EXERCISE 8

TO AGGLUTINATE ANTIGENS FROM ROOT NODULES

Identification of a particular Rhizobium strain, by direct use of

the bacteroids in the nodules, is described. This direct method

eliminates the time consuming steps of isolating the strain in

pure culture prior to its use as an antigen in an agglutination

reaction.

Key steps/objectives

1) Develop antiserum of B. japonicum)

2) Prepare Leonard jars

3) Prepare broth inoculum

4) Pregerminate soybean seeds

5) Plant and inoculate pregerminated soybean seeds

6) Harvest plants for nodules

7) Perform agglutinations with bacteroid antigens

8) Read and record agglutinations

(a) Developing antisera

(Key step 1)

Inoculate B. japonicum strain TAL 379 onto YMA-flats.  Harvest

culture and make antigen preparation for the development of

antibodies for agglutination as described in Exercise 6. Other

strains of B. japonicum, for which antisera are available, may be

substituted in place of TAL 379.

(b) Culturing soybean plants nodulated with a serologically

marked strain of B. japonicum

(Key steps 2, 3, 4, 5, and 6)

Prepare Leonard jars.  This should be done well ahead of

experiment.

Inoculate 100 ml of YM-broth in a 250 ml Erlenmeyer flask with a

loopful of TAL 379 from an agar slant.  This should be initiated

at least 7 days before planting the Leonard jars to give

sufficient time for the growth of the culture.

Surface sterilize soybean seeds as described in Appendix 10. 

Plate seeds on water-agar plates for germination.  Do not invert

plates for soybean and other large seeded legumes. Pregerminate

seeds 1-2 days before planting and inoculation in Leonard jars.

Plant three germinated soybean seeds in each Leonard jar. 

Inoculate each seed with 1 ml of TAL 379 broth culture.  Plant

four jars.

Harvest and wash the soybean nodules after 30-35 days of plant

growth.  Separate the nodules from the roots.  Pack and seal the

washed nodules in small polyethylene bags (size 100 mm x 100 mm

and 0.04 mm thickness).  Use one bag per plant and label.  Bags

of the specified size or slightly smaller can be purchased

commercially or can be made in the laboratory if the polyethylene

material and a bag sealer are available.

(c) Separating bacteroid-antigens from nodules for agglutination

(Key step 7)

Fill a 1 liter beaker with approximately 500 ml of water and

bring it to a boil; then control the heating source to produce

gentle boiling.  Immerse one bag of nodules in the boiling water

for 3-5 min, then remove the bag and cool.  Save the remaining

bags of nodules for Exercise 9.

Cut open the plastic bag with scissors.  Using forceps, transfer

the nodules to the agglutination tray, one nodule per well.  Have

one nodule in each well, beginning at well A-1 through A-10

(refer to the well identification system on the agglutination

tray).  Leave wells B-1 through B-10 empty; these wells will be

used for agglutinations with antigens separated in the series of

wells in row A.

With a Pasteur pipette, place 6 drops (0.03 ml drop-1) of saline

into each well containing a nodule.  (Excess heat-treated nodules

can be stored frozen and thawed later for use in agglutination

without losing the ability of the bacteroid-antigens to

agglutinate).

Gently press out (do not homogenize) the nodule contents into the

saline in the wells with fine forceps or a round-ended glass rod

(4 mm diameter).  Gently stir the exuding nodular contents into

the saline and push the resulting nodule tissue against the wall

of the well.  Rinse the rod and wipe dry for each nodule.

Suitable flat toothpicks can be substituted for forceps or glass

rods. Toothpicks are used once and discarded.

With a fresh Pasteur pipette, transfer 3 drops of the antigen

from well A-1 to B-1.  Rinse the pipette thoroughly with hot

water by sucking the hot water into the pipette and emptying the

pipette contents in another beaker.  Next, transfer (with the

same pipette) 3 drops from well A-2 to B-2.  With alternate

rinsing of the pipettes between transfers, transfer the antigen

A-3 to B-3, A-4 to B-4, and so on until A-10 to B-10.  (In these

transfers, the same pipette is used each time as the nodules were

formed by one strain, TAL 379.  If the nodules were formed by

strains from a mixed inoculum, each antigen transfer must be done

with a fresh Pasteur pipette).  Variable Finn pipettes with

disposable tips are excellent substitutes for Pasteur pipettes,

especially when large numbers of nodules need to be identified,

since a fresh tip can be used for each nodule.

(d) Agglutinating the bacteroid-antigens with homologous

antiserum

(Key steps 7 and 8)

Prepare a 1/25 dilution of antiserum TAL 379 by diluting 0.4 ml

of the stock antiserum in 9.6 ml of saline.

Dispense the diluted antiserum by placing 3 drops in each of the

wells B-1 through B-10.  Place 3 drops of the 1/25 diluted

antiserum and 3 drops of saline in well B-11 (serum control). 

Set up the antigen-saline control in well B-12 by placing 3 drops

of antigen from well A-1 followed by the addition of 3 drops of

saline.  Mix the reactants in the wells with a round-ended glass

applicator, starting at well B-10 and proceeding towards well

B-1.  Use the same applicator for mixing the contents in the

wells.  Rinse and wipe dry the applicator between each mixing. 

The contents in the wells may also be mixed by holding the plate

loosely in a level position with both hands and tapping the side

of the plate with a free forefinger.  Avoid spilling during this

operation.

Cover the tops of the wells containing the reactants with a strip

of cellophane tape.  This will prevent evaporation during

incubation.  Leave a tab of tape to assist removal.

Place the trays at 37C for 2 h in an incubator.  At the end of

this time transfer the trays to 4C in a refrigerator and leave

overnight.

Record the appearance of the positive agglutinations by

comparison with the antigen-saline control.  (The titer of the

antiserum at which the agglutinations occurred would be 1/25 x

3/6 = 1/50.)

This method has been used on nodules of Glycine max, Centrosema

pubescens, Vigna unguiculata, and Phaseolus lunatus with good

results.  These legumes have nodules of similar size.  With other

species of legumes, the volume of saline to be used in squashing

the nodule to extract the bacteroid antigen has to be determined

by trial and error.  The volumes of the bacteroid-antigen and the

serum in the wells also have to be determined before large

numbers of nodules are identified by agglutination.  The use of

thick suspensions of the bacteroid antigen should be avoided

because unreacted antigen produces turbidity resulting in

ambiguity in the recognition of positive agglutinations.  The

ultimate purpose of manipulating the volumes of saline and serum

to be used during the agglutination is to regulate the density of

the antigen close to 1 x 109 bacteroids ml-1.

Figure 8.1.  Identification of nodule bacteroids by

agglutination in an agglutination tray.

Requirements

(a) Developing antisera

See Exercise 6

(b) Culturing soybean plants nodulated with serologically marked

strains of B. japonicum

Sterilizing solutions (See Appendix 10)

Sterile water (500 ml)

Sterile 250 ml wide-mouthed flask

Soybean seeds

Water-agar plates (three)

100 ml broth culture of B. japonicum (TAL 379)

Sterile 5 ml pipettes

Sterilized gravel (mulch for Leonard jars)

Leonard jars (four)

Isopropyl alcohol in spray bottle

Spirit lamp, matches

Forceps

Scissors, polyethylene bags

(c) Separating bacteroid-antigen from nodules

Beaker (1 l)

Scissors, fine forceps

Sterile Pasteur pipettes (or variable Finn pipettes with

disposable tips if available)

Bunsen burner

Round-ended glass rod

Sterile saline (250 ml)

Tissue paper

Agglutination tray (rigid polystyrene "U" plate)

Nodules containing bacteroids of B. japonicum TAL 379

Nodules containing bacteroids of B. japonicum TAL 378

(d) Agglutinating the antigens with homologous antiserum

Antiserum (TAL 379)

Calibrated Pasteur pipettes

Antigen from (c)

Glass applicator

Cellophane tape

Incubator (37C), refrigerator (4C)