ANALYTICAL METHODS 373 



where the initial primary spoilage substance is lactic acid, pH is a simple, 

 fairly reliable freshness test. 



Of the many specific tests for single chemical substances used as a 

 measurement of freshness, that for trimethylamine^ has been most widely 

 used. While the trimethylamine measured during spoilage of fish con- 

 tributes little to the fishy odor, with some species of fish there is a fairly 

 consistent increase in trimethylamine with spoilage. The test has proved 

 to be useful principally with certain low-oil content marine species such 

 as cod and haddock. Among the many other specific tests which have 

 been used for fish freshness are those for histamine^, hydrogen sulfide ^ 

 acetoin', and certain free amino acids^. 



Many of the chemical freshness tests are successful in correlating with 

 deteriorative changes occurring at the middle and late stages of spoilage 

 where such spoilage is usually fairly obvious from organoleptic odors. 

 Few, if any, of the tests provide a sensitive measure of early deteriorative 

 changes before onset of the obvious spoilage changes. 



Total bacterial counts are sometimes carried out as an indication of 

 spoilage. While such total counts do include non-spoilage bacteria along 

 with those which are responsible for spoilage, plate counts are neverthe- 

 less useful, at least in cases where relative rather than absolute values 

 are to be used. Standard plate count methods such as are used for other 

 foods are employed for fish. Where a measure of spoilage is desired, plate 

 incubation temperatures of 20 or 25°C should be used rather than 37.5°C 

 which is suitable mainly w^here counts are concerned with sanitary 

 practices. 



Methods for Fish Oils 



When fish oils are to be extracted for subsequent examination, it is 

 important to use extraction procedures which will not result in oxidation 

 or polymerization during extraction. The method of Bligh and Dyer^, 

 employing a cold shaking extraction with a chloroform-methanol mixture 

 is useful for this purpose. 



The oxidative changes in fish oils which lead to rancidity can be 

 followed by measuring total oxygen adsorbed (by use of a Warburg 

 apparatus or by weight increase^"), by measuring the amount of inter- 

 mediate peroxide content^^, or by measuring the amount of certain end 

 products^^ 



The first of these methods (oxygen adsorption) is of an accelerated 

 type, whereby the rate at which oxygen is adsorbed by the oil is meas- 

 ured. The other methods are tests for products formed by oxidation of 

 the oils. They also can be applied to measurement of the rate of oxygen 

 adsorption by aerating the oils and, at suitable intervals, running the 

 test in question. Tests of this type, where the rate of oxygen uptake is 



