SUMIDA ET AL.: ILLUSTRATING 



35 



need to be accurately drawn. An effective balance between show- 

 ing important characters for identification and three-dimen- 

 sional reahsm of the egg is required to maintain clarity. Several 

 illustrations of the egg at different stages of development and 

 from different perspectives are helpful in demonstrating key 

 characters such as embryonic pigmentation, myomeres, and po- 

 sition of the oil globule(s) in the yolksac. Adherence to a con- 

 sistent illustrative style is primarily critical for a developmental 

 series of eggs. As with fish larvae, pen and ink drawings provide 

 the most practical technique for illustrating fish eggs, but the 

 specific style of illustrating and details shown depend upon the 

 character of the egg and its stage of development. Many kinds 



of illustrative styles and techniques are found in the literature 

 (see Ahlstrom and Moser, 1980 and references cited therein) 

 and examination of these is most helpful in effectively illus- 

 trating a particular type of fish egg. 



(B.Y.S.) National Marine Fisheries Service, 8604 La Jolla 

 Shores Drive, La Jolla, California 92038; (B.W.) Gulf 

 Coast Research Laboratory, East Beach Drive, Ocean 

 Springs, Mississippi 39564; (W.L.) Department of 

 Fisheries, Humboldt State University, Arcata, Cal- 

 ifornia 95521. 



Clearing and Staining Techniques 



T. POTTHOFF 



THE clearing of tissues and the staining of cartilage and bone 

 are indispensable in the study of larval and juvenile fishes. 

 At the National Marine Fisheries Service Miami Laboratory 

 modifications of the clearing and differential cartilage-bone 

 staining technique proposed by Simons and Van Horn (1971) 

 and Dingerkus and Uhler (1977) are used. The modifications 

 are in part based upon an unpublished manuscript by W. R. 

 Taylor and G. C. Van Dyke from the National Museum of 

 Natural History, Washington, D.C. A wide size range of fish 

 from 3 mm NL to larger than 500 mm SL can be cleared and 

 stained. The technique works well for all sizes, but adjustments 

 in the various solution soaking times are made dependent on 

 fish size (Table 5). 



Method 



F/.Ya/ZoA!. —Specimens are fixed in 1 0-15% marble chip buffered 

 formalin. Samples previously fixed in formalin of lower than 

 10-15% concentration and specimens presently in alcohol or 

 fixed in alcohol should be refixed in 10-15% formalin for 

 best results. Eighty to 90% of all larvae of different perciform 

 families fixed in alcohol totally disarticulated during clearing 

 and staining. In juvenile and adult fish > 100 mm SL the flesh 

 is routinely removed from the left side before or after fixation. 



Dehydration— This is an important step, because even small 

 amounts of water interfere with the staining of cartilage. Place 

 specimen from the formalin into solution of 50 parts of 95% 

 cthanol and 50 parts distilled water. Do not wash or soak spec- 

 imens with water during transfer from formalin to alcohol. 

 After one day for larvae < 20 mm SL and two days for specimens 

 20-80 mm SL and three to five days for specimens >80 mm 

 SL transfer from 50% ethanol into absolute ( 100% or 200 prooO 

 ethyl alcohol. If absolute ethanol is not available, 190 proof or 

 95% ethanol can be substituted for the absolute, although stain- 

 ing of cartilage will not be as intense. A second change of ab- 

 solute alcohol is desirable in larger than 20 mm SL specimens. 

 Leave larvae <20 mm SL for one day in the absolute alcohol 



and juveniles 20-80 mm SL for 2 days. Adult and juvenile fish 

 80-200 mm SL should be kept in absolute ethanol for 3 days 

 and fish >200 mm SL should be soaked for one week. An 

 intermediate absolute alcohol change should be given to all 

 specimens with longer than one day soaking time. 



Cartilage staining. — This is accomplished by placing specimens 

 in an acidified alcohol solution of the alcian blue stain. For best 

 results 70 parts of absolute alcohol should be mixed with 30 

 parts of acetic acid 99% glacial. To every 100 ml of acidified 

 alcohol 20 mg of alcian blue powder should be added. The above 

 solution should be used on larvae and juveniles from 3 mm NL 

 to 80 mm SL. For larger fish, a staining solution of 60 parts 

 absolute alcohol and 40 parts of acid with 30 mg of alcian blue 

 for every 100 ml of acidified alcohol should be used. Fish larvae 

 and juveniles <80 mm SL should be left in the alcian staining 

 solution no longer than 24 hours. Larger juveniles and adults 

 should be stained no longer than 36 hours. Specimens >500 

 mm SL can remain 48 hours in the alcian staining solution. 

 After the specified time in the alcian solution the stain is per- 

 manently fixed in the cartilage and cannot be removed with any 

 chemicals used in the clearing and staining process. Staining 

 solution can be used twice for staining larvae but should be 

 discarded after staining a juvenile or adult fish. 



Neutralization. — This process raises the pH within the specimen 

 thus allowing proper subsequent bleaching. The higher pH pre- 

 vents further calcium loss from the bones for better alizarin red 

 stain. To neutralize the specimen remove it directly from the 

 alcian staining solution and place it in a saturated sodium borate 

 solution for 12 hours for specimens <80 mm SL and for 48 

 hours for larger specimens. For the juveniles and adults that 

 soak for 48 hours, change the sodium borate solution once. 



Bleaching (an optional .s/cpA — Larvae with little pigment on 

 their body (e.g., Scombridae) should not be bleached. Larvae 

 covered with pigment (e.g., Istiophoridae) and all juveniles and 

 adults must be bleached. Prepare bleaching solution by mixing 



