Bacteriological 



A quadrangular section of the skin was removed aspeticelly from the 

 flesh of the fisho With sterile instruments a 20 gram sample of muscle was 

 excised and weighed into a sterile, tared petri disho This fish flesh was 

 then transferred into a sterile Waring Blendor, 180 mis of sterile buffer 

 solution l/were added, and the mixture was thoroughly macerated <, Samples of 

 this mixture were used for all subsequent bacteriological tests „ 



Tryptone glucose extract agar was employed for plate counts, and 

 standard lactose broth was used for determining the M»PoN« of coliforms So So 

 agar, MacConkey's agar, and bismuth sulfite agar were used for determining the 

 presence of enteric pathogens Tetrathionate broth was used as an enrichment 

 mediunio The Most Probable Number method was used for determining the number 

 of anaerobes, employing Difco anaerobe medium„ All samples were incubated 

 at 37° Co Plate counts and the M.P.N. determinations of anaerobes were made 

 at the end of I4O hours incubation The K.PJ. of coliforms was made according 

 to the procedures outlined in Standard Methods for the Examination of Water 

 and Sewage (1936)° Representative colonies that appeared on tryptone glucose 

 extract agar were transferred to agar slants for further identif icationo 

 Coliform colonies found on Levine's eosine methylene blue agar were also 

 transferred for further ident if icationo All colonies were purified on tryptone 

 glucose extract agar plates previous to identif icatioru Smears were made from 

 the anaerobe medium, stained by the Gram method, and examined for the presence 

 of organisms resembling the genus Clostridium^ 



Toxicological 



A modification of the method used by Macht and Spencer (19^1) for 

 testing the toxicity of fish muscle was employedo A 50 gram sample of muscle 

 was removed in the manner identical to that employed in obtaining the sample 

 for bacteriological examination This was placed in a chemically clean 

 Waring Blend or, 100 ml of sterile o percent saline were added, and the 

 mixture was thoroughly macerated, A portion of the mixture was placed in a 

 glass tube and centrifuged until a clear liquid supernate was obtained,, Gonad 

 tissue was prepared in a similar mannero The sample weight of the gonad tissue 

 varied, however, the proportion of saline used was the same as that used in 

 the preparation of the muscle tissue extracto The clear supernate was used 

 for the tests. 



Three white mice, each weighing between 20 and 25 g, was each 

 injected intraperitoneally with 1 ml of the saline extract made from the raw 

 tissue o These mice were then observed closely for 1 hour and after that at 

 periodic intervals o In those cases where all injected mice died, the saline 

 extract of the raw sample was heated to 80° Co for g hour (an arbitrary 

 temperature and time) and another series of 3 mice was injected with 1 ml 

 of the heated extracto In those cases where the mice exhibited a positive 

 reponse to the intraperitoneal injection of the extract, the raw tissue was 

 fed orally to another group of animals For the oral feeding tests, three mice 

 that had been fasted for 2U hours prior were used„ No necropsies were performed. 



l/ The method for preparation of the buffer solution is as follows? Dissolve 

 3I4 g of KHpPOL in approximately 500 ml of distilled water . Adjust to 

 pH 7o2 with IN NaOHo Dilute to 1 liter. Add 1„25 ml of this stock buffer 

 solution to each liter of distilled watero 



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