**2.1 Samples from environment (water, soil, swamp, and environmental surfaces)**

One milliliter of water (or 1 g of swamp, soil, and metal rust) should be suspended in 9 ml of anoxic Postgate liquid medium. The tubes should be brim-filled with medium and closed to provide anaerobic conditions. Another option to provide anaerobic conditions is to add in tube 1 ml sterile liquid paraffin. The schema of sampling is presented in **Figure 1**.

#### **2.2 Samples from feces of human or animals**

It is thought that the species of SRB, their composition, and the number found in the intestinal lumen differ from that of the composition and number of

**25**

water (1 l).

separately.

square (in cm<sup>2</sup>

**Figure 1.**

large intestine of animals.

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

etc.), the temperature of medium should be +40°C.

*The scheme of sampling from different environmental biotopes.*

For isolation of SRB from biofilms, 10<sup>−</sup><sup>5</sup>

**3. Medium and cultivation conditions**

done for recalculation of CFU of SRB released from cm<sup>2</sup>

microorganisms on the surface of the intestinal mucosa [2, 9, 28, 34]. Similar to environmental samples (see **Figure 1**), fecal samples from human or animals should be fresh and directly suspended in anoxic modified Postgate liquid medium (pH 7.5, *temperature* to +37°C). One gram of feces suspends in 9 ml of the modified Postgate liquid medium [3, 35]. The same quantity of feces should be taken for determining the dry matter and recalculation of colony-forming units (CFU) per 1 g of dry matter. Before this procedure, the medium should be heated in thermostat to +37°C temperature. If the samples from domestic or wild birds (chickens, geese, ducks,

**2.3 Samples from intestine (biopsy or sections of the large intestine of animals)**

Intestinal SRB with other intestinal bacteria can form biofilms on the surface of the epithelial cells of the large intestine [34]. These biofilms include species of *Desulfovibrio* genus and the species of *Bacteroides*, *Pseudomonas*, *Clostridium*, *Escherichia,* or other intestinal microorganisms. Such biofilms are often resistant to antimicrobial substances [36]; therefore it is an interesting area of the study.

acid) should be added to the modified Postgate liquid medium for releasing SRB from a biofilm. A fresh piece of biopsy should be weighed, and its approximate

liquid medium (pH 7.5, *temperature* to +37°C). This calculation of square must be

The same procedure can be applied for isolation of SRB from sections of the

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

*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

) must be calculated and added to 9 ml of the modified Postgate

M EDTA (ethylenediaminetetraacetic

of a biofilm.

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

**Figure 1.**

*Microorganisms*

detail are necessary.

mesophilic SRB species.

The goal of chapter is to describe:

animals and from biopsy material

analysis of the 16S rRNA gene

• Generalization of this research

selections is described in Section 3.

of sampling is presented in **Figure 1**.

**2.2 Samples from feces of human or animals**

**2. Selection of samples**

**surfaces)**

• Media, isolation, purification, and cultivation conditions

• Morphological diversity and physiological and biochemical properties

plant [12–14] and also present in the human and animal intestines [15–19]. The main species of intestinal SRB, *Desulfovibrio* genus, are often isolated from patients with inflammatory bowel disease (IBD) and healthy subjects [15, 20–25]. Other species of SRB, *Desulfomicrobium*, *Desulfobulbus*, *Desulfobacter*, *Desulfomonas*, and *Desulfotomaculum* were also seldom isolated from human and animal feces [1, 23, 26]. An increased number of SRB are often detected in patients with periodontitis [18]; inflammatory bowel diseases, including ulcerative colitis; and many other diseases [27–31]. Some scientists also suggest that SRB may be the cause of some forms of colon cancer, given the fact that these microorganisms produce hydrogen sulfide affecting the intestinal cell metabolism causing various diseases [32, 33]. That is why the isolation of SRB new strains, their purification from other microorganisms, and study of SRB cultural, physiological, biochemical, and genetical properties in

It should be also noted that many species may be uncultured, so it is important to apply molecular and genetic methods such as Illumina sequencing. This method can give a clear picture of SRB diversity in the detected sample. However, in this chapter, the focus will be on isolation, purification, and cultivation of cultured

• Methods of sample selections from water, soil, swamp, and feces of human or

• Identification based on physiological and biochemical properties and sequence

As was noted, the SRB can be present in a sulfate-rich environment. The samples selected from the different ecotopes should be directly placed in anoxic modified Postgate liquid medium [3]. The composition of the medium and conditions of

**2.1 Samples from environment (water, soil, swamp, and environmental** 

One milliliter of water (or 1 g of swamp, soil, and metal rust) should be suspended in 9 ml of anoxic Postgate liquid medium. The tubes should be brim-filled with medium and closed to provide anaerobic conditions. Another option to provide anaerobic conditions is to add in tube 1 ml sterile liquid paraffin. The schema

It is thought that the species of SRB, their composition, and the number found in the intestinal lumen differ from that of the composition and number of

**24**

*The scheme of sampling from different environmental biotopes.*

microorganisms on the surface of the intestinal mucosa [2, 9, 28, 34]. Similar to environmental samples (see **Figure 1**), fecal samples from human or animals should be fresh and directly suspended in anoxic modified Postgate liquid medium (pH 7.5, *temperature* to +37°C). One gram of feces suspends in 9 ml of the modified Postgate liquid medium [3, 35]. The same quantity of feces should be taken for determining the dry matter and recalculation of colony-forming units (CFU) per 1 g of dry matter. Before this procedure, the medium should be heated in thermostat to +37°C temperature. If the samples from domestic or wild birds (chickens, geese, ducks, etc.), the temperature of medium should be +40°C.

#### **2.3 Samples from intestine (biopsy or sections of the large intestine of animals)**

Intestinal SRB with other intestinal bacteria can form biofilms on the surface of the epithelial cells of the large intestine [34]. These biofilms include species of *Desulfovibrio* genus and the species of *Bacteroides*, *Pseudomonas*, *Clostridium*, *Escherichia,* or other intestinal microorganisms. Such biofilms are often resistant to antimicrobial substances [36]; therefore it is an interesting area of the study.

For isolation of SRB from biofilms, 10<sup>−</sup><sup>5</sup> M EDTA (ethylenediaminetetraacetic acid) should be added to the modified Postgate liquid medium for releasing SRB from a biofilm. A fresh piece of biopsy should be weighed, and its approximate square (in cm<sup>2</sup> ) must be calculated and added to 9 ml of the modified Postgate liquid medium (pH 7.5, *temperature* to +37°C). This calculation of square must be done for recalculation of CFU of SRB released from cm<sup>2</sup> of a biofilm.

The same procedure can be applied for isolation of SRB from sections of the large intestine of animals.
