Quantifying Vibrio aestuarianus in Seawater and Oysters

The presence of GFP-tagged bacteria and bacterial counts were measured in 100 μl of sampled seawater by flow cytometry (Coulter Epics XL cytometer, Beckman®, and CyFlow Cube 6 Robby Partec®) on 10,000 events or after 5 min with a fixed threshold of FL1 fluorescence. This method enabled measurement of a concentration of V. aestuarianus ≥10³ bacteria/mL. V. aestuarianus DNA amounts were measured by qPCR detection methods (8 (link)) in oyster tissues (50 mg) or seawater (100 μL) after DNA extraction via QiaAmp® tissue kit procedures (Qiagen®) and total-DNA adjustment to 5 ng/μL when needed. A standard curve was built by means of serially diluted titrated genomic extracts (12 (link)). Assays were performed on MX3000 and MX3005 machines (Agilent®) with the Brilliant III Ultrafast kit (Stratagene®).

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Lupo C., Travers M.A., Tourbiez D., Barthélémy C.F., Beaunée G, & Ezanno P. (2019). Modeling the Transmission of Vibrio aestuarianus in Pacific Oysters Using Experimental Infection Data. Frontiers in Veterinary Science, 6, 142.

Publication 2019

Epics XL cytometer MX3000 MX3005

Assays Bacteria Bacterial counts Flow cytometry Fluorescence Genomic Oyster Tissue

Corresponding Organization :

Other organizations : Ifremer, Laboratoire de Génie des Procédés – Environnement – Agro-alimentaire

Top 5 similar protocols

Variable analysis

independent variables

Presence of GFP-tagged bacteria

dependent variables

Bacterial counts measured in 100 μl of sampled seawater
V. aestuarianus DNA amounts measured by qPCR detection methods in oyster tissues (50 mg) or seawater (100 μL)

control variables

Fixed threshold of FL1 fluorescence used for flow cytometry measurements
DNA extraction via QiaAmp® tissue kit procedures (Qiagen®)
Total-DNA adjustment to 5 ng/μL when needed for qPCR
Standard curve built by means of serially diluted titrated genomic extracts
Assays performed on MX3000 and MX3005 machines (Agilent®) with the Brilliant III Ultrafast kit (Stratagene®)

controls

Positive control: Serially diluted titrated genomic extracts used to build a standard curve for qPCR
Negative control: Not explicitly mentioned

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