Non-vital staining, especially with rose Bengal, has been widely used in ecological studies to differentiate between the tests of dead (unstained) foraminifera from those presumed to be living at the time of collection (stained). Doubts have been expressed about staining methods because of the possibility that dead individuals may retain undecayed protoplasm for weeks or months after death; when stained, such individuals would be recorded as living. To assess the importance of such false positives, it is necessary to examine rates of mortality, and the modes of generation of empty tests, i.e., whether due to reproduction, growth stages (leaving empty tests during growth) or death. It can be argued that reproduction, ontogeny, and death through predation lead to tests devoid of protoplasm. Whereas reproduction may affect only a small proportion of the population of each species (due to high pre-reproductive mortality), predation in oxygenated environments may be responsible for the major part of that pre-reproductive mortality. In oxygenated environments, disease or adverse environmental conditions are most likely to lead to dead individuals having tests containing protoplasm. In dysaerobic/anoxic environments, predation by macrofauna may be excluded, so foraminifera die through other causes and thus more tests with dead protoplasm may be potentially available for staining. Therefore, for most other environments, the problem of staining dead individuals is almost certainly overstated. Furthermore, from comparative studies, it seems that the most commonly used technique (staining with rose Bengal) is as reliable as others. Now that new vital staining techniques, especially the use of fluorescent probes, are being introduced, it is timely for further objective comparative studies of all techniques to be made in order to evaluate data already gathered and to develop the best strategies for future ecological studies according to whether they are field-based or experimental.

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