424
E. A. FREUNDT E l AL.
the cover glasses. Agar is melted rapidly and allowed to stand for a few
minutes so that air bubbles can disperse. One millilitre is pipetted over
the 4.5 x 2.5 cm area marked on each microscope slide to give a layer of
agar 1 mm deep. For the macro-test, 7 ml of agar is pipetted into the ring,
giving a depth of 1.8 mm. The agar sets in a few minutes and the rings can
be removed. Before the wells are punched, slides should be transferred to
4°C in a suitable container with moist filter paper to avoid drying out. T h e
wells should not be punched until the slides or plates are required for use
and must be sealed by rapidly pipetting hot agar in and out. Wells are
filled with antigen or antiserum so that the fluid is just level with the agar.
Recharging the wells should be avoided since this practice may result in
displacement and multiplication of precipitin bands, especially if concentrations equal to or higher than those originally used are added.
Reactions are allowed to develop at room temperature and are usually
optimal between 24 and 48 h. Thereafter, the reactions often become too
diffuse to record. If the reactions develop too rapidly, it may be advantageous to conduct the test at 4°C. Plates and slides should not be subjected to changes of temperature, since this tends to produce artifacts.
Gels should be inspected at regular intervals and the results recorded,
preferably photographically, at different stages.
For storage, the gels can be dried and stained. Slides or plates are
washed for 2 days in saline containing the concentration of NaCl used in
the gel with two changes each day, and finally in distilled or deionised
water for a further day. The agar is covered with filter paper and dried on
to the glass in a warm place over a period of several days. Staining is by
immersion for 20-30 min in Amidoschwarz (Naphthalene black) 10B, or
Ponceau fuchsin (Section 2). Gels are decolourised in at least two changes
of decolourising solution until the background is clear.
Preliminary micro-tests are set up to select suitable reference antisera
and to establish that these sera do not react with constituents of the medium
(Section 2). The concentration of antigen which gives optimal precipitation
with the homologous antiserum is also determined. T h e actual test, in
which homologous and heterologous antigens (peripheral wells) are compared against a reference antiserum (central well), is then carried out.
Although reference sera are pretested for the presence of antibodies
against constituents of the medium, it is desirable that one of the peripheral
wells should contain the growth medium as a control. As a further control,
another slide is set up with antigens in the same positions, but with preimmunisation serum in the central well. I n the first series of tests, the
arrangement of the heterologous antigens in relation to the homologous
antigen is necessarily arbitrary, but in subsequent tests cross-reacting and
homologous antigens can be placed in adjacent wells.
E. A. FREUNDT E l AL.
the cover glasses. Agar is melted rapidly and allowed to stand for a few
minutes so that air bubbles can disperse. One millilitre is pipetted over
the 4.5 x 2.5 cm area marked on each microscope slide to give a layer of
agar 1 mm deep. For the macro-test, 7 ml of agar is pipetted into the ring,
giving a depth of 1.8 mm. The agar sets in a few minutes and the rings can
be removed. Before the wells are punched, slides should be transferred to
4°C in a suitable container with moist filter paper to avoid drying out. T h e
wells should not be punched until the slides or plates are required for use
and must be sealed by rapidly pipetting hot agar in and out. Wells are
filled with antigen or antiserum so that the fluid is just level with the agar.
Recharging the wells should be avoided since this practice may result in
displacement and multiplication of precipitin bands, especially if concentrations equal to or higher than those originally used are added.
Reactions are allowed to develop at room temperature and are usually
optimal between 24 and 48 h. Thereafter, the reactions often become too
diffuse to record. If the reactions develop too rapidly, it may be advantageous to conduct the test at 4°C. Plates and slides should not be subjected to changes of temperature, since this tends to produce artifacts.
Gels should be inspected at regular intervals and the results recorded,
preferably photographically, at different stages.
For storage, the gels can be dried and stained. Slides or plates are
washed for 2 days in saline containing the concentration of NaCl used in
the gel with two changes each day, and finally in distilled or deionised
water for a further day. The agar is covered with filter paper and dried on
to the glass in a warm place over a period of several days. Staining is by
immersion for 20-30 min in Amidoschwarz (Naphthalene black) 10B, or
Ponceau fuchsin (Section 2). Gels are decolourised in at least two changes
of decolourising solution until the background is clear.
Preliminary micro-tests are set up to select suitable reference antisera
and to establish that these sera do not react with constituents of the medium
(Section 2). The concentration of antigen which gives optimal precipitation
with the homologous antiserum is also determined. T h e actual test, in
which homologous and heterologous antigens (peripheral wells) are compared against a reference antiserum (central well), is then carried out.
Although reference sera are pretested for the presence of antibodies
against constituents of the medium, it is desirable that one of the peripheral
wells should contain the growth medium as a control. As a further control,
another slide is set up with antigens in the same positions, but with preimmunisation serum in the central well. I n the first series of tests, the
arrangement of the heterologous antigens in relation to the homologous
antigen is necessarily arbitrary, but in subsequent tests cross-reacting and
homologous antigens can be placed in adjacent wells.