Aquaculture 238 (2004) 75 – 82

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Improved growth of Flavobacterium psychrophilum

using a new culture medium
Catalina Cepeda *, Sonia Garcı́a-Márquez, Ysabel Santos
Departamento de Microbiologı́a y Parasitologı́a, Facultad de Biologı́a, Instituto de Acuicultura,
Universidad de Santiago de Compostela, Campus Sur, 15782 Santiago de Compostela, Spain
Received 14 April 2004; accepted 4 May 2004

Abstract

The comparative study of different media used for cultivation of Flavobacterium psychrophilum
showed that tryptone – yeast extract – salts supplemented with glucose, named FLP medium, was the
most appropriate for the growth of this fish pathogen. By means of the endpoint dilution method, it
was found that the bacterium grew more efficiently on the improved FLP medium than on the other
four solid media assayed. This medium was also the most effective for the recovery of the pathogen
from tissues of diseased fish. Culture of F. psychrophilum in FLP broth was also more rapid and
provided high cell yields. In addition, FLP is a low-cost and easy-to-prepare medium.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Flavobacterium psychrophilum; Culture medium; Growth; Rainbow trout fry syndrome (RTFS);
Cold-water disease (CWD)

1. Introduction

Flavobacterium psychrophilum (formerly Cytophaga psychrophila, Flexibacter psy-

chrophilus) (Bernardet et al., 1996) is the causative agent of ‘‘bacterial cold-water
disease’’ (CWD) and ‘‘rainbow trout fry syndrome’’ (RTFS) in salmonid fish and
occasionally in other fish species (Lehmann et al., 1991; Iida and Mizokami, 1996).
The disease is common in most areas of the world and causes considerable economic
losses (Dalsgaard, 1993).
Detection and identification of this pathogen in fish samples based on bacterial
isolation is not always easy because of the lack of effective media. The solid media most

* Corresponding author. Tel.: +34-981563100x13340; fax: +34-81596904.

E-mail address: mpsantos@usc.es (C. Cepeda).

0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2004.05.013
76 C. Cepeda et al. / Aquaculture 238 (2004) 75–82

commonly used for the isolation of F. psychrophilum from diseased fish and for the
characterization of the bacterium are Anacker and Ordal agar (AOA)(Anacker and
Ordal,1959) and several modifications of this medium (Wakabayashi and Egusa, 1974;
Shieh, 1980; Baxa et al., 1986; Bernardet and Keroault, 1989; Obach and Baudin-
Laurencin, 1991; Holt et al., 1993; Toranzo and Barja, 1993; Daskalov et al., 1999). Other
media devised for the isolation of this pathogen are tryptone– yeast extract – agar (TYEA)
and tryptone – yeast extract – salts agar (TYESA) or improved standard formulations
(Michel et al., 1999).
In liquid media, F. psychrophilum grows best in Shieh (Shieh, 1980), modified
Cytophaga (Wakabayashi and Egusa, 1974), Cytophaga broth supplemented with carbo-
hydrates and skimmed milk (CBCM) (Daskalov et al., 1999), tryptone-enriched Anacker
and Ordal (Bernardet and Keroault, 1989) added with horse serum and trace elements
(Michel et al., 1999) and tryptone– yeast extract –salts broth (TYESB) (Holt, 1987).
Although these media appear to be convenient for isolation and cultivation of the F.
psychrophilum, these are not routinely used in diagnostic and research laboratories and for
biomass production. Moreover, preparation of some of these culture media is difficult and
slow because several ingredients must be separately sterilized by filtration before it
addition as enrichment factors.
The aim of the present work was to develop an improved medium for the recovery of
the bacterium from diseased fish and for the production of F. psychrophilum biomass in
liquid culture for use in vaccine preparation.

2. Materials and methods

2.1. Bacteria and growth conditions

In this study, 13 strains of F. psychrophilum, isolated from different fish species, were
used (Table 1). The identity of F. psychrophilum isolates was confirmed using previously
described morphological, physiological and biochemical tests, APIZYM (bioMerieux,
France) and PCR-based methods (Santos et al, 1992; Cepeda and Santos, 2000). Other
bacteria isolated from fresh water and fish (Flavobacterium columnare, Flavobacterium
succinicans, Flavobacterium flevensis, Flavobacterium johnsoniae) were also included.
The bacteria were maintained frozen at 70 jC in the commercial cryopreservative
medium Microbankk tubes (ProLab Diagnostics, Ontario, Canada). The bacterial strains
were cultured immediately before use on the different media evaluated and incubated at 18
jC during 24 –72 h.

2.2. Bacteriological media

In this study, five different media were compared for bacterial recovery (solid media)
and for biomass production (liquid media): modified Anacker and Ordal agar (MAOA)
[Bacto Peptone (Difco), 5 g l 1; yeast extract (Cultimed, Panreac Quı́mica, Barcelona,
Spain), 0.5 g l 1; sodium acetate (Sigma, St. Louis, USA), 0.01 g l 1; agar (Cultimed), 15
g l 1] and broth (MAOB) (Bacto Peptone, 5 g l 1; yeast extract, 0.5 g l 1; sodium
 C. Cepeda et al. / Aquaculture 238 (2004) 75–82 77

Table 1
Comparison of the growth of F. psychrophilum and other bacteria in several culture agar media
Strains Origin Culture media
MAOA MAOAG TYESA FLPA CACM
F. psychrophilum
5a 5 5 6 5
NCIMB1947 Oncorhynchus kitsutch, USA 10 10 10 10 10
4 4 5 6 5
PT4.1 Oncorhynchus mykiss, Spain 10 10 10 10 10
LNP PO1/88b O. mykiss, France 10 3
10 4
10 5
10 6
10 5

TG28/86b O. mykiss, France 10 2

10 2
10 3
10 4
10 3
2 2 3 5 4
YUMBO O. mykiss, Spain 10 10 10 10 10
2 2 2 5 4
NCIMB 2282 O. kitsutch, USA 10 10 10 10 10
MT1274c O. mykiss, UK 10 2
10 2
10 2
10 4
10 4

FPC 828d O. kitsutch, Japan 10 4

10 4
10 4
10 5
10 4
3 3 5 6 3
AA 3.1 Anguilla anguilla, Spain 10 10 10 10 10
3 4 5 6 5
Trouw229 O. mykiss, Spain 10 10 10 10 10
4 4 4 6 5
Trouw224 O. mykiss, Spain 10 10 10 10 10
MT1276c O. mykiss, UK 10 5
10 5
10 4
10 5
10 5
5 5 5 6 5
Tours 5/I Cyprinus carpio, France 10 10 10 10 10

F. columnare
4 4 5 5 5
NCIMB 2281 Oncorhynchus nerka 10 10 10 10 10

F. jonhsoniae
6 5 5 5 5
CECT 5015 Grass, UK 10 10 10 10 10

F. flevensis
2 5 5 5 2
NCIMB 12056 Fresh water, Netherlands 10 10 10 10 10

F. succinicans
5 5 5 5 5
NCIMB 2277 Oncorhynchus tshawytscha, USA 10 10 10 10 10
NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
CECT, Colección Española de Cultivos Tipo, Valencia, Spain.
a
The endpoint dilution indicates the last dilution of the culture which produced visible growth.
b
Strain supplied by J.-F. Bernardet, INRA, Jouy-en-Josas, France.
c
Strain supplied by T. Hastings, FRS Marine Laboratory, Aberdeen, UK.
d
Strain supplied by H. Wakabayashi, Department of Aquatic Bioscience, Graduate School of Agricultural and
Life Sciences, University of Tokyo, Japan.

acetate, 0.01 g l 1) (Toranzo and Barja, 1993), modified Anacker and Ordal agar and
broth supplemented with D(+)-glucose (Panreac Quı́mica) (0.5 g l 1) (MAOAG,
MAOBG), tryptone –yeast extract –salts agar [Bacto Tryptone (Difco), 4.0 gl 1; yeast
extract, 0.4 g l 1; CaCl2
2H2O (Panreac Quı́mica), 0.2 g l 1; MgSO4
7H2O (Panreac
Quı́mica), 0.5 g l 1; Agar 10.0 g l 1] and broth [Bacto Tryptone (Difco), 4.0 g l 1; yeast
extract, 0.4 g l 1; CaCl2
2H2O (Panreac), 0.2 g l 1; MgSO4
7H2O (Panreac), 0.5 g l 1]
(TYESA, TYESB) (Holt et al., 1993), tryptone – yeast extract– salts agar and broth
supplemented with glucose (0.5 g l 1) (FLPA, FLPB) and Cytophaga agar [Bacto
Tryptone (Difco), 0.5 g l 1; yeast extract, 0.5 g l 1; beef extract (Oxoid) 0.2 g l 1;
sodium acetate, 0.2 g l 1; Agar 9.0 g l 1] and broth [Bacto Tryptone (Difco), 0.5 g l 1;
yeast extract, 0.5 g l 1; beef extract (Oxoid) 0.2 g l 1; sodium acetate, 0.2 g l 1]
78 C. Cepeda et al. / Aquaculture 238 (2004) 75–82

supplemented with carbohydrates [D(+)-glucose; D(+)-galactose (Sigma); L-rhamnose

(Sigma)] and skimmed milk (Sigma) (CACM, CBCM) (Daskalov et al., 1999). The
media were adjusted to pH 7.2– 7.4 and sterilized by autoclaving (121 jC, 15 min). For
the preparation of CACM and CBCM, 10% (w/v) solutions of carbohydrates and
skimmed milk were separately sterilized by filtration (0.22 Am pore size, Millipore)
and added to cooled media.

2.3. Growth in solid media

The ability of the strains to grow on solid media MAOA, MAOAG, TYESA, FLPA
and CACM was evaluated by the endpoint dilution method (Daly and Stevenson, 1985).
Briefly, bacterial strains grown on each media were diluted in autoclaving sterilized
phosphate buffered saline [PBS, NaCl (Panreac Quı́mica), 8.0 g l 1; KCl (Panreac
Quı́mica), 0.2 g l 1; Na2HPO4(Panreac Quı́mica), 1.44 g l 1; KH2PO4 (Panreac
Quı́mica), 0.24 g l 1, pH 7.4] and adjusted to an initial density of 1.2108 cells ml 1
(McFarland scale no. 4). Then, tenfold dilutions were prepared and 0.1 ml of each dilution
were spread onto the various media. The plates were incubated at 18 jC and daily
examined up to 96 h of inoculation. Growth was recorded as the endpoint dilution defined
as the last dilution of the culture at which growth was recorded on seeded plates. Each test
was carried out in triplicate.

2.4. Growth in liquid media

To study the growth response in liquid media of F. psychrophilum, strains NCIMB 1947
from the National Collection of Industrial and Marine Bacteria and the strains Trouw 224
and Trouw 229 isolated from diseased trout in Spain were used. The bacteria were pre-
cultured in 25 ml MAOB, MAOBG, TYESB, FLPB and CBCM, and 2 ml of these
cultures were then inoculated into 200 ml of each broth medium. Inoculated media were
incubated with shaking at 100 rpm at 18j C. The growth was monitored by measuring the
absorbance at 580 nm over a 72-h period and by counting the number of viable bacteria in
agar plates. All experiments were repeated three times.

2.5. Naturally infected fish

All the media were also tested for the recovery of F. psychrophilum under field
conditions. For this purpose, 17 samples from epizootics occurring between 2000 and
2002 were taken from different trout farms suffering continuous disease problems
associated with F. psychrophilum. For bacterial isolation, samples of kidney and spleen
of diseased fishes were taken under aseptic conditions and streaked directly onto plates
of MAOA, MAOAG, TYESA, FLPA and CACM. Colonies were counted after
incubation (18 jC, 96 h) and representative strains from each medium were isolated
in pure culture. The taxonomic position of the bacterial strains was determined using
morphological, physiological and biochemical tests, API ZYM (Biomerieux) (F. psy-
chrophilum strains) and API 20E (other Gram-negative isolates) as previously described
(Bernardet and Keroault., 1989; Santos et al., 1993; Santos et al., 1992). The identity of
 C. Cepeda et al. / Aquaculture 238 (2004) 75–82 79

F. psychrophilum strains was also confirmed by PCR-based methods (Cepeda and

Santos, 2000).

3. Results

3.1. Recovery of F. psychrophilum on solid media

The growth abilities of F. psychrophilum, as well as other bacteria, on the different solid
media assayed in laboratory conditions are given in Table 1. FLPA proved to be the most
effective medium, allowing the recovery of F. psychrophilum at the highest dilution
(10 6 – 10 5), followed by the other two media containing tryptone (CACM, TYESA).
The bacterium grew less well in media containing peptone as nitrogen source (MAOAG,
MAOA). The results varied slightly with strains of the bacterium studied. In all the media
tested, the colonies recovered appeared with marked evidence of yellow pigmentation, but
in FLPA, the colonies showed the largest size.
Growth of F. succinicans, F. johnsoniae, F. flevensis and F. columnare was similar in all
the media tested.

3.2. Growth of F. psychrophilum in broth cultures

Fig. 1 shows the growth curves obtained for F. psychrophilum strain Trouw 224
cultured in the different broth media. Similar growth curves were obtained with the other
strains assayed (Trouw 229 and NCIMB 1947). Growth in liquid media was consistently
higher in FLPB followed by TYESB and CBCM. In these media, the absorbance and the
number of viable bacteria peaked over 25 h after inoculation. The number of bacteria
recovered by plating indicated that the culture in FLPB reached a stationary phase when
the viable count was approximately 5109 CFU/ml, in comparison with a cell count of

Fig. 1. Growth of F. psychrophilum strain Trouw 224 in five different liquid media.
80 C. Cepeda et al. / Aquaculture 238 (2004) 75–82

2108 and 3107 in TYESB and CBCM, respectively. The culture in MAOBG and
MAOB produced the lowest cell yield.

3.3. Field studies

The comparative study of the media under field conditions showed FLPA as the most
effective medium for the recovery of F. psychrophilum from disease fish. In fact, in 60% of
the cases of ‘‘cold-water disease’’ diagnosed during 2000 and 2002 the bacterium was
always isolated in FLPA and occasionally in other media. F. psychrophilum was recovered
from these disease fish in pure culture and in mixed culture with other Gram-negative
bacterial strains of the genera Aeromonas and Enterobacter.

4. Discussion

The culture of the fish pathogen F. psychrophilum is difficult and requires a long
incubation period (more than 72 h). In this study, we have tried to find an effective,
low-cost and easy-to-prepare medium for the cultivation of F. psychrophilum. With this
aim, and considering the published results in relation with the importance of salts and
carbohydrates for the growth of F. psychrophilum (Song et al., 1988; Holt et al., 1993;
Daskalov et al., 1999), we have developed an improved medium with the same
composition of TYES but supplemented with glucose. Although previous studies
(Michel et al., 1999; Lorenzen, 1993; Obach and Baudin-Laurencin, 1991) have proven
the value of serum as a enrichment factor for the successful culture of F. psychrophilum,
in an effort to reduce costs, we did not use this expensive ingredient. Otherwise, in
order to develop a medium that could be commercialised, skimmed milk was not
included in the new formulation to avoid the use of components that must be separately
sterilized by filtration.
The media used in the study were compared as convenient liquid media for mass-
producing cells and suitable solid media for isolation and routine culture of F. psychro-
philum. The culture medium FLP established in this work was the more appropriate in all
cases. This can be attributed to a successful combination of tryptone, salts and glucose.
Our results demonstrated that the use of tryptone (TYES, FLP and CCM media) instead of
peptone (MAO and MAOG) clearly enhances F. psychrophilum growth and confirmed the
value of the reinforcement of tryptone content for the culture of this pathogen (Michel et
al., 1999; Bernardet and Keroault, 1989). We also corroborated the improved growth
response of F. psychrophilum in medium containing salts described by other authors (Holt
et al., 1993). The presence of salts in the media also increases the chance of survival of F.
columnare, another filamentous bacterium that is very sensitive to osmotic pressure
changes (Song et al., 1988). Descriptions of the utilization of carbohydrate by F.
psychrophilum have been confusing. Although F. psychrophilum has been described as
a bacterium that does not produce acids from carbohydrates (Holt et al., 1993; Austin and
Austin, 1993), our results and those of Daskalov et al. (1999) have proven that the
incorporation of carbohydrates in the culture media (agar or broth) increases the growth
rate of this fish pathogen. Glucose could be implicated in the formation of extracellular
 C. Cepeda et al. / Aquaculture 238 (2004) 75–82 81

polysaccharide as has been indicated for F. columnare (Shieh 1980) or it could change the
physical – chemical characteristics of the culture medium favouring the viability of F.
psychrophilum. Further studies are necessary to clarify the role of glucose.

5. Conclusion

In conclusion, although all the media tested can be used in the laboratory for the routine
culture of F. psychrophilum, FLP agar medium is recommended for a fast recovery of the
bacterium from stored cultures and for isolation of this bacterial species from environ-
mental samples. Culture in FLP broth provides high cell yields in a short culturing period
which is of great importance in the study of the pathogenesis of the disease and for large-
scale production of vaccines.

Acknowledgements

The authors are grateful to Trouw España (Cojobar, Burgos, Spain) for kindly
providing bacterial strains.

References

Anacker, R.L., Ordal, E.J., 1959. Studies on the myxobacterium Chondrococcus columnaris: I. Serological
typing. J. Bacteriol. 78, 25 – 32.
Austin, B., Austin, D.A., 1993. Bacterial Fish Pathogens: Diseases in Farmed and Wild Fish, second ed. Ellis
Horwood, Chichester.
Baxa, D.V., Kawai, K., Kusuda, R., 1986. Characteristics of gliding bacteria isolated from disease cultured
flounder, Paralichthys olivaceous. Fish Pathol. 21, 251 – 258.
Bernardet, J.F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K., Vandamme, P., 1996. Cutting a Gordian knot:
emended classification and description of the genus Flavobacterium, emended description of the family
Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (Basonym, Cytophaga aquatilis Strohl
and Tait 1978). Int. J. Syst. Bacteriol. 46, 128 – 148.
Bernardet, J.F., Keroault, B., 1989. Phenotypic and genomic studies of Cytophaga psychrophila isolated from
diseased rainbow trout (Oncorhynchus mykiss) in France. Appl. Environ. Microbiol. 55, 1796 – 1800.
Cepeda, C., Santos, Y., 2000. Rapid and low-level toxic PCR-based method for routine identification of Fla-
vobacterium psychrophilum. Int. Microbiol. 3, 235 – 238.
Dalsgaard, I., 1993. Virulence mechanisms in Cythophaga psychrophila and other Citophaga-like bacteria
pathogenic for fish. Annu. Rev. Fish Dis., 127 – 144.
Daly, J.G., Stevenson, R.M.W., 1985. Charcoal agar, a new growth medium for the fish disease bacterium
Renibacterium salmoninarum. Appl. Environ. Microbiol. 50, 868 – 871.
Daskalov, H., Austin, D.A., Austin, B., 1999. An improved growth medium for Flavobacterium psychrophilum.
Lett. Appl. Microbiol. 28, 279 – 299.
Holt, R.A., Rohovec, J.S., Fryer, J.L., 1993. Bacterial cold water disease. In: Inglis, V., Roberts, R.J., Bromage,
N.R. (Eds.), Bacterial Diseases of Fish, Blackwell, Oxford, pp. 3 – 23.
Holt, R.A., 1987. Cythophaga psychrophila the causative agent of bacterial cold water disease in salmonids fish.
PhD thesis, Oregon State University, Corvallis, OR.
Iida, Y., Mizokami, A., 1996. Outbreaks of coldwater disease in wild ayu and pale chub. Fish Pathol. 34, 89 – 90.
Lehmann, J., Mock, D., Stürenberg, F.J., Bernardet, J.F., 1991. First isolation of Cytophaga psychrophila from a
systemic disease in eel and cyprinids. Dis. Aquat. Org. 10, 217 – 220.
82 C. Cepeda et al. / Aquaculture 238 (2004) 75–82

Lorenzen, E., 1993. The importance of the brand of the beef extract in relation to the growth of Flexibacter
psychrophilus in Anacker & Ordal’s medium. Bull. Eur. Assoc. Fish Pathol. 13, 64 – 65.
Michel, C., Antonio, D., Hedrick, R.P., 1999. Production of viable cultures of Flavobacterium psychrophilum:
approach and control. Res. Microbiol. 150, 351 – 358.
Obach, A., Baudin-Laurencin, F., 1991. Vaccination of rainbow trout (Oncorhynchus mykiss) against the visceral
form of cold-water disease. Dis. Aquat. Org. 12, 13 – 15.
Santos, Y., Huntly, P.J., Turnbull, A., Hasting, T.S., 1992. Isolation of Cytophaga psychrophila (Flexibacter
psychrophilus) in association with rainbow trout mortality in United Kingdom. Bull. Eur. Assoc. Fish Pathol.
12, 209 – 210.
Santos, Y., Romalde, J.L., Bandı́n, I., Magariños, B., Núñez, S., Barja, J.L.Y., Toranzo, A.E., 1993. Usefulness of
the API-20E system for the identification of bacterial fish pathogens. Aquaculture 116, 111 – 120.
Shieh, H.S., 1980. Studies on the nutrition of a fish pathogen, Flexibacter columnaris. Microbios Lett. 13,
129 – 133.
Song, Y.L, Fryer, J.L., Rohovec, J.S., 1988. Comparison of six media for the cultivation of Flexibacter columnaris.
Fish Pathol. 23 (2), 91 – 94.
Toranzo, A.E., Barja, J.L., 1993. Fry mortality syndrome (FMS) in Spain. Isolation of the causative bacterium
Flexibacter psychrophilus. Bull. Eur. Assoc. Fish Pathol. 13, 30 – 32.
Wakabayashi, H., Egusa, S., 1974. Characteristics of myxobacteria associated with some freshwater fish diseases
in Japan. Bull. Jpn. Soc. Sci. Fish. 40, 751 – 757.