corroborates Dawson's findings but indicates 

 that some dyes, particularly Trypan blue and 

 Trypan red, become increasingly toxic when 

 allowed to stand in solution for extended 

 periods of time. On one occasion a 0.25 per- 

 cent solution of Trypan blue aged 3 weeks at 

 room temperature caused over 50 percent 

 mortality when used in a regular staining 

 operation. A fresh solution was mixed from 

 the same lot of stain, and mortality was 

 reduced to less than 8 percent. In discussing 

 the use of benzidine dye solutions (including 

 solutions of Trypan blue and Trypan red) as 

 vital dyes. Foot (1950) states that not only 

 are electrolytes (such as sea water) unsafe 

 as solvents, but it is equally dangerous to 

 allow the solutions to age, because the sus- 

 pended dye particles may become agglutinated 

 and render the solutions highly toxic* This 

 may have been the cause of some high mor- 

 talities experienced when using aqueous solu- 

 tions of the Trypan dyes. 



No attempt has been made to determine the 

 rate at which Trypan blue and Trypan red 

 solutions become toxic with age. It is sug- 

 gested, however, that neither dye be used if 

 it has been in solution more than 3 days. Fast 

 green FCF apparendy remains nontoxic in 

 solution over a long period of time. No 

 observations have been made with Niagara 

 sky blue 6B. 



Tolerance to Stains 



was recorded as the difference in survival 

 between the groups injected with stain solu- 

 tions and those injected with distilled water. 

 The procedure was repeated once. Final values, 

 computed from two trials at each staining 

 level, are plotted in figure 8. Fast green FCF 

 is clearly the least toxic of the three stains 

 tested. Trypan red is more toxic than fast 

 green FCF but noticeably less toxic than 

 Trypan blue. 



In addition, four groups of 25 stained shrimp 

 each were held 90 days to determine If any 

 long-term effect could be shown as a result 

 of staining. Results of this experiment indi- 

 cated that most mortality from staining oc- 

 curred within the first 24 hours. 



OOS 01 15 2 25 0.3 0.33 0.4 



QUANTITY OF AQUEOUS OYE SOLUTION 



(MU R«commiria«d Qoonfiiy J 



Figure 8.— Tolerance of pink shrimp to injections of 

 varying quantities of three vital dyes in 0.5 percent 

 solution. 



To evaluate the relative toxicity of the above 

 stains, 18 groups of 50 pink shrimp each (all 

 specimens about 95 mm. in total length) were 

 selected and treated with varying amounts of 

 0.5 percent solutions. Each group was injected 

 with a different amount of the stain solution 

 and held in circulating sea water for 24 hours. 

 In all, six levels ("doses") were tested. As 

 controls, identical groups were injected with 

 equivalent volumes of sterile distilled water 

 and held with the stained animals. The degree 

 of intolerance to increasing amounts of dye 



* Benzidine dyes do not go into solution when placed 

 in water but remain as colloidal suspensions (Evans 

 and Schulemann. 1914). 



Optimum Staining Levels 



Experiments with pink shrimp in the size 

 range 70 to 150 mm, total length were con- 

 ducted to determine the amount of dye required 

 to achieve the most distinct and permanent 

 mark. These shrimp were stained with various 

 solutions ranging in quantity from 0.020 to 

 0. 250 ml. and observed for a 90-day period. 

 Analysis of results yielded the values in table 

 1, which were found most suitable from the 

 standpoints of minimizing staining mortality 

 and maximizing distinctness and permanency 

 of marks. Similar studies have not been made 

 with brown or white shrimp. 



10 



