669
genic streptococci (Kitao et al. 1979 ; Kitao 1982 ) and Edw. tarda (Amandi et al.
1982 ). Essentially, a drop of bacterial suspension ( ca . 10
8 cells/ml, prepared in
0.85 % (w/v) saline) is added to a microscope slide. This is followed by adding a
drop of antiserum (use a range of dilutions), with gentle mixing for 2 min. A positive response is indicated by clumping of the cells. The reaction may also be carried
out in microtitre wells, using serial dilutions of antisera (Toranzo et al. 1987 ). Of
course, it must be emphasised that the reliability refl ects the specifi city of the
antiserum.
• Precipitin reactions and immunodiffusion
The value of these tests for diagnosis has been overshadowed by other techniques, such as FAT and whole cell agglutination. For detailed discussion, the reader
should consult Kimura et al. ( 1978 ) and Smibert and Krieg ( 1981 ).
• Complement fi xation
This technique probably has greater value for fi sh virology than for fi sh bacteriology; it has been described by Ahne ( 1981 ).
• Antibody-coated latex particles
The so-called latex agglutination test has found widespread use for diagnosis of
ERM (e.g. Hansen and Lingg 1976 ), furunculosis and vibriosis (including Hitra
disease). Commercial kits have found success, despite some cross reactions
(Romalde et al. 1995 ). However, the technique may be readily adapted for most
bacterial fi sh pathogens. As originally described for Aer. salmonicida by McCarthy
( 1975a , b ), the test involves the use of globulins from hyper-immune serum (titre =
>1:5000) and sensitised latex. The globulins are precipitated by the addition of saturated ammonium sulphate to the antiserum, and the precipitated proteins are sedimented by centrifugation. They are subsequently re-dissolved in 0.9 % (w/v) saline,
dialysed overnight at 4 °C against three changes of saline, and, after centrifugation,
the supernatant, which contains the globulins, is stored at – 20 °C until required.
The latex particles (0.81 μm diameter; Difco) are sensitised in globulin solution at
37 °C for 2 h. For the test, 200 μl of the antigen (bacterial suspension in glycinebuffered saline, i.e. 0.73 % (w/v) glycine and 1 % (w/v) NaCl; pH 8.2; supplemented
with 1 % (w/v) Tween 80) is mixed for 2 min with an equal volume of sensitised
latex on a clean glass plate. A positive result is indicated by clumping of the latex.
The technique may be used for pure or mixed cultures and tissue. Thus, positive
diagnoses may ensue from tissues unsuitable for culturing, e.g. fi sh stored at −20 °C
for 14 days, 5 °C for 7 days, or from formalin-fi xed material (McCarthy 1975a , b ).
As before, positive and negative controls are necessary.
• Co-agglutination with antibody-sensitised staphylococci
Reported for Aer. salmonicida and Ren. salmoninarum , this technique is similar
to the latex test (Kimura and Yoshimizu 1981 , 1983 , 1984 ). Essentially, Sta. aureus
(ATCC 12598) is suspended in 0.5 % formalin-PBS for 3 h at 25 °C to inactivate the
cells, washed three times in fresh PBS. The cells are mixed with antiserum in the
Bacteriological Examination
genic streptococci (Kitao et al. 1979 ; Kitao 1982 ) and Edw. tarda (Amandi et al.
1982 ). Essentially, a drop of bacterial suspension ( ca . 10
8 cells/ml, prepared in
0.85 % (w/v) saline) is added to a microscope slide. This is followed by adding a
drop of antiserum (use a range of dilutions), with gentle mixing for 2 min. A positive response is indicated by clumping of the cells. The reaction may also be carried
out in microtitre wells, using serial dilutions of antisera (Toranzo et al. 1987 ). Of
course, it must be emphasised that the reliability refl ects the specifi city of the
antiserum.
• Precipitin reactions and immunodiffusion
The value of these tests for diagnosis has been overshadowed by other techniques, such as FAT and whole cell agglutination. For detailed discussion, the reader
should consult Kimura et al. ( 1978 ) and Smibert and Krieg ( 1981 ).
• Complement fi xation
This technique probably has greater value for fi sh virology than for fi sh bacteriology; it has been described by Ahne ( 1981 ).
• Antibody-coated latex particles
The so-called latex agglutination test has found widespread use for diagnosis of
ERM (e.g. Hansen and Lingg 1976 ), furunculosis and vibriosis (including Hitra
disease). Commercial kits have found success, despite some cross reactions
(Romalde et al. 1995 ). However, the technique may be readily adapted for most
bacterial fi sh pathogens. As originally described for Aer. salmonicida by McCarthy
( 1975a , b ), the test involves the use of globulins from hyper-immune serum (titre =
>1:5000) and sensitised latex. The globulins are precipitated by the addition of saturated ammonium sulphate to the antiserum, and the precipitated proteins are sedimented by centrifugation. They are subsequently re-dissolved in 0.9 % (w/v) saline,
dialysed overnight at 4 °C against three changes of saline, and, after centrifugation,
the supernatant, which contains the globulins, is stored at – 20 °C until required.
The latex particles (0.81 μm diameter; Difco) are sensitised in globulin solution at
37 °C for 2 h. For the test, 200 μl of the antigen (bacterial suspension in glycinebuffered saline, i.e. 0.73 % (w/v) glycine and 1 % (w/v) NaCl; pH 8.2; supplemented
with 1 % (w/v) Tween 80) is mixed for 2 min with an equal volume of sensitised
latex on a clean glass plate. A positive result is indicated by clumping of the latex.
The technique may be used for pure or mixed cultures and tissue. Thus, positive
diagnoses may ensue from tissues unsuitable for culturing, e.g. fi sh stored at −20 °C
for 14 days, 5 °C for 7 days, or from formalin-fi xed material (McCarthy 1975a , b ).
As before, positive and negative controls are necessary.
• Co-agglutination with antibody-sensitised staphylococci
Reported for Aer. salmonicida and Ren. salmoninarum , this technique is similar
to the latex test (Kimura and Yoshimizu 1981 , 1983 , 1984 ). Essentially, Sta. aureus
(ATCC 12598) is suspended in 0.5 % formalin-PBS for 3 h at 25 °C to inactivate the
cells, washed three times in fresh PBS. The cells are mixed with antiserum in the
Bacteriological Examination