674
Biolog-GN, Enterotubes and RapidID 32 systems have made an inroad into routine
diagnostic laboratories. Whereas some systems, e.g. Biolog-GN, are clearly adaptable to- and useful for environmental isolates, others have been developed specifi -
cally for a given use, usually medically important bacteria. The consequence is that
the supporting identifi cation schemes may be inappropriate for fi sh pathogens.
Many workers have used API 20E, which was developed for medically important
enteric bacteria that grew overnight at 35–37 °C, for the identifi cation of fi sh pathogenic bacteria, but the system weighs heavily on the use of sugar fermentation reactions, which may be infl uenced by the presence of plasmids. Of course, it is
necessary to modify the API 20E protocol for use with marine bacteria by suspending cultures in 2–3 % (w/v) saline rather than distilled water, and the inoculated test
strips were incubated at 25 °C for up to 48 h. There may be some issues, for example, the API 20E profi le(s) for T. maritimum and Ps. anguilliseptica may be indistinguishable. Also, Y. ruckeri may be confused with Haf. alvei. Nevertheless, it is
accepted that the API 20E and API 20NE systems have a role in the diagnosis of
bacterial fi sh pathogens, and the manufacturer’s computer-based identifi cation
matrices are regularly updated to account for new information. In addition, APIZYM is very useful for diagnosing Ren. salmoninarum (See Table 13.1 ), which
gives a characteristic profi le:
− + − + − + − − + − + + − − − + − − + − −
/
Biolog-GP has been regarded as effective for the identifi cation of some Grampositive bacterial fi sh pathogens when compared with the results of 16S rRNA gene
sequencing, correctly identifying 18 Lac. garvieae and 10 Str. iniae isolates but not
Lac. piscium (the type strain did not grow on the recommended medium), V. salmoninarum (misidentifi ed as V. fl uvialis ) and Str. parauberis (misidentifi ed as Ent. faecalis ) (Verner-Jeffreys et al. 2011 ).
Diagnoses may also be achieved also by means of diagnostic schemes based on
reactions in conventional phenotypic tests. The procedure may be automated, as
with the Abbott Quantum II system (Teska et al. 1989 ), and involves spectrophotometric readings at 492.6 nm, with a sample cartridge containing 20 inoculated biochemical chambers. Alternatively, specially constructed diagnostic tables may be
used, such as presented in Tables 13.1 , 13.2 and 13.3 .
• Colony morphology and pigmentation
This should be recorded from ‘young’ colonies, i.e. shortly after growth is initially detected. The presence of aerial hyphae may be assessed with a stereomicroscope. The presence of pigment should be assessed from basal medium
supplemented with 5–10 % (w/v) skimmed milked powder (Oxoid).
• The Gram-staining reaction
With smears from young cultures, this reaction serves also to determine the presence of rods, cocci, mycelia, microcysts and endospores. For convenience, we recommend the use of commercially available staining and decolorising solutions,
13 Diagnosis
Biolog-GN, Enterotubes and RapidID 32 systems have made an inroad into routine
diagnostic laboratories. Whereas some systems, e.g. Biolog-GN, are clearly adaptable to- and useful for environmental isolates, others have been developed specifi -
cally for a given use, usually medically important bacteria. The consequence is that
the supporting identifi cation schemes may be inappropriate for fi sh pathogens.
Many workers have used API 20E, which was developed for medically important
enteric bacteria that grew overnight at 35–37 °C, for the identifi cation of fi sh pathogenic bacteria, but the system weighs heavily on the use of sugar fermentation reactions, which may be infl uenced by the presence of plasmids. Of course, it is
necessary to modify the API 20E protocol for use with marine bacteria by suspending cultures in 2–3 % (w/v) saline rather than distilled water, and the inoculated test
strips were incubated at 25 °C for up to 48 h. There may be some issues, for example, the API 20E profi le(s) for T. maritimum and Ps. anguilliseptica may be indistinguishable. Also, Y. ruckeri may be confused with Haf. alvei. Nevertheless, it is
accepted that the API 20E and API 20NE systems have a role in the diagnosis of
bacterial fi sh pathogens, and the manufacturer’s computer-based identifi cation
matrices are regularly updated to account for new information. In addition, APIZYM is very useful for diagnosing Ren. salmoninarum (See Table 13.1 ), which
gives a characteristic profi le:
− + − + − + − − + − + + − − − + − − + − −
/
Biolog-GP has been regarded as effective for the identifi cation of some Grampositive bacterial fi sh pathogens when compared with the results of 16S rRNA gene
sequencing, correctly identifying 18 Lac. garvieae and 10 Str. iniae isolates but not
Lac. piscium (the type strain did not grow on the recommended medium), V. salmoninarum (misidentifi ed as V. fl uvialis ) and Str. parauberis (misidentifi ed as Ent. faecalis ) (Verner-Jeffreys et al. 2011 ).
Diagnoses may also be achieved also by means of diagnostic schemes based on
reactions in conventional phenotypic tests. The procedure may be automated, as
with the Abbott Quantum II system (Teska et al. 1989 ), and involves spectrophotometric readings at 492.6 nm, with a sample cartridge containing 20 inoculated biochemical chambers. Alternatively, specially constructed diagnostic tables may be
used, such as presented in Tables 13.1 , 13.2 and 13.3 .
• Colony morphology and pigmentation
This should be recorded from ‘young’ colonies, i.e. shortly after growth is initially detected. The presence of aerial hyphae may be assessed with a stereomicroscope. The presence of pigment should be assessed from basal medium
supplemented with 5–10 % (w/v) skimmed milked powder (Oxoid).
• The Gram-staining reaction
With smears from young cultures, this reaction serves also to determine the presence of rods, cocci, mycelia, microcysts and endospores. For convenience, we recommend the use of commercially available staining and decolorising solutions,
13 Diagnosis