87
healthy chinook salmon, and eventually recovered it again from the resultant kidney
lesion. Continuing this pioneering work, Ordal and Earp ( 1956 ) supplemented
Dorset egg medium with 0.05–1.0 % (w/v) L-cysteine, tryptone and yeast extract,
and succeeded in isolating the pathogen in 3–4 weeks following incubation at
17 °C. The outcome of this work was the formulation of cysteine blood agar with
which Koch’s postulates were fulfi lled (Appendix in Chap. 12 ; Ordal and Earp
1956 ). Foetal calf serum was substituted for human blood in a modifi cation proposed by Evelyn et al. ( 1973 ). This was developed further by removing sodium
chloride and substituting peptone for tryptone and beef extract (KDM2; Appendix
in Chap. 12 ; Evelyn 1977 ); this medium is now used commonly for growth of the
BKD organism. In a parallel development, Wolf and Dunbar ( 1959 ) used MuellerHinton agar supplemented with 0.1 % (w/v) L-cysteine hydrochloride (MHC) to
culture the pathogen, although success with this medium did not occur with Smith
( 1964 ). However, Bullock et al. ( 1974 ) confi rmed the value of MHC, although this
has been subsequently contended by Evelyn ( 1977 ). Serum-rich KDM2 was considered to be superior to serum-defi cient MHC, indicating the benefi t of serum for the
cultivation of the BKD organism. This was further supported by Paterson et al.
( 1979 ), who supplemented MHC with 10 % (v/v) foetal calf serum, and successfully
used the medium for isolating the pathogen from Atlantic salmon. Daly and
Stevenson ( 1985 ) proposed replacing serum with charcoal, which serves as a detoxicant (Appendix in Chap. 12 – charcoal agar). However, these media, being
extremely rich in composition, are generally suitable for the growth of many aerobic, heterotrophic bacteria. Moreover, fast-growing organisms may rapidly outcompete and overgrow the slower-growing BKD organism. A solution was proposed by
Evelyn ( 1977 ), who advocated the use of a drop-plating technique (essentially, this
is analogous to dilution plates, which dilute out potential interference by fast-growing heterotrophs). In a later report, Evelyn ( 1978 ) recommended the use of peptone
(0.1 % w/v)-saline (0.85 % w/v) as a diluent to remove any inhibiting factors against
the pathogen, which may be present in kidney tissue (Evelyn et al. 1981 ; Austin
1986 ). Some inconsistencies in the performance of KDM2 were attributed to variation in the composition of the commercial peptone (Evelyn and Prosperi-Porta
1989 ). To overcome this inconsistency when single lots of peptone were not available, two possible modifi cations were suggested. Firstly, a “nurse” culture technique was reported. This technique accelerated the growth of the BKD organism,
and increased the sensitivity at which the pathogen could be detected. The technique, which was based on satellitism or cross feeding, involved inoculating the
nutritionally fastidious pathogen next to a non-fastidious feeder – the nurse organism. Evelyn et al. ( 1989 ) placed drops of a dense suspension of a stock culture of the
BKD organism (= the nurse organism) onto the centre of KDM2 plates. Samples,
suspected of containing the pathogen, were placed as 25 μl drops around the periphery of the nurse culture. With incubation, the nurse culture grew rapidly, presumably modifi ed the conditions in the KDM2, and thus enhanced the growth of the
pathogen in the periphery. For example, colonies of the BKD organism were observable after incubation for 19 days, compared to 25 days for the conventional approach.
The second modifi cation involved supplementing KDM2 agar with a small amount
Renibacterium salmoninarum
healthy chinook salmon, and eventually recovered it again from the resultant kidney
lesion. Continuing this pioneering work, Ordal and Earp ( 1956 ) supplemented
Dorset egg medium with 0.05–1.0 % (w/v) L-cysteine, tryptone and yeast extract,
and succeeded in isolating the pathogen in 3–4 weeks following incubation at
17 °C. The outcome of this work was the formulation of cysteine blood agar with
which Koch’s postulates were fulfi lled (Appendix in Chap. 12 ; Ordal and Earp
1956 ). Foetal calf serum was substituted for human blood in a modifi cation proposed by Evelyn et al. ( 1973 ). This was developed further by removing sodium
chloride and substituting peptone for tryptone and beef extract (KDM2; Appendix
in Chap. 12 ; Evelyn 1977 ); this medium is now used commonly for growth of the
BKD organism. In a parallel development, Wolf and Dunbar ( 1959 ) used MuellerHinton agar supplemented with 0.1 % (w/v) L-cysteine hydrochloride (MHC) to
culture the pathogen, although success with this medium did not occur with Smith
( 1964 ). However, Bullock et al. ( 1974 ) confi rmed the value of MHC, although this
has been subsequently contended by Evelyn ( 1977 ). Serum-rich KDM2 was considered to be superior to serum-defi cient MHC, indicating the benefi t of serum for the
cultivation of the BKD organism. This was further supported by Paterson et al.
( 1979 ), who supplemented MHC with 10 % (v/v) foetal calf serum, and successfully
used the medium for isolating the pathogen from Atlantic salmon. Daly and
Stevenson ( 1985 ) proposed replacing serum with charcoal, which serves as a detoxicant (Appendix in Chap. 12 – charcoal agar). However, these media, being
extremely rich in composition, are generally suitable for the growth of many aerobic, heterotrophic bacteria. Moreover, fast-growing organisms may rapidly outcompete and overgrow the slower-growing BKD organism. A solution was proposed by
Evelyn ( 1977 ), who advocated the use of a drop-plating technique (essentially, this
is analogous to dilution plates, which dilute out potential interference by fast-growing heterotrophs). In a later report, Evelyn ( 1978 ) recommended the use of peptone
(0.1 % w/v)-saline (0.85 % w/v) as a diluent to remove any inhibiting factors against
the pathogen, which may be present in kidney tissue (Evelyn et al. 1981 ; Austin
1986 ). Some inconsistencies in the performance of KDM2 were attributed to variation in the composition of the commercial peptone (Evelyn and Prosperi-Porta
1989 ). To overcome this inconsistency when single lots of peptone were not available, two possible modifi cations were suggested. Firstly, a “nurse” culture technique was reported. This technique accelerated the growth of the BKD organism,
and increased the sensitivity at which the pathogen could be detected. The technique, which was based on satellitism or cross feeding, involved inoculating the
nutritionally fastidious pathogen next to a non-fastidious feeder – the nurse organism. Evelyn et al. ( 1989 ) placed drops of a dense suspension of a stock culture of the
BKD organism (= the nurse organism) onto the centre of KDM2 plates. Samples,
suspected of containing the pathogen, were placed as 25 μl drops around the periphery of the nurse culture. With incubation, the nurse culture grew rapidly, presumably modifi ed the conditions in the KDM2, and thus enhanced the growth of the
pathogen in the periphery. For example, colonies of the BKD organism were observable after incubation for 19 days, compared to 25 days for the conventional approach.
The second modifi cation involved supplementing KDM2 agar with a small amount
Renibacterium salmoninarum