POTTHOFF: CLEARING AND STAINING 



37 



lution with the addition of heat (Miller and Van Landingham, 

 1969). With this method the bones are not disarticulated, but 

 some bone distortion was experienced. 



Variables affecting results.— The results of the clearing and 

 staining procedure are not always satisfactory because of known 

 and unknown variables. Results can never be predicted with 

 certainty. The known variables are: ( 1 ) Time and ambient tem- 

 perature the organism is subjected to between death and fixation. 

 The longer an organism remains unpreserved after death and 

 the higher the temperature, the less the tissues will clear. For 

 best results, specimens should be killed in the fixative, or if that 

 is not possible, they should be kept cool or frozen before fixation. 

 (2) Effect of fixative and preservative. Marble chip buffered 

 formalin is a good fixative for larval fish if specimens are re- 

 moved from it after 24 hours. Buffered formalin as a preser- 

 vative destroys first the stain uptake in cartilage. Bone decalcifies 

 as buffered formalin becomes acid over a longer time period 

 and decalcified bone will not stain. Therefore, it is best to fix 

 specimens in 10% formalin and then to preserve them in 70- 

 95% ethanol. Specimens fixed and preserved in ethanol should 

 be re-fixed in formalin before clearing and staining. (3) Time in 

 a preservative. The longer a specimen has been preserved, the 

 less predictable the clearing and staining outcome will be. Some 

 fish larvae from the Dana collection in the 1920's were cleared 

 and stained. The results were startling for both Formalin and 

 alcohol preserved material because some specimens cleared and 

 stained well, but most were unfit for study. 



Other vanables which affect the results of clearing and staining 

 exist, but are not understood. No matter how carefully one 

 adheres to the procedures, the clearing and staining results are 

 not predictable. 



Interpretation of results. — Frequently specimens will remain 

 opaque and overstain with alcian or alizarin for unknown rea- 

 sons. This makes viewing of cartilage and bone structure diflicult 

 or impossible. Such specimens can be used for study of fin ray 

 development and for fin ray counts. 



Cartilage or bone does not always stain but can be made 



visible in cleared preparations by changing light conditions at 

 the microscope and manipulating the substage mirror. Cartilage 

 appears reticulated in structure whereas bone is structurally clear 

 and hyaline. 



Erroneous conclusions can be made if one solely relies on 

 color to determine cartilage and bone. In general, cartilage will 

 appear blue and bone red, but often alcian blue is taken up by 

 bones and rarely alizarin red by cartilage. For instance, devel- 

 oping fin rays often appear blue. 



Generally larger developed cartilage structures will stain bet- 

 ter than small developing ones. Thus, in the same specimen one 

 may find brightly blue stained cartilage, pale blue cartilage, and 

 cartilage with no stain at all. Therefore, special care is indicated 

 when viewing newly developed cartilage. 



The ossification onset in cartilage is difficult to determine. A 

 thin layer of bone forming all around the cartilage can be de- 

 tected by examining the outer edges of the cartilage structure: 

 a shiny hyaline line forms there, probably only a cell layer thick. 



Investigators are often discouraged by clearing and staining 

 results, particularly when their sample is small. In a larval de- 

 velopmental series I usually clear and stain 200 to 400 speci- 

 mens, and I am able to study each aspect and area of devel- 

 opment that I wish to examine because of the large sample size 

 at hand. For example, in a specimen in which the pectoral fin 

 support area is unclear and stained poorly the caudal area may 

 be clear and stained well. Thus, this specimen is utilized only 

 for caudal development, whereas in another specimen the pec- 

 toral area may be clearer and better stained. Thus, with a large 

 sample size, the uncertainties and vagaries of the clearing and 

 staining procedure are overcome. 



Application of clearing and staining.— Cleanng and staining is 

 helpful in identification offish larvae when external characters 

 are inadequate. It also aids systematic and phylogenetic studies 

 of larvae to adult fishes. This subject has been discussed in detail 

 by Dunn (1983b). 



National Marine Fisheries Service, Southeast Fisheries 

 Center, Miami Laboratory, 75 Virginia Beach Drive, 

 Miami, Florida 33149. 



Radiographic Techniques in Studies of Young Fishes 

 J. W. Tucker, Jr. and J. L. Laroche 



RADIOGRAPHY is useful for obtaining skeletal informa- 

 tion in studies of fish taxonomy and morphology. Al- 

 though clearing and staining provides more detail, radiography 

 has other advantages. It produces an easily stored, long-term 

 record of the skeleton and does not permanently alter the con- 

 dition of the specimen. In many cases, counts can be obtained 

 more accurately from radiographs than from the specimens 



themselves. If an x-ray unit and darkroom are available, ra- 

 diography is usually faster and easier than clearing and staining. 

 The time saved may be of value in studies of population vari- 

 ation, in which many specimens must be examined. Radiog- 

 raphy has also been used to monitor decalcification of larvae 

 stored in formalin (Tucker and Chester, in press), and has been 

 suggested for use in toxicological studies to check large numbers 



