Isolation and enumeration of phosphate solubilising bacteria from different habitats

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BACKGROUND

Bio fertilizers are the product that is comprise of specific living microbes that are beneficial and are added to the oil as inoculants of microbes. They are very popular due to its non-hazardous, environmental friendly and chemical free nature. Various microorganisms like Azolla, phosphate solubilising microbes, cyanobacteria, and endophytic diazotrophs are utilized for making bio fertilizers. A major portion of the soil phosphate is present in form of insoluble phosphorus and thus can’t be used by plants. Large portion in chemical fertilizer remains unavailable after its application in the oil. It is because of the strong bond that is formed between phosphorus with magnesium and calcium in alkaline pH and in acidic soils with iron and calcium. This element moves very slowly in the soil and do not respond to speedy take up by plants. This results in formation and growing of phosphorus depleted zones in places of roots and oil in rhizosphere. Phosphorus deficiency can also form small leaves and slow growth of plants. Plant growth promoting bacteria are those bacteria present on the soil and rhizosphere that can render high benefit to the growth of the plant trough various mechanisms and phosphorus solubilising capability of microorganisms is one of the most important factor associated with plant phosphorus nutrition. High cost and bad impact of chemical fertilizers has promoted the use of plant promoting bacteria in most of the agricultural practices.

Phosphate solubilising microorganisms play vital role in providing phosphorus to the plants. They are slowly becoming vital organisms for improvement of soil. The main objective of this test is to isolate phosphate solubilising bacteria.

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REQUIREMENTS

Soil sample: The soil samples should be collected from various habitats.

Medium: Pikovskay solid medium

Others: Plates

   Flask

   Inoculating loop

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PROCEDURE

Homogenization of soil sample: Mix the soil sample homogeneously in double distilled water in the ratio 1:2 and subject it to centrifugation for about 5 minute at 250C at 5000rpm.1

Isolation: After homogenization is done, take 1 g of each sol sample and serially dilute it upto 10-4 dilution. Then from the dilution plate 10-3 and 10-4 take 1 ml and plate on Pikovskaya agar medium by pour plate method.2 This medium is specifically used for isolation of PSB microorganisms. Incubate these plates under aerobic conditions at 320C for about 9-10 days.3

Screening and selection of PSB: After incubation period, count the colonies with clear zones that indicate number of phosphate solubilising bacteria and this is colony forming unit per gram of soil.4 In each plate watch out for single, well separated colonies with clear zones and streak it on Pikovskay solid medium utilizing quadrant streak method. Repeat this process till you get pure culture with high p solubilisation.5 Those isolates that showed perfect and clear zones is inoculated in nutrient broth and incubated 280C for 72 hours at 100 rpm for isolation and DNA quantification. Morphology of the isolates can be studied by doing gram staining, starch hydrolyses and catalase testing.6

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CONCLUSION

This is one of the reliable and best methods for isolating phosphate solubilising method.

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REFERENCES

  1. Arpana N, Kumar SD, Prasad TN. Effect of seed inoculation, fertility and irrigation on uptake of major nutrients and soil fertility status after harvest of late sown lentil. J Applied Biol 2002; 12:23-6.
  2. Pikovskaya RI. Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Microbiologiya 1948; 17:362-70.
  3. SundaraRao WVB, Sinha MK. Phosphate dissolving organisms in the soil and rhizosphere. J Indian J AgricSci 1963; 33:272-8.
  4. De Freitas JR, Banerjee MR, Germida JJ. Phosphate-solubilising rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L.). Boil Fert Soil 1997; 24:358-64.
  5. Eckford MO. Thermophilic bacteria in milk. American J Hygiene 1927; 7:201-2.
  6. Blazevic DJ, Ederer GM. Principles of biochemical tests in diagnostic microbiology, New York, USA: Willey and Co.; 1975. p. 13-45.