Method: How to quantify Azotobacter chroococcum, Rhizobium leguminosarum, Bacillus megaterium, Frateuria aurantia in soils, organic fertilizers and other products and media.
1. 1 g of a stock of each product is mixed with 99 ml of sterile distilled water.
2. From this, 1 ml is taken and diluted in 9 ml sterile distilled water containing test tubes, then serially diluted up to 7 times (from 0.1 to 0.0000001).
3. From each tube, 0.1 ml is spread on the selective solid media in Petri dishes (one sample's dilution per a dish): 1) Jensons medium, Waksman-77 or nutritive solid agar (for Azotobacter chroococcum), 2) Nutrient Agar or Pikovskaya's medium (for Bacillus megaterium) and 3) Yeast Extract Mannitol Salt Agar (for Rhizobium leguminosarum), 4) Mannitol Egg Yolk Polymyxin (MYP) Agar (for Frateuria aurantia) respectively, and incubated overnight at 37ºC.
4. Numbers of bacterial colonies grown overnight is scored. Only dishes with the colonies 1) located randomly over media with low densities are used for counting and 2) having the characteristic morphology and color (anticipated for each selective media) are used for scoring.
5. Quantity of each bacterial species in the product stock is calculated according to the formula below:
N= 100 x n / d, where N is a quantity of bacteria of each species in 1 g of the product sample, 100 is the original dilution of the sample, n is number of colonies grown in a dish with the solid selective media, and d is a serial dilution of the sample (from 0.1 to 0.0000001).
The counting data for each bacterial species in each sample are collected in triplets and averaged respectively.
*The procedure is performed by a certified microbiologist in the certified laboratory.
References:
1. Kuppuraj Raja Sekar, Natchimuthu Karmegam. Earthworm casts as an alternate carrier material for biofertilizers: Assessment of endurance and viability of Azotobacter chroococcum, Bacillus megaterium and Rhizobium leguminosarum. Scientia Horticulturae 124 (2010) 286–289.
2. S. Umesha, M. Srikantaiah, K. P. Vishwajith, K. S. Prasanna, M. Divya. Microbial Population and Nutrient Uptake of Maize as Influenced by Biofertilizers and Different Sources of Organics under Field Conditions. Environment & Ecology 33 (1B) : 448—452, January—March 2015. ISSN 0970-0420
3. D. Barrie Johnson, Stewart Rolfe, Kevin B. Hallberg and Eigil Iversen. Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine. Environmental Microbiology (2001) 3(10), 630-637.