MBARI procedures for automated nutrient analyses using a modified Alpkem Series 300 Rapid Flow Analyzer

This report documents the methods used at the Monterey Bay Aquarium Research Institute (MBARI) for analyzing seawater nutrient samples with an Alpkem Series 300 Rapid Flow Analyzer (RFA) system. The methods have been optimized for the particular requirements of this laboratory. The RFA system has been used to analyze approximately 20,000 samples during the past two years. The methods have been optimized to run nutrient analyses in a routine manner with a detection limit of better than -±1% and a within run precision of -±1% of the full scale concentration range. The normal concentration ranges are 0-200 ~M silicate, 0-5 ~M phosphate, 0-50 ~M nitrate, 0-3 ~M nitrite, and 0-10 ~M ammonium. The memorandum is designed to be used in a loose-leaf binder format. Each page is dated and as revisions are made, they should be inserted into the binder. The revisions should be added into the binder. Retain the old versions in order to maintain a historical record of the procedures. (88 pages)

PCR-SSCP and Isoelectric Focusing as Screening Methods for Identification of (Sub-) Tropical Fish Species

To ensure the authentication of fishery products lacking biological characters, rapid species identification methods are required. Two DNA- and protein-based methods, PCR-SSCP (polymerase chain reaction - single strand conformation polymorphism) of a 464 bp segment of the cytochrome b – gene and isoelectric focusing (IEF) of water-soluble proteins from fish fillets, were applied to identify fillets of (sub-) tropical fish species available on the European market. Among the samples analysed were two taxonomically identified species from the family Sciaenidae and one from Sphyraenidae. By comparison of DNA- and protein patterns of different samples, information about intra-species variability of patterns, and homogeneity of batches (e.g. fillet blocks or bags) can be obtained. PCR-SSCP and IEF may be useful for pre-checking of a large number of samples by food control laboratories. Zusammenfassung Zur Sicherstellung der Authentizität von Fischerei-Erzeugnissen ohne biologische Merkmale sind schnelle Verfahren zur Speziesidentifizierung hilfreich. Zwei Methoden der DNA- bzw. Protein-Analyse wurden eingesetzt, um Filets (sub-) tropischer Fischarten, die auf dem europäischen Markt angeboten werden, zu identifizieren. Bei diesen Methoden handelt es sich um die PCR-SSCP (Polymerase-Kettenreaktion – Einzelstrang-Konformationspolymorphismus) – Analyse der PCR-Produkte und die IEF (isoelektrische Fokussierung) der wasserlöslichen Fischmuskelproteine. Unter den untersuchten Proben waren zwei taxonomisch bestimmte Arten aus der Familie Sciaenidae und eine Spezies aus der Familie Sphyraenidae. Durch Vergleich der DNA- bzw. Proteinmuster lassen sich Informationen über die intra-spezifische Variabilität solcher Muster und die Einheitlichkeit von Partien (beispielsweise Filetblöcke oder Filetbeutel) gewinnen. PCR-SSCP und IEF können in Laboratorien der Lebensmittelüberwachung als Vortest gerade bei hohen Probenzahlen sinnvoll eingesetzt werden.

Study of white spot disease in four native species in Persian Gulf by histopathology and PCR methods

After serious disease outbreak, caused by new virus (WSV), has been occurring among cultured penaeid shrimps in Asian countries like China since 1993 and then in Latin American countries, during June till July 2002 a rapid and high mortality in cultured Penaeus indicus in Abadan region located in south of Iran with typical signs and symptoms of White Spot Syndrome Virus was confirmed by different studies of Histopathology, PCR, TEM, Virology. This study was conducted for the purpose of determination of prevalence(rate of infection)/ROI and grading severity (SOI) of WSD to five species: 150 samples of captured shrimps and 90 samples of cultured ones; Penaeus indicus, P. semisulcatus, P. merguiensis, Parapenaopsis styliferus, and Metapenaeus affinis in 2005. 136 of 240 samples have shown clinical and macroscopical signs & symptoms including; white spots on carapase (0.5-2 mm), easily removing of cuticule, fragility of hepatopancreas and red color of motility limbs. Histopathological changes like specific intranuclear inclusion bodies (cowdry-type A) were observed in all target tissues (gill, epidermis, haemolymph and midgut) but not in hepatopancreas, among shrimps collected from various farms in the south and captured ones from Persian Gulf, even ones without clinical signs. ROI among species estimated, using the NATIVIDAD & LIGHTNER formula(1992b) and SOI were graded, using a generalized scheme for assigning a numerical qualitative value to severity grade of infection which was provided by LIGHTNER(1996), in consideration to histopathology and counting specific inclusion bodies in different stages(were modified by B. Gholamhoseini). Samples with clinical signs, showed grades more than 2. Most of the P. semisulcatus and M. affinis samples showed grade of 3, in the other hand in most of P. styliferus samples grade of 4 were observed, which can suggest different sensitivity of different species. All samples were tested by Nested PCR method with IQTm 2000 WSSV kit and 183 of 240 samples were positive and 3 1evel of infection which was shown in this PCR confirmed our SOI grades, but they were more specified.