Enrichment and Isolation of Rhizobium

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Method

A. Making the medium
A total of 3 liters of Yeast Mannitol Agar (YMA) [Microbial Media, p. 1007] was made: 
 

Agar	15.0g
Mannitol	10.0g
K2HPO4	0.5g
Yeast Extract	0.4g
Mg2SO4.7H2O	0.2g
NaCl	0.1g

Components were poured into a large flask and the volume was brought to 1 L by adding distilled water. The flask opening was covered with foil, and medium was gently heated to boiling with the stir bar mixing the solution. The flask was autoclaved for 15 minutes at 15 psi at 121 C. Plates were poured and allowed to cool overnight.

B. Isolation/Confirmation techniques
 

Soybean plant inoculated with Rhizobium last year was obtained from the greenhouse with the permission of Dr. Kenworthy of at UMCP. Brown nodules ~7mm wide and a little bit of root were cut from the root system of the soybean plant.

Clover with white nodules growing on roots were cut from a clover patch outside of the Microbiology Building on UMCP campus. These samples were kept in some soil in a Ziploc bag punctured with several holes for air, stored in a dark place for two days before nodules were processed.

 

Cut Soybean root nodule

[Click here for source]

 

Nodules were cleaned and prepared according to Part B of Final protocol

Large number of Yeast Mannitol Agar plates were streaked with either soybean or clover samples, using sterile technique. The plates were inverted and left to grow at room temperature (20-25 C) for two or three days.

Gram stains were performed on the two different clover colonies and the soybean colonies. Gram stains were done after the first and second streaking of the plates. A capsule stain was performed on the soybean and clover samples. Wet mounts were prepared for soybean and clover samples. Movie clips showing motility were made for the clover sample. Catalase and oxidase tests were performed for soybean and clover samples. Dextrose tests were performed for a soybean and a clover sample. One soybean plate and two clover plates (one yellow and white clover colony sample) were placed in the anaerobe chamber for three days.