668
Nevertheless, tentative diagnoses, especially of asymptomatic infections (Busch
and Lingg 1975 ; Hansen and Lingg 1976 ; Johnson et al. 1974 ) may result from use
of polyclonal antibodies in any of a multiplicity of serological procedures, including
FAT, whole-cell (slide) agglutination, precipitin reactions, complement fi xation,
immunodiffusion, antibody-coated latex particles (this is akin to human pregnancy
testing), co-agglutination using antibody-coated staphylococcal cells, passive haemagglutination, immuno-India ink technique (Geck) or ELISA (e.g. Saeed and
Plumb 1987 ). The serological techniques are discussed below:
• Fluorescent antibody technique (FAT)
There are two variations to this test, namely the direct and indirect methods. FAT
has found use for the diagnosis of many fi sh diseases, especially in laboratories
(Eurell et al. 1978 ), for which it is regarded as a highly effective procedure
(Kawahara and Kusuda 1987 ). For example, α- and ß-haemolytic streptococcal isolates may be readily differentiated by FAT (Kawahara and Kusuda 1987 ).
For the direct method (see Bullock et al. 1980 and Smibert and Krieg 1981 for
further details), fl uorescein isothiocyanate is conjugated with whole or with the IgG
fraction of the antiserum. Twofold dilutions (1:5 and 1:8) are prepared in PBS
(0.1236 % (w/v) Na 2 HPO 4 , anhydrous; 0.018 % (w/v) NaH 2 PO 4 .H 2 0; 0.85 % (w/v)
NaCl; pH 7.6) and used to standardise the ‘conjugate’. Thus, a bacterial suspension
(containing ca . 10
8 cells/ml in PBS) is pipetted onto grease-free microscope slides,
air-dried and fi xed at 60 °C for 2 min (or fi xed in 95 % ethanol for 1 min., and then
air-dried). The ‘conjugate’ is pipetted onto the slide and left in a moist chamber for
5 min. to react (room temperature, i.e. 15–20 °C, is adequate). Subsequently, the
excess antiserum is removed by draining, before the slide is thoroughly rinsed in
PBS (for ca . 10 min). The slide is air-dried, and the smear covered with a drop of
buffered glycerol (Difco; at pH 9.0) before overlayering with a coverslip. This
should be quickly examined with a fl uorescence microscope. The optimum dilution
of conjugate is determined, from the doubling dilutions, by rating the degree of fl uorescence from 0 to 4+ (after Jones et al. 1978 ). The ‘use’ dilution of the conjugated
antiserum is 50 % of the highest dilution, which gives maximum fl uorescence. With
this information, the diagnosis may proceed for unknown cultures. However, it is
always important to include positive and negative controls.
For iFAT (see Laidler 1980 ), bacterial smears are prepared and fi xed (as above).
Doubling dilutions of rabbit antiserum are prepared, and 20 μl aliquots added to the
bacterial smears. These are placed in a moist chamber, left for 30 min to react, and
washed for 30 min in two changes of PBS. After air-drying, the smears are covered
with a suitable dilution of fl uorescein-labelled sheep anti-rabbit globulin, incubated
for 30 min in the moist chamber, rinsed thoroughly in PBS, air-dried and mounted
in buffered glycerol. Examination, with a fl uorescence microscope should proceed
as quickly as possible.
• Whole-cell agglutination
This is a quick and easy technique, which provides much useful data. Whole cell
agglutination is used widely as a diagnostic tool, for example with the fi sh patho13 Diagnosis
Nevertheless, tentative diagnoses, especially of asymptomatic infections (Busch
and Lingg 1975 ; Hansen and Lingg 1976 ; Johnson et al. 1974 ) may result from use
of polyclonal antibodies in any of a multiplicity of serological procedures, including
FAT, whole-cell (slide) agglutination, precipitin reactions, complement fi xation,
immunodiffusion, antibody-coated latex particles (this is akin to human pregnancy
testing), co-agglutination using antibody-coated staphylococcal cells, passive haemagglutination, immuno-India ink technique (Geck) or ELISA (e.g. Saeed and
Plumb 1987 ). The serological techniques are discussed below:
• Fluorescent antibody technique (FAT)
There are two variations to this test, namely the direct and indirect methods. FAT
has found use for the diagnosis of many fi sh diseases, especially in laboratories
(Eurell et al. 1978 ), for which it is regarded as a highly effective procedure
(Kawahara and Kusuda 1987 ). For example, α- and ß-haemolytic streptococcal isolates may be readily differentiated by FAT (Kawahara and Kusuda 1987 ).
For the direct method (see Bullock et al. 1980 and Smibert and Krieg 1981 for
further details), fl uorescein isothiocyanate is conjugated with whole or with the IgG
fraction of the antiserum. Twofold dilutions (1:5 and 1:8) are prepared in PBS
(0.1236 % (w/v) Na 2 HPO 4 , anhydrous; 0.018 % (w/v) NaH 2 PO 4 .H 2 0; 0.85 % (w/v)
NaCl; pH 7.6) and used to standardise the ‘conjugate’. Thus, a bacterial suspension
(containing ca . 10
8 cells/ml in PBS) is pipetted onto grease-free microscope slides,
air-dried and fi xed at 60 °C for 2 min (or fi xed in 95 % ethanol for 1 min., and then
air-dried). The ‘conjugate’ is pipetted onto the slide and left in a moist chamber for
5 min. to react (room temperature, i.e. 15–20 °C, is adequate). Subsequently, the
excess antiserum is removed by draining, before the slide is thoroughly rinsed in
PBS (for ca . 10 min). The slide is air-dried, and the smear covered with a drop of
buffered glycerol (Difco; at pH 9.0) before overlayering with a coverslip. This
should be quickly examined with a fl uorescence microscope. The optimum dilution
of conjugate is determined, from the doubling dilutions, by rating the degree of fl uorescence from 0 to 4+ (after Jones et al. 1978 ). The ‘use’ dilution of the conjugated
antiserum is 50 % of the highest dilution, which gives maximum fl uorescence. With
this information, the diagnosis may proceed for unknown cultures. However, it is
always important to include positive and negative controls.
For iFAT (see Laidler 1980 ), bacterial smears are prepared and fi xed (as above).
Doubling dilutions of rabbit antiserum are prepared, and 20 μl aliquots added to the
bacterial smears. These are placed in a moist chamber, left for 30 min to react, and
washed for 30 min in two changes of PBS. After air-drying, the smears are covered
with a suitable dilution of fl uorescein-labelled sheep anti-rabbit globulin, incubated
for 30 min in the moist chamber, rinsed thoroughly in PBS, air-dried and mounted
in buffered glycerol. Examination, with a fl uorescence microscope should proceed
as quickly as possible.
• Whole-cell agglutination
This is a quick and easy technique, which provides much useful data. Whole cell
agglutination is used widely as a diagnostic tool, for example with the fi sh patho13 Diagnosis