Enrichment and Isolation of Rhizobium

Protocol

Materials

Agar, K2HPO4, Mannitol, Yeast extract, MgSO4·H2O, NaCl, beakers, flasks, graduated cylinders, innoculating loops, test tubes, aluminium foil, pair of scissors, distilled water, bleach, alcohol, ziploc bags, tweezers, scaples, glass rods, petri dishes, slides, Iodine, safranin, Crystal violet, Maneval's solution, Congo red, Oxidase, Hydrogen Peroxide and dextrose tubes.

Making the medium

A total of 3 liters of Yeast Mannitol Agar(YMA) was made:

Agar	15.0g
Mannitol	10.0g
K2HPO4	0.5g
Yeast Extract	0.4g
Mg2SO4·H20	0.2g
NaCl	0.1g

1. Components were poured into a large flask and the volume was brought to 1L by add distilled water
2. Heat & mix for dissolving chemical
3. Autoclave

Preparation of bacteria sample

1. Plant inoculated with Rhizobium was obtained
2. Brown nodules with a little bit of root were isolated from root
3. Rinsed with water
4. Nodule were immersed in 1% chlorine bleach solution in sterile petri dishes for 15 min
5. All the materials are used to manipulate the nodules have to be washed by 70% alcohol
6. Bleach solution was poured off
7. Nodules were immersed in 70% alcohol
8. The lid was closed and the dish was swirled for about a minute
9. The solution was again poured off
10. Nodules were immersed in distilled water
11. The dish was swirled with the lid closed for a minute. This step was repeated two more times for rinsing
12. The tweezer sitting in alcohol was passed over flame for sterilization
13. The largest root nodules were transferred to separate drops of water on a petri dish and crushed
14. A large number of Yeast Mannitol Agar plates were streaked with samples using sterile technique.
15. The plates were inverted and storage at RT for 2~3 days