Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms - which stain is suitable?

BACKGROUND: There is confusion over the definition of the term “viability state(s)” of microorganisms. “Viability staining” or “vital staining techniques” are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. DISCUSSION: Many terms describe “vitality states” of microorganisms, however, several of them are misleading. Authors define “viable” as “capable to grow”. Accordingly, staining methods are substitutes, since no staining can prove viability. The reliability of a commercial “viability” staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the “viability” kit are dependent on the stains’ concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique. To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research. Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. SUMMARY: – The nomenclature regarding “viability” and “vitality” should be used carefully. – The manual of the commercial “viability” kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature. – Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting. – As microbiological parameter the Plating Efficiency should be used for comparison. – Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.