed with the growth medium at a volume fraction of 80:20]. As an option, additional dilutions of the test seawater in a geometric series may be tested to determine ECx based on the regression analysis. 8.3 Preparation of the test medium First, test seawater shall be collected at an appropriate site (e.g. surface water, mining wastewater, etc.). A filtrate shall be obtained using an appropriate filtering device and a filter with a pore size of < 0,22 μm. The stock solutions of nutrients, metals and tris shall be added to the filtrate so that their concentration is the same as that in ASW-SN. The mixture shall then be diluted to 80 % volume fraction with the growth medium. As the test medium, aliquots of the diluted filtrate shall be dispensed into three test tubes of appropriate dimensions and optical properties for DF measurements (see Annex B).