TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.4
Double Diffusion Test In
Gels (Ouchterlony)

The gel double diffusion test (Ouchterlony) uses agar gels in which
preparations of viral antigens and specific antibodies are placed in
adjacent wells in a plate containing agar gel. The serological relationship
between viruses can be more precisely determined with this than with any
other method, except immuno-electrophoresis. In Ouchterlony, the
number of precipitation lines between reacting substances in opposing
wells corresponds to the number of antigenic components in a given
antigen-antibody system. However, this is one of the least sensitive
serological tests because the formation of precipitation lines depends on
high and equivalent concentrations of specific antigens and antibodies.

Materials and Methods

The following materials are necessary:

− Agarose
 − Sodium azide (NaN3)
− Sodium chloride (NaCl)
− Distilled water
− Plastic petri dishes (100 x 15 mm)
− Cork borer punch (5 mm diameter)
− Test tubes
− Rubber bulb
− Pasteur pipettes
−
2
Gauze pads (2.5 cm )
− Felt tipped pen or crayon
− Erlenmeyer flasks (125 ml and 250 ml)
− Test tube (100 ml)
− Burner
− Vacuum pump
− Kitasato flask (125 ml)

1. Gel preparation

Gel mixture
0.8 g agarose
0.2 g NaN3 +
100 ml distilled water

Put agarose and water in an Erlenmeyer flask stopped with cotton. Heat
the mixture in a water bath until the agarose dissolves. Cool to
approximately 40oC. Add sodium azide. Mix carefully to prevent the
formation of air bubbles. Dispense 15 ml of the solution on each petri
dish and partially cover until gel solidifies. Remove condensed water from
lids.

2. Sample preparation

Grind leaves in a mortar and using the tip of a pestle push sap to one
side. Cover macerated tissue with a damp gauze pad. Remove the juice
with a Pasteur pipette. Transfer to a labeled test tube.

3. Preparation of antiserum

Saline solution
0.85 g NaCl
100 ml distilled water
Mix well

Prepare the recommended antiserum dilution in saline solution. If the

optimum dilution is not known, try several titers (dilutions at 1/2, 1/4, 1/16,
1/32, etc.).

4. Preparation of the test plate

a) Use cork borer punch to make wells in the gel according to a

desired pattern. A 5-mm cork borer is normally used with 3 mm
spacing between wells. The agarose disks are removed by using a
vacuum system consisting of a Pasteur pipette connected through

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 a hose to a Kitasato flask, and a vacuum pump. If this equipment
is not available, use a dissection needle.

b) First, place the samples in the peripheral wells. Next, place the
antiserum in the center well. Use a clean Pasteur pipette for each
sample and antisera.

5. Incubation of the test plate

Place plates in a wet chamber. Use a hermetically sealed plastic

container with a piece of damp paper inside and a support to prevent the
place from getting wet.

6. Readings

The first reading is made after 2 hours and the second reading at 18
hours. For easier reading, use a dark box consisting of a wooden support
with an opening at the top big enough to support the dish firmly in place.
Place a fluorescent light inside the box.

Figure 1. M: test sample; V: positive control; As-V: antiserum against virus.

A: Reaction of antiserum and the corresponding control but not
with sample. B: Identity reaction between sample and control. C:
Partial identity reaction. D: Non-identity reaction.

Recommended Literature
Ball, E. 1974. Serological Test for the Identification of Plant Viruses.
Published by the American Phytopathological Society Inc. Plant
Virology Committee, USA.

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