Materials and Reagents

Sterile inoculation loop with incorporated needle (NUOVA APTACA, catalog number: 6001/SG/CS )
14-ml polypropylene round-bottom tubes (Corning, Falcon^®, catalog number: 352059 )
50-ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339652 )
Safe-lock 2-ml tubes (Eppendorf, catalog number: 0030120094 )
Sinorhizobium meliloti 1021
2,3,5-triphenyl-2H-tetrazolium chloride (TTC) (Sigma-Aldrich, catalog number: T8877 )
1,3,5-Triphenyltetrazolium formazan (Sigma-Aldrich, catalog number: 93145 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D5879 )
Tryptone (Sigma-Aldrich, catalog number: T9410 )
Yeast extract (Sigma-Aldrich, catalog number: Y0375 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C3306 )
Na₂HPO₄ (Acantor^® Performance Materials, J.T. Baker, catalog number: 4062-01 )
NaH₂PO₄ (Acantor^® Performance Materials, J.T. Baker, catalog number: 3818-05 )
TYR broth medium (5 g/L tryptone, 3 g/L yeast extract, 6 mM CaCl₂) (see Recipes)
Sodium phosphate buffer (pH 7.5) (see Recipes)

Equipment

Incubator room (at 30 °C)
Rotary shaker
Spectrophotometer (Beckman Coulter, catalog number: DU800 )
Cuvettes for spectrophotometry application in the visible spectrum (Kartell, catalog number: 1938 )
Micro-centrifuge (SCILOGEX D3024 High Speed Micro-Centrifuge) (Scilogex, catalog number: 912015139999 )
Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraeus^TM Megafuge^TM 16R )
Oven (at 65 °C)
Balance (Mettler Toledo, catalog number: B204-S )

Procedure

Growth of bacterial cells
1. Pure cultures of S. meliloti 1021 stored at -80 °C were taken by sterile loops, inoculated in 15-ml round-botton tubes containing 2.0 ml of TYR medium and incubated for 24 h at 30 °C on a rotary shaker (200 rpm).
2. Aliquots of the cultures (0.15 ml) were transferred into 50-ml conical centrifuge tubes containing 15 ml of fresh TYR medium (OD₆₀₀ = 0.2) and the optical density measured at 600 nm using a spectrophotometer and cuvettes for spectrophotometry application in the visible spectrum.
3. When the cultures reach the exponential growth phase (OD₆₀₀ = 0.7), which requires about 4 h of incubation, stop the growth and split the cultures in 10 ml for biomass evaluation and 1.5 ml for TTC reduction measurement.
TTC assay
1. Centrifuge the 10 ml bacterial cultures in 15-ml polypropylene round-bottom at 5,000 x g for 20 min (at room temperature) and discard the supernatant.
2. Incubate the cells at 65 °C in an oven for 4 h until dryness and weigh the cells with a balance.
3. Centrifuge the 1.5 ml bacterial cultures in 2-ml Eppendorf tubes at 8,000 x g for 5 min (at room temperature) and discard the supernatant.
4. Wash the bacteria with 1 ml of 50 mM sodium phosphate buffer (pH 7.5), centrifuge at 8,000 x g for 5 min (at room temperature) and discard the supernatant.
5. Resuspend the cells in 1 ml of 50 mM sodium phosphate buffer (pH 7.5) containing 24 mM 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) and incubate at 30 °C for 1 h on a rotary shaker at 200 rpm (Figure 1).
  
  Figure 1. Picture of the cell suspensions after 1 h of incubation at 30 °C
6. Collect the cells by centrifugation at 8,000 x g for 5 min (at room temperature) and discard the supernatant (Figure 2).
  
  Figure 2. Picture of the bacterial cells after the centrifugation step
7. Resuspend the cells thoroughly in 1 ml 99.5 % DMSO (at room temperature) to dissolve the produced formazan.
8. Spin down the bacterial cells by one-minute centrifugation at 13,000 x g (at room temperature) and collect the supernatants.
9. Prepare formazan standard solutions at different known concentrations dissolving the powder in 99.5 % DMSO.
10. Read the absorbance of the samples (supernatants) and formazan standards at 510 nm using a spectrophotometer. Use the 99.5 % DMSO as blank control.
11. Plot the calibration curve of the formazan standards (mg/ml) against the absorbance of the solutions at 510 nm.
12. Determinate the amount of formazan produced from reactions with the bacterial cells directly from the formazan calibration curve.
13. Normalize the data to cell biomass (g) corresponding to 1.5 ml bacterial cells and determine the amount of formazan produced per g cells.

Data analysis

Perform at least five biological replicates, each conducted at different times.
An example of the excel Formazan standard curve is described in Table 1.
The readings of some experimental samples and the amount of formazan they produced are reported in Table 2 and Table 3.

Table 1. Formazan standards and their relative absorbance at 510 nmv

Optical densities were determined using a spectrophotometer and data were plotted as shown in Figure 3.

Figure 3. Formazan standard curve. The optical density at 510 nm was determined for a range of Formazan standards from 0.0075-0.3 mg/ml. The linear regression line was added by using the Excel 2011 for Mac (14.1.0 version). The equation for the regression line and the correlation coefficient are shown on the graph.

Table 2. Amount of formazan produced by S. meliloti 1021 cells grown up to exponential phase

The amount of formazan reported in this table was calculated by interpolating the standard solution points reported in Table 1 and elaborated in Figure 1.

Table 3. Amount of formazan produced by S. meliloti 1021 cells grown up to stationary phase

The amount of formazan reported in this Table was calculated by interpolating the standard solution points reported in Table 1 and elaborated in Figure1.

Recipes

TYR broth medium (1 L)
5 g tryptone
3 g yeast extract
Add ddH₂O to 1 L. Sterilize by autoclaving
After the solution has cooled add 12 ml sterile 0.5 M CaCl₂
Sodium phosphate buffer (pH 7.5)

Prepare 1 M NaH₂PO₄ solution by dissolving 1.2 g in a final volume of 10 ml double distilled water
Prepare 1 M Na₂HPO₄ solution by dissolving 1.42 g in a final volume of 10 ml double distilled water
Mix 1.6 ml 1 M NaH₂PO₄ solution and 8.4 ml 1 M Na₂HPO₄ solution and dilute to 200 ml with double distilled water to prepare 50 mM sodium phosphate buffer, pH 7.5

Acknowledgments

The method for the TTC assay was adapted from Tachon et al. (2009). This work was partially supported by a dedicated grant from the Italian Ministry of Economy and Finance to the National Research Council for the project CISIA ‘Innovazione e Sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare - Legge No. 191/2009’. This work was also partially supported by the European Commission for funding the ABSTRESS project (FP7 KBBE-2011-289562). The authors declare that they have no conflict of interests.

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