typically are used in solutions maintained 

 at temperatures not exceeding 37" or, at a 

 maximum, 39° C. , which are temperatures that 

 are obviously optimal, or nearly so, for 

 all animal parasites potentially infective, 

 at the stage of recovery, for homothermous 

 hosts. In the event that it is known or 

 assumed, a priori , that the parasite is 

 infective for a poikilotherm, the tempera- 

 ture of the digest solution is changed 

 accordingly. 



The present investigation concerns 

 the use of peptic digestion in extensive 

 queUititative studies which are not concerned 

 with the recovery Of viable parasites. Under 

 the conditions of the present investigation, 

 certain advantages are evident in what may 

 be termed high-temperature peptic digestion. 

 Economy was important, and therefore the 

 use of minimal concentrations of enzyme was 

 desired. In addition, time for quantifica- 

 tion was considered of utmost importance. 



For enzyme reactions, time and tem- 

 perature are interdependent variables. The 

 actual temperature at which an enzyme re- 

 action proceeds at a maximum rate depends 

 on many factors such as the purity and 

 concentration of the enzyme and substrate, 

 the presence of activators or inhibitors, 

 and the method of following the reaction. 

 As has been well established (Baldwin, 

 1953; Neilands and Stumpf, 1955), the so- 

 called optimum temperature is a function of 

 the time chosen for rate measurement. For 

 any given set of experimental conditions, 

 it is possible to determine the temperature 

 at which the greatest amount of chemical 

 change is catalyzed by £in enzyme under that 

 particular set of conditions. Owing to the 

 susceptibility of enzymes to thermal inac- 

 tivation, at temperatures lower than the 

 optimum the enzyme will be relatively more 

 stable Eind the reaction will proceed more 

 slowly but will continue longer, resulting 

 in greater concentrations of end products. 

 At temperatures greater than the optimum, 

 the reaction will proceed more rapidly, but 

 the catalytic properties of the enzyme will 

 be more rapidly destroyed (Baldwin, 1953). 



A series of experiments were carried 

 out to determine the optimum temperature 

 for peptonization which would require a 

 minimum of time and a minimum of enzyme con- 

 centration. The results of these experi- 

 ments demonstrated that with 5 milliliters 

 of 0.25 percent pepsin solution per gram of 



flesh, 15 to 20 minutes at 50° - 54° C. was 

 sufficient for peptonization of the flesh 

 and complete separation of worms from the 

 musculature. At successive temperatures 

 lower than 50° C, the time for complete 

 peptonization was lengthened so that it 

 prohibited accumulating mass data; at suc- 

 cessive temperatures greater than 54° C. , 

 thermal inactivation of the enzyme inter- 

 fered with the digestion. 



In addition to the simplicity and 

 rapidity of the method, the high-temperature 

 digestion procedure is efficient in that 

 the worms are easily recognized, and recov- 

 ery of all the larvae is facilitated. Fur- 

 thermore, personal error and fatigue are 

 not likely to affect the results as much as 

 in the case of the more tedious procedure 

 of dissection cind manual isolation. 



The procedure is economical in that 

 the necessary equipment is simple and inex- 

 pensive. The principal cost is that of 

 pepsin, which was found to average slightly 

 less than $0.0005 per gram of flesh digested. 

 In the present investigation, the fish were 

 small, and the average weight of the fillets 

 was 400 grams; the cost of pepsin per fil- 

 let was $0.20, or $0.40 per fish. If only 

 the ventral portions of the fillets had 

 been used for the enumeration studies, the 

 cost per fish would have been approximately 

 $0.18. 



The procedure also offers the advan- 

 tage that it lends itself to multiple 

 determinations. During the course of the 

 investigation, it was found that 2 people, 

 working with a total of 5 digestion vessels, 

 could prepare the necessary solutions and 

 treat and enumerate the parasites in 50 

 fillets during a normal working day. Thus, 

 a single operator ceji examine four times 

 as many fish in a unit of time as with the 

 dissection method. 



References 



BALDWIN, ERNEST 



1953. Dynamic aspects of biochemistry. 

 Cambridge University Press, Great 

 Britain, 2nd Edition, pp. 17-19. 



BIRD, ALAN F. 



1957. Chemical composition of the nema- 

 tode cuticle. Observations on indi- 

 vidual layers and extracts from these 



