**4. Culture dependent approaches**

## **4.1 Plate count**

Standard culture technique involves selective plating, direct counting viable colonies/cells and characterization of microorganisms on artificial or synthetic growth media in laboratory condition [12]. The major limitation of the culture dependent techniques is that we are unable to study the microbes which cannot be grown on artificial media. In spite of putting efforts in improving the culture media which can copy the natural conditions for the growth of the microbes, it was seen that the majority of the fraction of microbes were still unculturable. The minimal media, culture dilutions and the extended incubations helps in recovery of few slow growing microbial species which were considered as uncultivable earlier, but the abundance of the microbes in soil, which can be seen under microscope, still remains untapped or untouched. A technique has been devised for the cultivation of uncultured microorganisms from different environments that involved encapsulation of cells in gel micro-droplets for large-scale microbial cultivation under low nutrient flux conditions [13].

### **4.2 Community level physiological profiling (CLPP)**

Garland and Mills developed a technique to assess the potential functional diversity of the bacterial population through Sole Source Carbon Utilization (SSCU) patterns [14]. Based on carbon utilization, BIOLOG (https://biolog.com) introduced the technique into ecological studies to estimate metabolic potential of microbial communities. The BIOLOG plate method was used for comparing metabolic activity of heterotrophic microbial communities from different habitats using redox chemistry. These plastic microtiter plates contain colorless tetrazolium dye with 95 different carbon substrates (e.g. carbohydrates, amino acids, amines, amides, carboxylic acids, etc.) in wells, and one well without any substrate used as control. Soil samples are shaken in a suitable solution; the soil solution is inoculated into the plate wells and incubated at constant temperature. Soil microorganisms oxidize the substrates in the microtiter plate wells and also reduce the tetrazolium dye to a violet formazan which is measured spectrophotometrically [15]. Currently BIOLOG has developed various microtiter plates for identification of aerobes, anaerobes, yeasts and filamentous fungi. This method is rapid, convenient and can help to identify over 2900 bacteria, fungi and yeast. But the drawback is the microbes which are cultivable and able to grow in high-nutrient conditions can only be studied. This method was used mainly to investigate microbial communities from

*Soil Metagenomics: Concepts and Applications DOI: http://dx.doi.org/10.5772/intechopen.88958*

bulk and rhizosphere soil and to estimate the impact of stressors on soil microbial communities [15]. The BIOLOG microplates were used for identifying the bacterial population in the contaminated soils around the Explosive Institute of Riyadh community (Saudi Arabia) which shown *Proteobacteria* to be the dominant phylum in all the five regions with Burkholderiaceae, Chromatiaceae and Pseudomonadaceae common and dominant families representing 49% of the relative abundance at the family level [16]. Compared to earlier microtiter plates, BIOLOG EcoPlate are more in use for Community Level Physiological Profiling (CLPP) as it contains three replicates of 31 different carbon sources and one control well per replicate (Biolog Inc., Hayward, CA, USA) [15].
