643
© Springer International Publishing Switzerland 2016
B. Austin, D.A. Austin, Bacterial Fish Pathogens,
DOI 10.1007/978-3-319-32674-0_12
Chapter 12
Isolation/Detection
Abstract There is no single technique suitable for the recovery of all known bacterial fi sh pathogens. Scientists need to use a combination of methods and incubation
conditions to achieve pure cultures. Even so, not all taxa are culturable: for example
Candidatus have not been cultured in vitro .
Keywords Media recipes • Isolation • Marine 2216E agar • TSA • BHIA
To an extent, the range of media to be used is governed by personal choice and
experience (Table 12.1 ). The formulae of commonly used media is included in an
appendix (Appendix “ Appendix – Media used for the isolation and growth of bacterial fi sh pathogens ”) at the end of the chapter. However, the incubation regimes
adopted in many laboratories needs some attention as 30 or 37 °C for 24–48 h is not
suffi cient to recover anything apart from the fastest growing organisms, which may
mean that the real pathogens are missed.
• For marine fi sh, it is advisable to include media prepared with seawater, e.g.
seawater/marine 2216E agar (for example, as supplied by Difco). Alternatively,
some media such as TSA may be supplemented with 1–2 % (w/v) sodium chloride, which is suitable for the isolation and growth of many heterotrophic marine
bacterial fi sh pathogens.
• For marine fi sh with damaged gills, low nutrient agar, such as cytophaga agar
(see Appendix “ Appendix – Media used for the isolation and growth of bacterial
fi sh pathogens ”) prepared in seawater, should be used.
• For freshwater fi sh, the routine use of TSA and/or BHIA (as supplied by Difco
or Oxoid) is recommended. Nutrient agar, which may be used, is more suited for
medical rather than fi sh bacteria.
Although most of the pathogens are aerobic, it is worthwhile remembering that
Cl. botulinum and Eu. tarantellae are anaerobic. For the former, Robertson’s meat
broth (Appendix “ Appendix – Media used for the isolation and growth of bacterial
fi sh pathogens ”) should be used for isolation, whereas with the latter, BHIA is satisfactory. For some aerobic organisms, i.e. Acinetobacter , atypical Aer. salmonicida
© Springer International Publishing Switzerland 2016
B. Austin, D.A. Austin, Bacterial Fish Pathogens,
DOI 10.1007/978-3-319-32674-0_12
Chapter 12
Isolation/Detection
Abstract There is no single technique suitable for the recovery of all known bacterial fi sh pathogens. Scientists need to use a combination of methods and incubation
conditions to achieve pure cultures. Even so, not all taxa are culturable: for example
Candidatus have not been cultured in vitro .
Keywords Media recipes • Isolation • Marine 2216E agar • TSA • BHIA
To an extent, the range of media to be used is governed by personal choice and
experience (Table 12.1 ). The formulae of commonly used media is included in an
appendix (Appendix “ Appendix – Media used for the isolation and growth of bacterial fi sh pathogens ”) at the end of the chapter. However, the incubation regimes
adopted in many laboratories needs some attention as 30 or 37 °C for 24–48 h is not
suffi cient to recover anything apart from the fastest growing organisms, which may
mean that the real pathogens are missed.
• For marine fi sh, it is advisable to include media prepared with seawater, e.g.
seawater/marine 2216E agar (for example, as supplied by Difco). Alternatively,
some media such as TSA may be supplemented with 1–2 % (w/v) sodium chloride, which is suitable for the isolation and growth of many heterotrophic marine
bacterial fi sh pathogens.
• For marine fi sh with damaged gills, low nutrient agar, such as cytophaga agar
(see Appendix “ Appendix – Media used for the isolation and growth of bacterial
fi sh pathogens ”) prepared in seawater, should be used.
• For freshwater fi sh, the routine use of TSA and/or BHIA (as supplied by Difco
or Oxoid) is recommended. Nutrient agar, which may be used, is more suited for
medical rather than fi sh bacteria.
Although most of the pathogens are aerobic, it is worthwhile remembering that
Cl. botulinum and Eu. tarantellae are anaerobic. For the former, Robertson’s meat
broth (Appendix “ Appendix – Media used for the isolation and growth of bacterial
fi sh pathogens ”) should be used for isolation, whereas with the latter, BHIA is satisfactory. For some aerobic organisms, i.e. Acinetobacter , atypical Aer. salmonicida