EXERCISE 9
RHIZOBIAL ANTIGEN-ANTIBODY REACTIONS IN GEL BY IMMUNODIFFUSION
Immunodiffusion in gel allows for the recognition of
antigenically identical strains and for differentiating between
closely related nonidentical strains.
Soluble and diffusible
heat-stable antigens of varying molecular size are studied in
this technique.
Key steps/objectives
1)
Inoculate YMA flats for antigen preparation
2)
Prepare gel in plastic Petri dishes
3)
Harvest cultures for antigen preparation
4)
Perform immunodiffusion
5)
Observe and record diffusion patterns
(a)
Preparing gel for diffusion
(Key step 2)
Place 100 ml of saline into a 250 ml Erlenmeyer flask.
Add 0.75
g of Difco Noble agar or Oxoid Ion agar No. 2 to the flask and
melt by steaming, autoclaving or heating in a microwave oven.
If
direct heat is applied to melt the agar, prevent charring of the
agar on the bottom of the flask by constant stirring and
controlling the heat.
To the melted agar, add 1 ml of a 2.5 %
(w/v) solution of sodium azide (a preservative), and swirl the
flask to ensure proper distribution of the sodium azide.
Pipette
25 ml of the hot gel into Petri dishes kept on a level surface.
Allow the agar to solidify.
A total of four plates with a gel
layer 4 mm in thickness should result.
Trace the outline of the bottom of a Petri dish on a sheet of
white paper.
Draw a hexagonal pattern of six circles (4 mm
diameters) equidistant (5 mm from edge to edge) from one another
in the center of the plate outline on this paper.
Draw a seventh
well in the center of the hexagonal pattern and shade in the
circles (Figure 9.1).
This pattern on the paper serves as a
template for cutting out wells from the gel.
Place a Petri dish (containing gel) on the template.
of circles should be visible through the gel.
gel using a 4 mm cork-borer.
The pattern
Cut wells into the
The cork-borer should be held
vertically when cutting the wells, otherwise wells with oblique
walls will result.
Carefully remove the gel plugs with pins or
other suitable implement or remove the plugs by suction using a
Pasteur pipette (with a slightly bent tip) attached to a suction
apparatus.
(A Pasteur pipette attached to an aspirator or vacuum
pump, with a "trap" in between for the gel plugs, is a suitable
suction apparatus.)
It will take some practice to produce plates with seven intact
wells.
A drop of molten agar may be necessary to seal off the
Figure 9.1.
Hexagonal pattern template for Petri dishes.
bottom of the well.
Sealing the well is usually not necessary
with the plastic Petri dishes but is essential for glass Petri
dishes.
The gel plates may be refrigerated if not required for
immediate use.
Make four sets (one set per Petri dish) of the
hexagonal pattern of wells.
Three or seven sets of wells can be
made per Petri dish with sufficient experience and care.
(b)
Preparing antigens
(Key steps 1 and 3)
Culture the following strains of Bradyrhizobium sp. on YMA flats:
TAL 651 (from Calopogonium mucunoides)
TAL 653, 655, and 855 (from Centrosema pubescens)
TAL 642 (from Lablab purpureus)
Harvest the cultures after 7 days of growth (Exercise 6) and
prepare antigen suspensions for immunodiffusion.
A final volume
of 1.0-1.5 ml of a dense antigen suspension containing
approximately 1 x 1010 cells ml-1 is desirable.
Divide the antigen suspension of each strain into two small
screw-capped tubes.
Small McCartney bottles are better
substitutes if these are available.
Heat treat one sample for 1
h at 100C by immersing the tube in boiling water.
Leave the
other sample unheated (untreated).
(c)
Setting up immunodiffusion reactions
(Key steps 4 and 5)
Place 2 drops (0.04 ml drop-1) of each heat-treated antigen in
their respective wells.
The position of the different antigens
for the diffusion is as shown in Figure 9.2.
Figure 9.2.
Well pattern for immunodiffusion.
Place the undiluted antiserum of TAL 655 in Well-7.
Similarly, set up another set of wells for immunodiffusion with
untreated (unheated) antigen.
Labelling on the bottom of the Petri dishes is essential to
facilitate the identification of the antigens in the wells.
Orientation of the dish can be established with a single line at
the 12 o'clock position and a diagrammatic record of the location
of each well.
Incubate the Petri dishes at room temperature in a
water-saturated atmosphere.
A saturated atmosphere is necessary
to prevent moisture loss from the gel.
Air-tight plastic boxes
can be improvised to provide this environment by placement of wet
paper towels on the inside prior to closing of the boxes.
Make observations at 24 and 48 h.
the form of drawings.
Record your observations in
Compare the diffusion patterns of the
heated and unheated antigens.
Interpret the diffusion patterns
for reactions of identity, partial identity, and nonidentity.
Heating can significantly alter the reactivity, concentration and
diffusibility of the somatic antigens leading to stronger and
well separated precipitin bands.
Figure 9.3.
Immunodiffusion reactions showing preciption bands.
Requirements
(a)
Preparing gel for diffusion
Autoclave, stove or microwave oven
Saline (100 ml)
Erlenmeyer flask (250 ml)
Sodium azide
Noble agar (purified agar) from Difco, Detroit, Michigan or
Oxoid Ion agar No. 2
Plastic Petri dishes (four)
Hexagonal pattern template
Cork-borer (4 mm)
(b)
Preparing antigens
Agar slant cultures of bradyrhizobia (TAL 642, 651, 653,
655, and 855) or other rhizobia
YMA slopes (five) in 500 ml flat medicine bottles
Screw-capped tubes (or small McCartneys)
Steam- or water-bath
(c)
Setting up immunodiffusion reaction
Pasteur pipettes
Rubber bulbs (1-2 ml capacity)
Air tight plastic boxes (or substitute of similar function)