give distinct, easily recognized markings. 

 Trypan red also gives a clear mark, but it 

 is less distinctive than the other two because 

 the color tends to blend with the natural color 

 of some shrimp. Trypan red may be an excel- 

 lent stain for white shrimp since it contrasts 

 with the shrimp's natural color. Unlike fast 

 green FCF and Trypan blue, however, it is 

 subject to some fading. 



Solvents and Dilution 



An important factor in successful shrimp 

 marking is proper handling of stain solutions. 

 Filtered sea water is the stain solvent most 

 often reported in attempts to mark marine 

 animals with vital dyes. Dawson (1957) used 

 distilled water, filtered sea water, glycerine, 

 mineral oil, and alcohol as stain solvents 

 when testing various stains as shrimp-marking 

 agents. In the four stains he reported as the 

 most potentially useful for marking shrimp, 

 a mixture of 90 percent filtered sea water 

 and 10 percent distilled water was used. He 

 concluded, however, that distilled water was 

 the best all-round solvent. Experience with 

 filtered sea water indicates it is generally 

 satisfactory as a solvent for fast green FCF 

 but unsatisfactory for use with Trypan blue. 

 Trypan red, or Niagara sky blue 6B. When 

 solutions with a sea-water base were used, 

 high mortalities frequently occurred with all 

 stains except fast green FCF. Moreover, 

 stains in solutions of natural or artificial 

 sea water did not always give equally distinct 

 marks from specimen to specimen. 



Menzel (1955) suggested that isotonic solu- 

 tions be used in marking shrimp. Experi- 

 mentation has shown that isotonic solutions 

 are not only unnecessary but, in fact, are 

 inferior to solutions prepared with distilled 

 water. As demonstrated by Williams (1960), 

 body fluids of Penaeus shrimp are very nearly 

 isotonic with respect to the surrounding me- 

 dium, and the animals demonstrate vigorous 

 osmoregulatory capacity. It is therefore rea- 

 sonable to assume that no ill effects will 

 develop from injections of small quantities 

 of sterile solutions which are hypotonic with 

 respect to body fluids. 



Sterile double-distilled water is recom- 

 mended as the best solvent for all stains 

 used as marking agents. It can be obtained 

 from any pharmaceutical house. 



Standardizing Solutions 



Conn (1923) suggests that all dye solutions 

 be defined in terms of dilutions of a saturated 

 solution. This seems reasonable although satu- 

 ration with any given quantity of solvent is not 

 always easily determined, especially when 

 dealing with suspensions and dark-colored 

 dyes. 



For marking shrimp, we need only be con- 

 cerned with a limited number of aqueous 

 stain solutions in concentrations of 0.5 percent 

 or less. All staining solutions reported herein 

 were made by first preparing a 0.5 percent 

 stock solution from dry stain and then making 

 the required dilutions. Recommended stock 

 solutions (0.5 percent) are prepared by dis- 

 solving the following amounts of dry stain ' in 

 100 ml. of sterile, double-distilled water 

 (65 OF.): 



Trypan blue (Harleco, Color 



Index No. 477) 0.67 g. 



Trypan red (Harleco, Color 



Index No. 438) 0.54 g. 



Fast green FCF (National 



Aniline, Color Index No. 



42053) 0.56 g. 



Stir mixtures until no large particles remain 

 and filter once through Whatman #1 filter 

 paper or equivalent. Unfiltered solutions have 

 been found unsatisfactory for marking shrimp. 



The staining action of certain dye solutions 

 is improved by aging (Conn, 1950). Dawson 

 (1957) found, however, that aged solutions 

 offered no advantage, so stains were usually 

 prepared just before use. Experience not only 



* For Trypan blue. Trypan red, and fast green FCF 

 of the grades discussed herein, acmal dye content of 

 the raw material (as specified on container labels) 

 was 79 percent, unknown, and 94 percent, respec- 

 tively. In tbc case of materials wbich specify a dif- 

 ferent percentage dye composition, appropriate adjust- 

 ments should be made in calculating the amoont of dry 

 material required. 



