Violet red bile agar

Portions of 10 g of fish white muscle were dissected aseptically from refrigerated fish specimens, mixed with 90 mL of 0.1% peptone water (Merck, Darmstadt, Germany) and homogenized in sterilized stomacher bags (AES, Combourg, France) as described elsewhere (Ben-Gigirey, Vieites Baptista de Sousa, Villa, & Barros-Velázquez, 1998; Ben-Gigirey, Vieites Baptista de Sousa, Villa, & Barros-Velázquez, 1999) . Serial dilutions from the microbial extracts were prepared in 0.1% peptone water.
Total aerobes were assessed by surface inoculation on plate count agar (PCA, Oxoid Ltd., London, UK) after incubation at 30ºC for 48 h. Psychrotrophs were also determined in PCA, after an incubation period of 7 days at 7-8ºC. Enterobacteriaceae were investigated by pour plating using Violet Red Bile Agar (VRBA) (Merck, Darmstadt, Germany) after an incubation period of 24 h at 37±0.5ºC. Bacteria exhibiting proteolytic or lipolytic phenotypes were determined on casein-agar or tributyrine-agar, respectively, after incubation at 30ºC for 48 h, as described elsewhere (Ben-Gigirey, Vieites Baptista de Sousa, Villa, & Barros-Velázquez, 2000) .
In all cases, microbial counts were transformed into log CFU g -1 muscle before undergoing statistical analysis. All analyses were conducted in triplicate.

Aliquots of fish muscle (10 g) were sampled aseptically, mixed with 90 mL of 0.1 % peptone water (Merck, Darmstadt, Germany) in sterile bags (Seward, London, UK) and homogenised in a masticator (AES, Combourg, France), as previously reported. 23, 24 Serial dilutions (10 -1 to 10 -8 ) of these extracts were also prepared in 0.1 % peptone water.
Total aerobes and psychrotrophs were enumerated on plate count agar (PCA) (Oxoid Ltd., London, UK) after incubation at 30 ºC, for 48 h and 7-8 ºC for 7 days, respectively. In both cases, all colonies were considered to be target bacteria.
Enterobacteriaceae were enumerated by the pour plate method in violet red bile agar (VRBA) (Merck, Darmstadt, Germany), after incubation at 37 ± 0.5 ºC, for 24 h. Only red colonies were counted. In all cases, culture media were prepared according to the manufacturer's instructions. Bacteria exhibiting a proteolytic phenotype were enumerated on casein agar after incubation at 30 ºC, for 48 h, as previously described. 25 Only white or yellow colonies, exhibiting a decolorized halo were counted.
Microbial counts were transformed into log CFU g -1 muscle, prior to statistical analysis. All analyses were carried out in triplicate.

Horse mackerel samples of 10 g of white muscle were taken aseptically, mixed with 90 mL of 0.1 % peptone water (Merck, Darmstadt, Germany) and further homogenised in sterilised stomacher bags (AES, Combourg, France) according to previous research [28] [29] . A 0.1 % peptone-water solution was employed to prepare serial dilutions from the different microbial extracts.
Total aerobes were investigated on plate count agar (PCA, Oxoid Ltd., London, UK) after incubation at 30 ºC for 48 h. Psychrotrophes were also investigated in PCA, being the incubation carried out at 7-8 ºC for 7 days. Enterobacteriaceae were analysed by pour plating using Violet Red Bile Agar (VRBA) (Merck, Darmstadt, Germany) after incubation for 24 h at 37 ± 0.5 ºC. Microorganisms showing a proteolytic or lipolytic phenotype were analysed on casein-agar medium and tributyrine-agar, respectively, after incubation at 30 ºC for 48 h, according to previous research [30] .
For all kinds of analyses, microbial counts were transformed into log CFU g -1 muscle values before undergoing the statistical analysis. All analyses were carried out in triplicate.

Samples of 10 g of fish muscle were dissected aseptically from refrigerated fish specimens, mixed with 90 mL of 0.1% peptone water (Merck, Darmstadt, Germany) and homogenised in sterilised stomacher bags (AES, Combourg, France) as previously described (Ben-Gigirey et al., 1998; Ben-Gigirey et al., 1999) . In all of the cases, serial dilutions from the microbial extracts were prepared in 0.1% peptone water.
Total aerobes were investigated by surface inoculation on plate count agar (PCA, Oxoid Ltd., London, UK) after incubation at 30ºC for 48 h. The anaerobe counts were also determined in PCA at 30±0.5ºC, except that an anaerobic atmosphere kit (Oxoid Ltd.) was placed together with the plates inside the anaerobiosis jar.
Psychrotrophs were also investigated in PCA, being the incubation carried out at 7-8ºC for 7 days. Enterobacteriaceae were investigated via pour plating using Violet Red Bile Agar (VRBA) (Merck, Darmstadt, Germany) after incubation at 37±0.5ºC for 24 h. In all of the cases, bacterial counts were transformed into log CFU g -1 muscle before undergoing statistical analysis. All of the analyses were conducted in triplicate.

Muscle samples (10 g) were dissected aseptically from chilled lobster krill specimens, mixed with 90 ml of 0.1 % peptone water (Merck, Darmstadt, Germany), and homogenised in sterilized stomacher bags (AES, Combourg, France) as previously described (Ben-Gigirey et al., 1998 , 1999) . In all cases, serial dilutions of the microbial extracts were prepared in 0.1 % peptone water.
Total aerobes were determined by surface inoculation on plate count agar (PCA, Oxoid Ltd., London, UK), after incubation at 30ºC for 48 h. The anaerobe counts were also determined on PCA at 30ºC, except that an anaerobic atmosphere kit (Oxoid) was placed together with the plates inside an anaerobic jar. Psychrotrophes were also determined on PCA but incubation was carried out at 7-8ºC for 7 d. Enterobacteriaceae were determined by pour plating using Violet Red Bile Agar (VRBA) (Merck, Darmstadt, Germany) after incubation at 37ºC for 24 h. Microorganisms exhibiting a proteolytic or lipolytic phenotype were determined on casein-agar medium or tributyrine-agar, respectively, after incubation at 30ºC for 48 h, as previously described (Ben-Gigirey et al., 2000) .
In all cases, bacterial counts were transformed into log CFU g -1 muscle before undergoing statistical analysis. All microbiological analyses were done in triplicate.