Dear Dyahruri,

You may follow the protocol depicted in the following publication:

Int J Food Microbiol. 2009 Feb 28;129(3):229-36. doi: 10.1016/j.ijfoodmicro.2008.11.028. Epub 2008 Dec 6.

Evaluation of different procedures for the optimized detection of Vibrio parahaemolyticus in mussels and environmental samples.

Blanco-Abad V1, Ansede-Bermejo J, Rodriguez-Castro A, Martinez-Urtaza J.

Author information

Abstract

Vibrio parahaemolyticus is a marine bacterium with a worldwide distribution and is frequently associated with human outbreaks of infection. Detection and isolation of V. parahaemolyticus from natural sources is often problematical because of limitations in the analytical procedures. We evaluated a combination of conventional and molecular protocols previously described for the investigation of V. parahaemolyticus, with the aim of identifying the best procedures for improved detection of this organism in environmental matrixes. A total of 259 samples of zooplankton (103), mussels (48) and seawater (108) were investigated by an Absence-Presence method (A/P), whereas 118 samples of zooplankton (70) and mussels (48) were analyzed by the Most Probable Number (MPN) method. All samples were processed by a two-step enrichment procedure, firstly with APW broth and then with SPB as selective secondary broth. Detection of V. parahaemolyticus was by direct-PCR and by plate culture on TCBS and CHROMagar Vibrio, after sample enrichment in APW and SPB. With the A/P method, V. parahaemolyticus was detected in 23.6% samples by direct-PCR, whereas only 11.2% samples were positive with the plate culture method. With the MPN method, V. parahaemolyticus was detected in 54.2% and 27.1% of the samples by direct-PCR and plate culture respectively; this indicated the existence of 31% false negative results with the A/P method. No significant differences between the use of a single (APW) or two-step enrichment (APW+SPB) were observed by direct-PCR with A/P or MPN, although a significant higher presence of V. parahaemolyticus was detected by plate culture in both protocols with the two-step enrichment procedure. In conclusion, direct-PCR after sample enrichment in APW broth was the most successful method for detection of V. parahaemolyticus with the A/P procedure and enumeration by MPN. Better detection was obtained with MPN than with the A/P protocol. Conversely, the plate culture procedure showed better results with the two-step enrichment protocol in which CHROMagar Vibrio was used as the selective agar.

http://www.ncbi.nlm.nih.gov/pubmed/19131137

Another publication which may be helpful is the following:

Culturing marine bacteria – an essential prerequisite for biodiscovery

Ian Joint, Martin Mühling, Joël Querellou

Microbial Biotechnology

Special Issue: Marine Omics. Editors: Laura Giuliano,

Michele Barbier and Frederic Briand

September 2010, Volume 3, Issue 5, pages 564–575

Abstract:

The potential for using marine microbes for biodiscovery is severely limited by the lack of laboratory cultures. It is a long-standing observation that standard microbiological techniques only isolate a very small proportion of the wide diversity of microbes that are known in natural environments from DNA sequences. A number of explanations are reviewed. The process of establishing laboratory cultures may destroy any cell-to-cell communication that occurs between organisms in the natural environment and that are vital for growth. Bacteria probably grow as consortia in the sea and reliance on other bacteria for essential nutrients and substrates is not possible with standard microbiological approaches. Such interactions should be considered when designing programmes for the isolation of

marine microbes. The benefits of novel technologies for manipulating cells are reviewed, including single cell encapsulation in gel micro-droplets. Although novel technologies offer benefits for bringing previously uncultured microbes into laboratory culture, many useful bacteria can still be isolated using variations of plating techniques. Results are summarized for a study to culture bacteria from a longterm observatory station in the English Channel. Bacterial biodiversity in this assemblage has recently been characterized using high-throughput sequencing techniques. Although Alphaproteobacteria dominated the natural bacterial assemblage throughout the year, Gammaproteobacteria were the most frequent group isolated by plating techniques. The use of different gelling agents and the

addition of ammonium to seawater-based agar did lead to the isolation of a higher proportion of Alphaproteobacteria. Variation in medium composition was also able to increase the recovery of other groups of particular interest for biodiscovery, such as Actinobacteria.

http://archimer.ifremer.fr/doc/00035/14631/11950.pdf

Hoping this will be helpful,

Rafik

 You can also take into account those bacteria growing in salt marshes like Salinibacteri ruber.These kind of microbes produce high carotenoids concentrations so the also contribute to improve the colour of the mussels.

Hi Everyone.. Thank you for all your valuable advise.. :)