A. N I T R A T E R E D U C T I O N
T h e reduction of nitrate has b e e n used as a means of evaluating the extent of bacterial contamination and of bacterial growth during fish spoilage ( T i l l m a n s et al, 1 9 2 1 ; Tillmans and Otto, 1 9 2 4 ) . No clear relationship b e t w e e n the amount of reduction and the state of spoilage was evident, and it was not found to b e an a c c u r a t e measure of bacterial growth.
B . O X Y G E N C O N S U M P T I O N
T h e oxygen uptake b y fish during spoilage has b e e n suggested as a freshness test b u t was found u n a c c e p t a b l e for this purpose (Tillmans and Mildner, 1 9 1 6 ; Tillmans et al, 1 9 2 1 ; Tillmans and Otto, 1 9 2 4 ) .
C. D Y E R E D U C T I O N T E S T S
Another means to obtain a measure of the extent of bacterial growth and of the bacterial content as a gauge of the degree of spoilage was the reduction of various dyes.
1. Methylene Blue
Tillmans et al ( 1 9 2 1 ) , Tillmans and Otto ( 1 9 2 4 ) , Okolov ( 1 9 3 6 ) , van de V e l d e ( 1 9 3 7 ) , and Rasmussen ( 1 9 5 0 ) all found some decrease in the reduction time with the onset of fish spoilage and the increase in bacterial numbers. Cavallone ( 1 9 5 9 ) found this m e t h o d unreliable as a freshness test of fish.
2. Resazurin
Resazurin, originally used to test the quality of raw milk, has b e e n used a n u m b e r of times as a measure of the bacterial population of perishable foods, including fish and shellfish. A m o n g the reports of its use are those b y W a l d b a u e r ( 1 9 3 1 ) , Mundinger and Wolff ( 1 9 3 3 ) ,
van de V e l d e ( 1 9 3 7 ) , Proctor and Greenlie ( 1 9 3 9 ) , and B a k e r et al ( 1 9 4 2 ) .
T h e color attained b y resazurin in milk of low bacterial content depends upon the redox potential value of the equilibrium set up b y the mixing of the reducing systems of the dye and milk and is not directly related to the n u m b e r of b a c t e r i a in the milk ( T h o r n t o n et al, 1 9 4 1 ) . Similar considerations could apply to other foods, including fish, and could help to explain the general observation that the reduction rate of the dye was of value as a measure of a high bacterial action b u t was of little significance for the early spoilage stages where the bacterial numbers and their products w e r e only beginning to increase. U n o and T o k u n a g a ( 1 9 5 4 ) studied the relationship b e t w e e n the reduction time of resazurin and the freshness of fish flesh. T h e y found this test useful for herring, b u t not for mackerel. T h e p H had a definite effect on the reaction. Cavallone ( 1 9 5 9 ) reports good results with Italian fresh-water fish.
3. Tetrazolium Derivatives
Colorless substituted tetrazolium ( T Z ) salts are reduced in neutral solutions b y living cells which then b e c o m e stained red b y the formation of formazan. T h e s e salts have b e e n used extensively for viability tests and in microbial and m e t a b o l i c studies. S h e w a n and Liston ( 1 9 5 7 ) reported on several years of investigation of the usefulness of this dye test for the purpose of assessing the quality of iced white fish. T h e y found that a special n e w derivative, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride, was m o r e useful as a sensitive indicator compared with the traditional T Z salts. T h e y described a t e c h n i q u e of using filter paper impregnated with the dye and either judging t h e color visually or, after extraction, spectrophotometrically. T h e general value of this test for whole fish and perhaps for fillets will b e c o m e clearer as more data b e c o m e available for various species stored under different conditions.
Moorjani and I y e n g a r ( 1 9 5 7 ) and Moorjani et al ( 1 9 5 7 ) measured the quality o f Indian fresh-water fish and studied the effect of p H , incubation period, and fish species on the reduction time. I t was claimed that this test was of less value in fishes with a high content of T M A O , since this s u b s t a n c e apparently exerted a poising effect on the redox potential of the system and thus tended to delay the reaction.
4. Other Dyes
Okolov ( 1 9 3 6 ) tested the reduction rate of indigo carmine as a measure of spoilage and concluded it was not as useful as methylene blue. I n 1937, van de V e l d e used the reduction of Janus green to a red
96 L I O N E L F A R B E R
color as a measure of spoilage, in addition to methylene b l u e and resazurin. H e found that the reduction time for all the dyes increased with the progress of spoilage.
D . B A C T E R I A L C O U N T S
1. Direct Count
T a r r ( 1 9 4 1 , 1 9 4 5 ) published a t e c h n i q u e designed to give a direct evaluation of the n u m b e r of b a c t e r i a in a sample. This m e t h o d was useful where the n u m b e r of b a c t e r i a was above a certain low level, which represented the limit of sensitivity of the procedure. T a r r also r e c o m m e n d e d a short incubation period to increase the sensitivity. T h e direct count included any nonviable organisms that m a y have b e e n present. Wittfogel ( 1 9 5 3 , 1955a, b ) used the direct count to assess the condition of marine fish. D i r e c t bacterial counts have b e e n used b y T a r r ( 1 9 4 4 a , 1 9 4 5 ) to measure the condition of fish and were advocated by him in a r e c e n t paper ( T a r r , 1 9 5 8 ) as other methods of a chemical nature fail to offer guidance as to freshness.
2. Total Viable Count
T h e most commonly used test for bacterial contamination is the total viable count and its many variations and modifications. This has been used b y m a n y individuals to obtain a measure of the condition of a sample of fish. T o t a l aerobic bacterial counts have b e e n used b y m a n y investigators to follow the deterioration of fish flesh and shellfish flesh, among w h o m are Griffiths and Stansby ( 1 9 3 4 ) , B o u r y and Schvinte ( 1 9 3 2 , 1 9 3 5 ) , Aleev et al. ( 1 9 3 6 ) , F i t z g e r a l d and Conway ( 1 9 3 7 ) , Salmon et al. ( 1 9 3 7 ) , van de V e l d e ( 1 9 3 7 ) ( w h o considered the count of little v a l u e ) , Proctor and Greenlie ( 1 9 3 9 ) , Castell et al. ( 1 9 4 8 ) , Rasmussen ( 1 9 5 0 ) , Luijpen ( 1 9 5 4 a ) , Partmann ( 1 9 5 4 ) , T a n i k a w a et al.
( 1 9 5 5 ) , F ä r b e r and L e r k e ( 1 9 5 6 a , b , 1957, 1 9 5 8 ) , Novak et al. ( 1 9 5 6 ) , F i e g e r et al. ( 1 9 5 8 ) , and Moorjani et al. ( 1 9 5 8 ) .
Sekine and Nakakubo ( 1 9 5 3 ) described a procedure for placing a sterilized p a r c h m e n t paper strip on the surface of the fish and then on the surface of a solid medium to obtain a measure of the bacterial contamination.
T h e type of bacterial flora and its relation to fish spoilage has b e e n studied b y Hunter ( 1 9 2 0 a , b , 1922a, b , c ) , S c h ö n b e r g ( 1 9 3 0 ) , S c h ö n b e r g and D e b e l i c ( 1 9 3 3 ) , T a r r ( 1 9 3 8 ) , Shewan ( 1 9 3 8 , 1944, 1 9 5 3 ) , Snow and B e a r d ( 1 9 3 9 ) , Notevarp et al. ( 1 9 4 2 ) , Kiser ( 1 9 4 4 ) , Kiser and B e c k w i t h ( 1 9 4 4 ) , Liston ( 1 9 5 7 ) , Nikkilä ( 1 9 5 5 ) , L e r k e et al ( 1 9 6 3 ) , and Adams et al. ( 1 9 6 4 ) .
Reviews covering the bacteriology of fish spoilage have b e e n
pub-lished b y Griffiths ( 1 9 3 7 ) , S h e w a n ( 1 9 4 9 ) , and T a r r ( 1 9 5 4 ) . Further
more, special chapters in V o l u m e I of this treatise deal with the bacteriological aspects of spoilage in sea-water fish b y S h e w a n ( V o l u m e I, C h a p t e r 1 4 ) , in fresh-water fish b y B r a m s t e d t and Auerbach ( V o l u m e I, Chapter 1 6 ) , and on shellfish b y F i e g e r and Novak ( V o l u m e I, C h a p t e r 1 5 ) .
E . E N Z Y M E A C T I V I T Y T E S T S
1. Catalase
Tillmans et al. ( 1 9 2 7 ) tested catalase activity as a criterion of spoilage and found it too variable or irregular to b e relied on. Poluektov ( 1 9 3 3 ) also reported that the increase in catalase activity of fish was too variable to b e used as a criterion o f spoilage. T o m i y a m a et al. ( 1 9 5 1 ) and T o m i y a m a and Yone ( 1 9 5 3 ) used the catalase activity of gill tissue and of surface washings of "kamaboko" ( s e e V o l u m e I I , Chapter 8 ) as a measure of bacterial activity and of the condition of the preparation.
T h e y reported that the catalase activity of t h e surface washings paralleled the growth of b a c t e r i a on the surface.
2. Peroxidase
Poleuktov ( 1 9 3 3 ) found the peroxidase activity of fish too variable to b e of use as a spoilage indicator. Okolov ( 1 9 3 6 ) , and Okolov and Shavskii ( 1 9 6 3 ) reported that the peroxidase activity could b e used as a criterion of the quality of salted fish and of the brines surrounding them.
3. Succinic Dehydrogenase
F u k u d a , in a series of papers ( 1 9 5 7 , 1 9 5 8 ) , reported on t h e estimation of freshness of fish b y the succinic dehydrogenase activity of the flesh and viscera. E v e n though the individual values fluctuated, there was in general a reduced activity with the decrease in freshness of the samples.
4. Other Enzymes
Tillmans et al. ( 1 9 2 7 ) found no c h a n g e in the activities of diastase or of proteolytic enzymes as spoilage progressed. F i e d l e r ( 1 9 4 1 a ) reported that there was an indication of a relationship b e t w e e n the age of fish and their autolytic proteolytic enzyme activity; enzymes in older fish r e a c t e d more slowly than those in younger fish. H e suggested that this m a y have a b e a r i n g on their subsequent k e e p i n g quality in cold storage.