**3. Medium and cultivation conditions**

The composition of modified liquid Postgate medium [3, 35] is the following (g/l): Na2SO4 (0.5); KH2PO4 (0.3); K2HPO4 (0.5); (NH4)2SO4 (0.2); NH4Cl (1.0); CaCl2 × 6H2O (0.06); MgSO4 × 7H2O (0.1); lactate, C3H5O3Na (2.0); yeast extract (1.0); FeSO4 × 7H2O (0.004); sodium citrate, C6H5O7Na3 × 2H2O (0.3); and distilled water (1 l).

*Separated solutions*: Mohr's salt solution [(NH4)2Fe(SO4)2 × 6H2O] (10%) and Na2S × 9H2O solution (1%) and 10 M solution of NaOH must be sterilized separately.

The modified liquid Postgate medium and solutions of Mohr's salt, sodium sulfide, and sodium hydroxide should be sterilized in autoclave (20 min, at 1 atm.). The sterilization provides sterile conditions and partial release of oxygen from the medium. The solution of sodium sulfide is hydrolyzed to hydrogen sulfide during autoclaving.

After sterilization, 10 ml/l of sterile Mohr's salt solution and 0.05 ml/l of sterile solution of sodium sulfide must be added to the medium. The addition of a small quantity (one drop) of sodium sulfide solution to the medium makes visible a black ring which confirms interactions of hydrogen sulfide and free Fe2+ released from Mohr's salt.

A sterile ascorbic acid solution also must be added to the medium, but it cannot be sterilized by autoclaving because it may partially decompose and lose its properties for redox potential. So, 20% ascorbic acid solution should be filtrated through membrane filters (0.2 μm) and added directly to the medium after sterilization. The final concentration of ascorbic acid in the medium should be 0.1 g/l, and the redox potential of the medium must be around −100 mV. Solution of hydrogen sulfide added to medium can also decrease a redox potential [3].

The redox and anaerobic conditions can be controlled by sodium resazurin as an indicator. In addition, FeS reduced and Na2S contained in the medium provides the necessary redox conditions for SRB cultures. The discoloration of sodium resazurin (redox potential of discoloration Eh = −100 mV) confirms the decrease of redox potential. A pH medium (7.5) provides by the addition of a sterile 10 M solution of NaOH.

The temperature of the media should be +25…+30°C for environmental samples, and + 37°C for intestinal samples (+40°C for samples from birds).

The tubes with samples must be completely filled up to the edges of the test tube with completed medium and closed with rubber stoppers. In another case, tubes can be filled up incompletely, but 1 ml of sterile liquid paraffin must be filled up to the top of the medium and closed with rubber stoppers.

As a control of the quality of the medium, known pure culture of SRB from some collections of microorganisms is recommended to also be used.

Cultivate in the thermostat at +25…+30°C, +37°C, or +40°C, depending on the origin of the sample, during for 1–5 days under anaerobic conditions. SRB from birds, animals, and humans mostly grow faster than environmental species.

Positive growth of SRB is indicated by observing a black FeS precipitate occurred in the bottom of the tube.
