The document describes the procedure to isolate rhizobium bacteria from root nodule samples. Key steps include washing and sterilizing root nodules, crushing the nodules, and streaking the crushed material onto YEMA media plates which allow for the growth of distinct rhizobium colonies that break the red color of the media and appear as white colonies. The procedure results in the observation of light yellow to pink homogeneous colonies of rhizobium growing on the media plates.

1. ISOLATION OF RHIZOBIUM
FROM ROOT NODULES

2. AIM :
To isolate rhizobium bacteria from the
given root nodule samples.

3. PRINCIPLE :
• The nitrogen fixation by free living nitrogen fixing bacteria is
carried out aerobically by Azotobacter species and micro
aerobically by Azospirillum species.
• Clostridium species fix the nitrogen anaerobically.
• Free living nitrogen fixers require an organic carbon source
for growth but can grow in a medium which is devoid of
nitrogen source.

4. MATERIALS REQUIRED :
Sample (Root nodules)
Glasswares
YEMA Media
Sterile water
Mercury chloride
Ethyl Alcohol
Motor and Pistal
Inoculation Loop

5. PROCEDURE :
• Immerse the root in the sterile water and
wash it to remove the soil.
• Cut the root nodules in the end and not in the
center.
• Cut the root nodules using the sterile blade.
• Immerse the root nodules in the sterile water
and wash it.

6. • Immerse the root nodules in the Mercury
chloride solution of (0.1%) and wash it for
5 minutes.
• Immerse the root nodules in the Ethyl
alcohol solution of (70%) and wash it for 3
minutes.
• Wash the root nodules in sterile water for
2 times.

7. • Crush the root nodules by using the
sterile motor and pistil.
• Then finally do the quadrant streaking
in the YEMA media by taking a single
loop of culture.
• Rhizobium bacteria breaks the red
colour and grows as a mucus white
colonies.
OBSERVATION :

8. RESULT :
From the given soil sample, we observe
light yellow to pink homogenous colonies
in YEMA media plate.

9. DISCUSSION :
Manitol is the fermentable sugar alcohol source.Magnesium provides
cations, essential for the growth of Rhizobia. Congo red inhibits
penicillin suspectives strain colonies of Rhizobia stand out as white
trandesscent, glistening and elevated with entire margins.