Limits of propidium iodide as a cell viability indicator for environmental bacteria

Abstract

Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram 2 Sphingomonas sp. LB126 and the gram 1 Mycobacterium frederiksbergense LB501T. Bacterial proliferation activities observed via DAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC 6 (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle. ' 2007 International Society for Analytical Cytology

Determination ofPseudomonas PutidaLive Cells with Classic Cultivation and Staining with “Live/Dead Baclight Bacterial Viability Kit”

“…It is possible PI to enter into the live dividing cells and to stain them red. This is in congruence with the research of Shi L et al on the limits of propidium iodide as a cell viability indicator for environmental bacteria (8). Some of red cells are moving actively, when they are directly observed on the glass slide.…”

“…Yamamoto proved that the toxic effect of ZnO nanoparticles depend on their size (0.1-0.8 μm) and concentration 0.4-100 mg/ml. High temperature treatment of ZnO particles leads to lower antibacterial activity (8). The crystalline structure and particle shape of the powder have little effect.…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Those cells were proven to be vital by cultivating on agar plates and CFU determination. Nevertheless, E. coli was found to not present elevated PI values in the same experiments suggesting that this false positive staining may not only be substrate but also species dependent (65). Nevertheless, PI is a good probe for detecting dead cell numbers in bacterial populations, which is proven by numerous publications (86)(87)(88)(89).…”

“…The compound is known to only stain dead cells and is therefore part of several viability kits which can be purchased from various companies. However, there are reports were PI was found to label highly reproductive cells as well (65). The review on cell wall reconstruction (see above) suggests a closeset of elongated cell wall reconstruction and porin construction during individual cell growth and PI entry probably through the shortly open cell wall structures.…”

Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures

“…Additionally, the cells still excluded propidium iodide, indicating that a certain degree of cytoplasmic membrane integrity remained. Such counter intuitive phenomena are not unique, PI straining during exponential phase has been observed in actinomycetes (Sebastine et al, 1999;Stocks & Thomas 2001) and mycobacteria (Shi et al, 2007). While it may be possible to construct explanations for the reasons that "live" cells fail to stain -"ve with bis-oxanol, or can stain +"ve with PI, one must also consider that such populations contain cells which are in fact CMP negative, with intact membranes, or that there can be a minimum in "viability" during exponential growth.…”