ISOLATION OF SYMBIOTIC NITROGEN FIXERS - RHIZOBIUM

 ISOLATION OF SYMBIOTIC NITROGEN FIXERS - RHIZOBIUM 

AIM: 

 To isolate the symbiotic nitrogen fixers- Rhizobium from root nodules of leguminous plants. 

PRINCIPLE: 

 Leguminous plants like cowpea, red gram, and black gram contain root nodules formed by rhizobium. Rhizobium in the soil enter into the roots of leguminous plant and form nodules and establish symbiotic association. Bacteria derive nutrients from the plants. The rhizobacteria fix nitrogen which is beneficial to the plant. Rhizobium is a symbiotic N2 fixer found to occur as bacteroids in the root nodules of leguminous plants. They can be easily isolated and cultured in vitro. Rhizobia are Gram- negative rods which are motile with bi-polar, sub-polar and peritrichous flagella.

root nodules of leguminous plant

 Biological Nitrogen Fixation is a natural process where certain bacteria and leguminous plants with nodules in their root systems are able to convert the nitrogen gas into a form that is usable for plant life. The ability to fix atmospheric nitrogen into a form that can be used for plant growth is confined to bacteria and cyanobacteria. Plants fix nitrogen only by virtue of associations with these simple organisms. The best-known associations are the symbioses of Rhizobium bacteria with legumes. Nitrogen fixing leguminous plants not 13 only supports plant growth independent of mineral nitrogen in the soil but also improve soil nitrogen status for associated crops by the residues of these plants. Rhizobium grows well on Yeast Extract Mannitol Agar (YEMA). Congo red added to the medium differentiates rhizobia that stand out as white, translucent, glistening elevated, small colonies with entire margin, in contrast to the red stained colonies of Agrobacterium and other bacteria. 

MATERIALS REQUIRED: 

• Root nodules 

• YEMA medium 

• Petri dishes 

• 70% ethanol 

• 0.1% mercuric chloride 

• Sterile tap water 

PROCEDURE: 

✓ Prepare and Sterilize YEMA medium. 

✓ Uproot the roots of leguminous plants and brought to the laboratory.

✓ Wash root systems in running tap water to remove adhering soil particle. 

✓ Select and wash healthy pink unbroken and firm root nodule.

 ✓ Immerse nodules in 0.1% mercuric chloride for 5minutes for surface sterilization. 

✓ Place nodules in 70% ethyl alcohol for 3 minutes.

 ✓ Wash nodules repeatedly by using sterile water. 

✓ Crush nodules using glass rod in 1mL of water.

 ✓ Make uniform suspension of nodule with help of sterile water. 

✓ Spread 0.1mL of each suspension from various dilutions on YEMA plates or Streak loop full of suspension. 

✓ Incubate all plates at 26 °C for 10 days. 

OBSERVATION: 

On YEMA medium Rhizobium grown as White mucoid colony. 

observation on YEMA media

RESULT: 

The symbiotic nitrogen fixers- Rhizobium has been isolated from the root nodules of the leguminous plants.

 DISCUSSION: 

Rhizobium is a symbiotic N2 fixer found to occur as bacteroids in the root nodules of leguminous plants. The rhizobacteria fix nitrogen which is beneficial to the plant. They can be easily isolated and cultured in vitro. Rhizobium grows well on Yeast Extract Mannitol Agar (YEMA). Congo red added to the medium differentiates rhizobia that stand out as white, translucent, glistening elevated, small colonies with entire margin, in contrast to the red stained colonies of Agrobacterium and other bacteria.