Demonstration of rhizobium from root nodules and its isolation

Demonstration of rhizobium from root nodules and its isolation

Aim:

To demonstrate the presence of rhizobium in root nodules by gram staining and isolate them on a nutrient medium.

Theory and Principle:

Leguminous plants like cowpea, red gram , 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.

Based on gram staining reaction bacteria can be divided into two large groups called gram positive and gram negative. Gram positive bacteria have thicker peptidoglycan compared to the gram negative bacteria. The lipid content of the cell wall of gram negative bacteria is higher than that of gram positive bacteria. Both gram positive and gram negative bacteria take up the primary stain crystal violet and stain red. When decolorized the porosity and permeability of the gram negative bacteria increases due to which the crystal violet iodine complex is given out by the gram negative bacteria. Further the gram negative bacteria takes up the counterstain safranin and stains red. Hence when bacteria are observed under microscope after gram staining the gram positive cells appear violet and gram negative cells appear red. Rhizobia are Gram- negative rods which are motile with bi-polar, sub-polar and peritrichous flagella

 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.

Requirements:

1. Root nodules (pink) of any leguminous plant

2. Congo red, Yeast Extract, Mannitol Agar (pH 6.8 – 7.0):

Mannitol : 10.0 g

K₂HPO₄ : 0.5 g

MgSO₄.7H₂O : 0..2 g

NaCl : 0.1 g

Yeast extract : 1. G

CaCO₃ : 3.0 g

Agar : 25.0 g

Congo red (1% aqueous)

2.5 ml (1.0 g in 100 ml)

Distilled water 1000.0 ml

3. Inoculation loop

4. Bunsen burner/laminar clean air flow hood.

5. Slides and glass rod.

6. Petri plates with YEMACR medium.

7. Sterile distilled water.

8. 95% alcohol and 0.1% HgCl₂.

Procedure:

1. Wash the root system under a slow stream of running tap water, taking care to see that the nodules are intact

2. Select pink nodules and remove them

3. Wash and keep the nodules in 95% ethanol for a minute, wash and transfer them to 0.1% HgCl₂.

4. Remove after five minutes and wash the nodules about four to five times with sterile distilled water

5. Place the nodule on a serile slide in a drop of sterile distied water and crush it either with a sterile glass rod or a flat tipped forceps

6. Remove a loopful of this cloudy suspension and streak inoculate on YEMACR plates and label.

7. Incubate in dark at 28°-30°C for 2-3 days and observe the colonies

.8. Make a smear of the remaining crushed material and gram stain and observe the gram negative bacilli. Even samples from the colonies can be gram stained.

Diagram:.

Observation

Gram’s stain

Colony characteristics of rhizobium on YEMA after incubation for 2-3 days at room temperature 

Size – 2-4 mm

Shape- circular

Colour – White

Margin – entire

Elevation – convex, raised

Opacity – semitranslucent

Texture – creamy

Consistency – mucilaginous

Gram nature – gram negative

Motility – actively motile

Results:

Gram staining of the root nodule exudate revealed the presence of gram negative rods.

The colony characteristics of rhizobia were studied after isolation on YEMA medium.

White, creamy, mucoid colonies were obtained.