Methods may be modified depending on combination of the criteria used for judgement of viability. Following are two examples of the methods.

　

3.4.1 Counting method using morphological change and mobility as criteria for judgement of viability:

　

3.4.1.1 For organisms of the L size group, samples prepared by the procedure described in the paragraph 2.1 and 2.4 must be observed under a compound or a stereo microscope. Among organisms in the samples, only viable ones judged by their appearance and color shall be counted. If judgment of the viability is difficult, use a biological microscope at a higher magnification. if there are organisms to be included in the S size group, keep a record for calculation of the number of S size organisms in the sample.

　

3.4.1.2 While the volume of the sample subjected to a one-time microscopy observation shall be arbitrary, microscopy must be continued multiple times, and the total volume of concentrated samples for microscopy shall be equivalent to or more than 1m terms of the original volume of the sample before concentration.

　

3.4.1.3 For organisms of the S size group, samples prepared by the procedure described in the paragraph 2.2 and 2.4 must be observed under a compound microscope. Among organisms in the samples, only viable ones judged by their appearance and colour shall be counted. Though the volume of the sample subjected to one-time microscopy is arbitrary, microscopy must be continued multiple times, and the total volume of concentrated samples for microscopy shall be equivalent to or more than 10ml in terms of the original volume of the sample before concentration. The sample volume for one-time microscopy (the volume of water transferred to the chamber) shall be determined so that microscopy does not affect the viability of aquatic organisms.

　

3.4.1.4 The above procedures shall be implemented as soon as possible after sampling, at least within one day. Organisms shall be kept at the same temperature as that during the test, and no chemical fixing process shall be applied.

　

3.4.2 Counting method using morphological change and a re-growth test as criteria for judgement of viablity.

　

3.4.2.1 For organisms of the L size group, samples prepared by the procedure described in the paragraph 2.1 and 2.4 must be observed under a compound or a stereo microscope. Observe the sample under microscope. If there is any individual of which viability cannot be judged, separate that individual by sucking it out using a pipett and inoculate it into a culture medium prepared by filtering sample water using a GF/F filter. Then observe the state of recovery of appearance, mobility and re-growth for one week.

　

3.4.2.2 For organisms of the S size group, samples prepared by the procedure described in the paragraph 2.2 and 2.4 must be observed under a compound microscope. Take same steps as for organisms in L size group described above.

　

3.4.2.3 The vessel containing the medium with the inoculated aquatic organisms shall be cultivated for the same temperature conditions at the treatment, and 12 hours of bright and dark cycles (60-90 μ mol photosynthetically available radiation m^-2 s^-1), if necessary.

　

3.4.2.4 The inoculation of organisms for culture and observation of result of re-growth shall be completed within each one-day on the same temperature condition. The reason for such quick arrangement is to suppress changes of physiological condition of object organisms during storage.