Differential Effect of Tetrazolium Dyes upon Bacteriophage Plaque Assay Titers

Hurst, Christon J.; Blannon, Janet C.; Hardaway, Rachel L.; Jackson, Wesley C.

doi:10.1128/aem.60.9.3462-3465.1994

Cited by 19 publications

(7 citation statements)

References 13 publications

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“…they occur consistently and exclusively in human faeces and sewage, do not multiply in the environment and do not have a faecal environmental source; sensitivity, i.e. they are present in greater numbers than the enteric viruses; they are at least as long-lived as the enteric viruses present in the environment and throughout the water treatment processes; their detection must be easy to perform by simple and inexpensive methods; and their characteristics in the aquifer should closely mimic those of the enteroviruses (Table 1) (Keswick et al 1984;Hurst et al 1994).…”

Section: Bacteriophagesmentioning

confidence: 99%

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters

Leclerc

Edberg

Pierzo

et al. 2001

Journal of Applied Microbiology

175

117

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Section: Bacteriophagesmentioning

confidence: 99%

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters

Leclerc

Edberg

Pierzo

et al. 2001

Journal of Applied Microbiology

175

117

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“…To our knowledge, there are only a few hints about suppression of bacterial metabolic activity by tetrazolium salts and their reduced components (5,17,29). We therefore decided to study possible inhibitory effects of CTC on bacterial metabolic activity in natural freshwater and brackish water samples in greater detail to test the validity of the commonly applied CTC method.…”

mentioning

confidence: 99%

Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride

Ullrich¹,

Karrasch²,

Hoppe³

et al. 1996

Appl Environ Microbiol

144

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The monotetrazolium redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used as a vital stain of actively respiring bacteria for several years. In this study, inhibitory effects on bacterial metabolism of this redox dye have been examined in a brackish water environment (Kiel Fjord, Germany) and a freshwater environment (Elbe River, Germany). As the results from time series experiments (1 to 10 h) show, bacterial growth and respiration of the investigated natural communities were clearly reduced by CTC supply. Compared with untreated controls (100%), CTC-treated samples showed distinctly lower heterotrophic bacterial plate counts (0 to 24 and 11.8 to 23.7%, respectively), bacterial production (0.9 to 14.1 and 1.1 to 9.6%, respectively), bacterial respiration (4.1 to 9.4 and 6.8 to 43.8% for several concentrations of 14 C-labeled glucose), and [ 14 C]glucose incorporation (0.2 to 4.2%). Additionally, toxicity of CTC was demonstrated by luminescence in a Microtox bioassay. CTC concentrations of 0.1 and 5.0 M required only 15 min for decreases of approximately 50 and 100%, respectively. The suppression of CTC on several bacterial metabolic processes suggests that determination by the CTC technique underestimates the actual number of active cells distinctly. This conclusion is confirmed by the comparison of generation times calculated on the basis of thymidine uptake data and active bacterial counts determined by the CTC assay and microautoradiography. While unrealistic short generation times (0.5 to 5 h) resulted from the CTC assay, the generation times calculated according to microautoradiography ranged within values (7 to 21 h) reported elsewhere for comparable aquatic environments. The inhibitory effect of CTC demonstrated in our experiments is an aspect with regard to the application of this tetrazolium dye for the estimation of active bacteria in natural aquatic environments which hitherto has not been considered.

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“…In order to enhance the morphology of phage plaques, tetrazolium salt dyes were used, however these are known to adversely affect phage kinetics and suppress plaque titers [48]. However, an automated microtiter plate reader has been described in literature using the OmniLog™ system to measure phage kinetics with the addition of tetrazolium salt .…”

Section: Discussionmentioning

confidence: 99%

Development and validation of a miniaturized host range screening assay for bacteriophages

Grey

Vaitekenas

et al. 2021

Preprint

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Antimicrobial resistance is a global health crisis, partly contributed by inappropriate use of antibiotics. The increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Current procedures assessing susceptibility and breadth of host range to bacteriophage are conducted using large-scale manual processes that are labor-intensive. The aim here was to establish and validate a scaled down methodology for high-throughput screening in order to reduce procedural footprint. Bacteriophages were isolated from wastewater samples and screened for specificity against 29 clinical Pseudomonas aeruginosa isolates and PA01 using a spot test (2 μL/ drop). Host range assessment was performed on four representative P. aeruginosa isolates using both double agar overlay assay on petri dishes and 24-well culture plates. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates were corroborated between the current standard practice of whole plate phage assay and 24-well phage assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

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Differential Effect of Tetrazolium Dyes upon Bacteriophage Plaque Assay Titers

Cited by 19 publications

References 13 publications

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters

Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride

Development and validation of a miniaturized host range screening assay for bacteriophages

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