Innovative Probes for Molecular Research 



PROTOCOL FOR LIVER ANALYSES 

 BREVETOXINS IN DOLPHIN LIVER 



Sp pri f"" kems related to tamfde mhwhtioe are otmibered: 



(1) RECEIPT: Sampbi chaD be received Toesday-Tbursday (shipped Monday-Wedoetday) ai the 

 Chira] CorporatioD Research and Developoeal Uboratory. The proper ^bippiag address is: 



Chira) Corporaiiao R&D Laboratory 



Uaivenity of Miami Rosenstid School of Marine and Atmoqiheric Scsence 



4600 Rickeabadcer Causeway 



Miami norida 33149 



ATTN: Daniel G. Bades 



The shipper should make every effort to retain portions of each sample shipped m case of Iocs of sh^nnent, 

 unavoidable loss of sample, or for eonfirmatioe by additioB labocatories. 



(2) SIZE: Livers shaD be of "«"«i«"'"" size 45-60 f, and iwfividual samples shaD be padcaged in separate 

 swirl packs or other DOs-water-solnUe p»'*^E™£ Other types of sample should be of similar size. 



(3) SHIPPING: Samples shaD be padced in dry ice, and shipping eootainers shaD be marked as 

 appropriate for infectious agents, as idrntiftcd by the lATA. A p p n yr i ate labels may include those pertaining to 

 toxins, dry ice, and infectious agents. 



(4) CODING: All samples should be eoded in a random £ashiofi, with copies of the coding retained 

 by the shipper. 



(5) SAMPLE QUALITY: Liver integrity is of principal importance in assays for brevetonns. No 

 guarantee can be made for analyses resulting froin decomposed, improperly p«'*ig^'<. or otherwise non-pristine 

 materials. 



(6) SAMPI ^. AGE All hough not qnantififid, cge may have Sipme bearing on the coocentratioas of 

 toads remaining. We do not set a time limit oe sam|rie storage for data is incompleie, but draw anentioo to it 

 as a possible complicating faaor in certain analyses. 



(7) CONTROLS: Appropriate eoded control amplrs should be submitted, mixed with authentic samples. 

 Two coofirmed cootrcds should be marked as CONTROL, and shipped with the first set of samples, for batft'T 

 confumation. 



Specific Pr o tocol Steps are Numbered: 



(1) Upon receipt, fiver samples are nnparkfd, logged, weighed, and placed m a -20^ freezer. We shall 

 analyze 10 samples per week, and based on shipments of this number, no sampk shaD be in our freezer for more 

 than one week. AD samples are treated as iafectioos agenu as defined by HHS publication No. (NIH) 88-8395. 



(2) AD protocols ntifize pristine ^assware which has been nordtased medficaDvfor the proien md h».; not 

 been used previousK. This is of predominant importance to prevent background lewds of tonn in re-used 

 glassware. AU soh«Dts are of rea^at grade or better. 



(3) Individual livers an placed m SOO mL gUkt beakers, 300 mL acetone is added, and each is covered with 

 a glass watchglass. Livers soak owemight at room temperature b acetone to dehydrate. The fiver suspension is 

 homo gen ized using a double blade Vtrtis hoaogenizer operated at 2S00 RPM m an ice bath. The acetone/bver 

 bomogeaate is powed off and is fihered throii^ a bochner funnel using Whatman #1 fiherpaper and eoltected 



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