EXPERIMENTAL FISH EMBRYOLOGY 385 



transfer the embryo (gently) with wide-mouthed and sterile pipette through sev- 

 eral changes of sterile medium. All glassware and solutions should be auto- 

 claved, instruments and pipette should be flamed before use. Steel instruments 

 are used exclusively. (Oppenheimer has reduced mortality from the usual 50% 

 to almost 0% by using aseptic precautions - private communication.) Sulfadiazine 

 (0. 002%) is safe to use in any of the culture media. 



POTENTIALLY SIMPLE METHOD FOR REARING GERM-FREE FISH* 



Aquatic animals, particularly fish that can be deprived of all detectable adherent orga- 

 nisms are highly useful in experiments involving nutrition, infection, and immunity. 

 Shaw and Aronson ('54) have been successful in dipping fish eggs of Tilapia macrocephala 

 into 0. 04% formaldehyde -aquarium water for 10 minutes, and then transferring them to 

 sterile water aseptically. Such eggs developed into germ-free fry, and survived for 

 several weeks after hatching, deriving their food from the large store of yolk. The fry 

 that died did not disintegrate, but were starved to death and gave rise to no bacteria. No 

 microorganisms could be cultured from the dead fry even in various nutrient broths or 

 thioglycoUate medium. No mold or fungus growths were found. Thus, this treatment 

 appeared to be effective in decontamination. Since Tilapia are normally raised on dried 

 food, which can be sterilized, the abundant eggs of this form may be cultured in a germ- 

 free environment for experimental purposes. 



a. Vital Staining of Fish Embryos 



Procedure: 



1. Prepare vital-dye stained cellophane. Grubler's Nile Blue Sulphate and 

 Neutral Red are to be used separately, and together. The cellophane (thin- 

 nest available) should be soaked in 1% aqueous solutions for a day or more, 

 then dried on clean white paper. The cellophane is preferable to the agar 

 because it remains in one piece and can be removed. Store the red, blue, 

 and the combination-stained cellophane in clean marked envelopes until used. 

 These vital dyes are not considered to be toxic. 



2. Pass the eggs (in their shells) through sterile media. Marine forms are to 

 be treated in double strength Holtfreter's and freshwater fornns in normal 

 Holtfreter's (Standard) Solution. When tap or fresh water are used the stain 

 penetrates too rapidly and may even damage some of the cells. 



3. Following the (Nicholas, 1927) technique described above, cut a minute win- 

 dow through the chorion of the egg to be studied. 



4. With watchmaker's forceps insert a small piece of stained cellophane through 

 the window and maneuver it into the described position with a fine hair loop. 

 The chorion will generally hold the cellophane in position, where it should 



be left for from 15 to 45 minutes. The penetration and diffusion of the dye 

 can be observed directly. The dye penetration is best in hypertonic media. 



5. After adequate staining has occurred, carefully remove the stained cellophane 

 with watchmaker's forceps, wash the egg with chorion in one change of ster- 

 ile medium and place it in a covered #2 Stender, containing the normal me- 

 dium, and at appropriate temperatures for that embryo. 



6. Observe during the next 36 to 48 hours, keeping the embryo at the lower 

 limit of viable temperatures if it is desired to prolong the early stages. The 

 record consists of a series of drawings of the changing position of the stained 

 areas, beginning immediately after applying the dye. (See Brummett 1954 

 for carbon marking procedure. ) 



* Courtesy of Dr. E. Shaw, Science 1957. 125:987-988. 



