Growth measurement of V. neptunius PP-145.98 strains was performed using 96-well microtiter plates. Each well contained 200 µL of CM9 medium (Lemos et al., 1988 (link)) supplemented with FeCl3 at 10 µM (PROBUS) or with the iron chelators ethylenediamine-di(o-hydroxyphenyl-acetic acid) (EDDA) at 5 µM or 2,2’–dipyridyl (dipyridyl) (Sigma) at 50 µM or 30 µM. Each well was inoculated with a 1:50 dilution of an overnight culture of the strain to be tested in TSB-1 at OD600 = 0.5. The plates were incubated at 25°C with shaking at 150 rpm. After 18 h of incubation, growth (OD600) was recorded in an iMACK Microplate reader (Bio-Rad). Bacterial cultures in CM9 with 30 µM 2,2’-dipyridyl and an OD600 ≈0.6 (after 6 h of incubation) were used to measure siderophore production using the chrome azurol-S (CAS) liquid assay (Schwyn and Neilands, 1987 (link)). Equal volumes of each cell free supernatant and CAS reagent were mixed and absorbance at 630 nm (A630) was measured in a UV-VIS spectrophotometer (Hitachi) after 15 min of incubation at room temperature.
Imack microplate reader
Manufactured by Bio-Rad
Sourced in Spain
The IMACK Microplate reader is a versatile laboratory instrument designed to measure the absorbance, fluorescence, and luminescence of microplate samples. It provides accurate and reliable data acquisition for a wide range of applications, including but not limited to, enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and nucleic acid quantification. The IMACK Microplate reader offers a compact and user-friendly design, ensuring efficient and streamlined operation in the laboratory environment.
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5 protocols using imack microplate reader
1
Growth and Siderophore Production of V. neptunius
2
Growth Promotion and Siderophore Assays for V. anguillarum
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Growth promotion assays were performed using 96-well microtiter plates. Each well contained 200 μL of CM9 medium (Lemos et al., 1988 (link)) supplemented with 10 μM FeCl3 to achieve iron excess conditions, or with the iron chelators EDDA [ethylenediamine-di(o-hydroxyphenyl-acetic acid)] 5 μM or 100 μM 2,2′-dipyridyl, to achieve iron restricted conditions. Each well was inoculated with a 1:50 dilution of a V. anguillarum overnight culture in TSB-1 adjusted to an OD600 = 0.5. The plates were incubated at 25°C or 18°C with shaking at 150 rpm. Growth (OD600) was recorded during 24 h in an iMACK Microplate reader (Bio-Rad). Bacterial cultures in CM9 with 50 μM 2,2′-dipyridyl and an OD600 ≈ 0.8 (after 6 h of incubation) were used to obtain supernatants and measure siderophore production using the chrome azurol-S (CAS) liquid assay (Schwyn and Neilands, 1987 (link)). For this purpose, equal volumes of cell free supernatants and CAS reagent were mixed and, after 15 min of incubation at room temperature, A630 was measured in a spectrophotometer (Hitachi).
3
Gallium Susceptibility of V. anguillarum
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To test the susceptibility
of V. anguillarum to gallium(III),
the RV22 vabF mutant strain was challenged to grow
in the presence of GaBr3, gallium complex32a , or Pcb-Ga3+ (2-Ga3+ ). Growth
was evaluated in 96-well microtiter plates containing 200 μL
per well as the final volume. An overnight culture of V. anguillarum was adjusted to an OD600 = 0.5, and a final dilution of 1:30 was inoculated in a CM9 minimal
medium supplemented with 30 μM 2,2′-dipyridyl. GaBr3, gallium complexes32a , and Pcb-Ga3+ (2-Ga3+ ) were tested at increasing concentrations
between 5 and 500 μM. The microplate was incubated at 25 °C
with shaking at 120 rpm. Growth was recorded for 18 h in an iMACK
Microplate reader (Bio-Rad). Each condition was assayed in duplicate
in the microplate, and three independent experiments were performed.
of V. anguillarum to gallium(III),
the RV22 vabF mutant strain was challenged to grow
in the presence of GaBr3, gallium complex
was evaluated in 96-well microtiter plates containing 200 μL
per well as the final volume. An overnight culture of V. anguillarum was adjusted to an OD600 = 0.5, and a final dilution of 1:30 was inoculated in a CM9 minimal
medium supplemented with 30 μM 2,2′-dipyridyl. GaBr3, gallium complexes
between 5 and 500 μM. The microplate was incubated at 25 °C
with shaking at 120 rpm. Growth was recorded for 18 h in an iMACK
Microplate reader (Bio-Rad). Each condition was assayed in duplicate
in the microplate, and three independent experiments were performed.
4
Piscibactin Analogues Antimicrobial Evaluation
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The biological activity of piscibactin analogues, 6 –9 , was evaluated in 96-well microtiter plates, using 200 µL as final volume. To determine the lowest concentration that allows growth, piscibactin and each analogue were used at the final concentrations of 20, 10, 2, and 0.2 µM from a stock solution prepared with methanol:milliQ water (1:1). From an overnight culture of RV22 ∆vabD, RV22 ∆vabD∆frpA and P. damselae subsp. piscicida, whose OD600 was adjusted to 0.5, a final dilution of 1:20 and 1:40, respectively, was inoculated in CM9 media supplemented with 75 µM 2,2ʹ-dipyridyl. After the addition of piscibactin and the tested analogues at the suitable concentrations, the plate was incubated at 25 °C with shaking at 120 rpm. The growth (OD600) was recorded for 18 h in an iMACK Microplate reader (Bio-Rad). Media supplemented with ferric chloride (10 µM FeCl3), with the chelating agent 2,2ʹ-dipyridyl (75 µM), and non-inoculated media were used as controls. The assay corresponding to each condition (iron excess or iron deficiency) and each bacterial strain was performed in duplicate in each plate. Three independent experiments were performed, and the results shown are the mean of the values obtained.
5
Siderophore Production in Iron-Restricted Conditions
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The ability to grow under iron-restricted conditions and the siderophore production of the parental and mutant strains were assayed in CM9 minimal medium [50 (link)] containing 2,2′-dipyridyl 90 µM as iron chelator. Bacteria were grown in TSB-1 for 5 h at 25 °C and cell concentration was adjusted to an optical density (OD600) of 0.5. This preinoculum was used to inoculate (1:40) CM9 medium in a 96-well microtiter plate (final volume of 200 µL per well). Plates were incubated for 18 h at 25 °C in an iMACK Microplate reader (Bio-Rad Laboratories, Madrid, Spain) taking OD600 measurements every 30 min. Media supplemented with FeCl3 10 µM and non-inoculated media were used as controls. All conditions were carried out in triplicate within each plate and all experiments were repeated four times, calculating standard deviations and means for each condition. Student’s t-test was used for statistical analysis using SPSS software (version 20; IBM SPSS Inc., Chicago, IL, USA).
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