Preservation of vitally stained zooplankton for live/dead sorting

Motility is an unreliable and unwieldly criterion for recognizing living zooplankton. Published accounts of the use of Neutral Red stain for live/dead differentiation prescribe sample preservation in formalin after colour intensification by acidification. However, Neutral Red is most soluble at these low (<6.8) pH values and it leaches from the material, thereby limiting the effective storage time. At pH higher than 8.0, Neutral Red is much less soluble, and we find that it can be ‘locked’ into the material by preservation in alkaline (pH 9.0) formalin. Under these conditions the stain is colourless but the intense magenta colour can be restored when required by acidification with acetic acid. This small modification greatly enhances the value of the vital staining technique, particularly when handling large numbers of samples or when working away from the laboratory.