Enrichment and Isolation of Rhizobium

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Introduction

Soybean root nodule [Click here for source]

Bacteria of genus Rhizobium are fast growing Gram-negative rods that are normally involved in nitrogen fixation for the atmosphere.  Rhizobia are commonly found in the root nodules of legumes; we obtained our samples from the root nodules of a greenhouse soybean plant inoculated with Bradyrhizobium japonicum (formerly called Rhizobium japonicum) and of naturally growing clovers on campus.  Our goal was to derive an enrichment culture protocol that allowed for optimal growth of Rhizobia from both samples.  The soybean nodule was manually inoculated with Rhizobium, so results from enrichment and isolation techniques for the soybean Rhizobium could serve as a basis for determining legitimate growth of Rhizobium on the clover sample, of which we weren’t sure of the presence of the bacteria.

Yeast mannitol agar (YMA) has been recommended (Microbial Media, www.nexusresearchgroup.com) for the isolation of Rhizobium, so we used this media for our protocol.  YMA contains yeast extract, which provides the bacteria with a nitrogen source, a necessary element for natural Rhizobium function.  YMA also contains mannitol, a major one of the many carbohydrates that Rhizobium can utilize for metabolism.  Our enrichment culture protocol also involves incubating the bacteria at room temperature (20-25 C) at standard room pH (6-8) and regular lighting for at least two days.  As this method follows Bergey’s description of optimum Rhizobium growth conditions (Bergey’s 5th  ed.), we expected our enrichment culture to be successful in growing the desired organism and having the organism achieve desired results via isolation techniques.