About Vital Stain
The Vital Stain Method
In U.S. ballast water management regulations, the current USCG requirement for analysis of the organisms in the 10 – 50 μm size class requires categorization of organisms as being “living” or “dead.” The Environmental Technology Verification (ETV) Staining Method (vital stain method) uses a combination of two fluorescein-based stains (FDA and CMFDA) to evaluate the status of organisms in the 10 – 50 μm size class in ballast water samples. The stains will penetrate into organisms, where functional esterases will convert them into fluorescent products that are retained by cellular membranes. Using epifluorescent microscopy, fluorescing organisms are enumerated as “living” individuals. Any motile organisms observed are also counted as “living.”
Questions Surrounding Reliability
There is a common misconception that the vital stain method is highly reliable, based on the results of one study (Steinberg et al., 2011). The study reported that false negative rates were very low for the four sites investigated. Motility was used to judge whether the staining was accurate, however, there are many species and stages of phytoplankton that are non-motile. The true false negative rates could have been significantly higher, but cannot be recalculated because the species were not disclosed.
Relative Accuracy & Repeatability of Methods
Substantial empirical data from tests using both the MPN and vital stain methods provides an important indication of the relative accuracy for a given test or organism species. The ideal situation is that a method is able to achieve 100% accuracy in classifying the organism correctly, but often methods do not achieve this standard. In some instances, a method can completely fail to accurately classify a given test or organism species and thereby diminish a methods ability to assess compliance with either USCG or IMO BWM Convention standards.
In the images below, the center of the target represents 100% accuracy in a given test or organisms species. 0% represents complete failure in a method’s accuracy for a given test or organism species.
The data for the MPN diagram presents a very conservative picture as the accuracy is presented and calculated on a species basis versus a number of organisms basis (“abundance basis”). The abundance basis calculation is a more relevant value as it is consistent with the USCG’s Ballast Water Discharge Standard (“number of organisms/mL” and not “number of species/mL”). If the data were to be presented and calculated on an abundance basis, all the data points would be at the center of the diagram as a much higher percentage of organisms are shown to grow out on an abundance basis versus a species basis.
Recent studies in ballast water applications have shown that the vital stain method is not completely reliable. For many species of phytoplankton, the fluorescence of stained living cells is not measurably higher than stained killed cells, so live and dead cells cannot be distinguished.
For some species, the fluorescence is lower in stained living cells than in stained killed cells – this opposes the expectations of the assay. In addition, staining intensities can be very different between species, making evaluations of mixed assemblages prone to error; dead cells of a bright species can have a higher fluorescence than live cells of a dim species, so errors in classification are guaranteed regardless of where the microscopist decides to set the classification threshold. There is no standard and no control for where a microscopist decides to set the fluorescence threshold for classification of organisms. This can lead to differences in results between samples, and more alarmingly, between test facilities.
A summary presentation of the recent work on vital stain’s lack of reliability and repeatability can be found here, and the corresponding full scientific paper recently accepted by the Journal of Phycology here.
A Common Misconception
There is a common misconception that the vital stain method gives a definitive “live” or “dead” status to an organism. In actuality, the method evaluates the presence of esterases within organisms and the integrity of the membrane. These properties do not define a cell as being “living” or “dead.”
This is an assay of some of the properties of a living organism (e.g., metabolism, structure), similar to how the MPN method is an assay of some of the other properties of a living organism (e.g., reproduction, growth).
Steinberg, M.K., E.J. Lemieux and L.A. Drake (2011). Determining the viability of marine protists using a combination of vital, fluorescent stains. Mar. Biol. 158: 1431–1437.
1. MPN: data extracted from ETV Method Development Experiments and land-based test reports (DHI-Denmark).
2. MPN Method Analysis: Cullen, J.J. and H.L. MacIntyre (2015). On the use of the serial dilution culture method to enumerate viable phytoplankton in natural communities of plankton subjected to ballast water treatment. J. Appl. Phycol. DOI 10.1007/s10811-015-0601-x.
3. MacIntyre 2016: Stain data extracted from MacIntyre H.L., and Cullen, J.J. 2016. Classification of phytoplankton cells as live or dead using the 2 vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate (FDA and CMFDA). J. Phycol. DOI 10.1111/jpy.12415-15-154.
4. NRL 2010: Stain data extracted from Multi-site validation of a method to determine viability of organisms ≥ 10 µm and < 50 µm in ships’ ballast water using two vital fluorescent stains (NRL, 2010).