FISHERIES RESEARCH BOARD OF CANADA TECHNICAL REPORT NO. 123

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1 FISHERIES RESEARCH BOARD OF CANADA TECHNICAL REPORT NO

2 FISHERIES RESEARCH BOARD OF CANADA Technical Reports FRS Technical Reports are research documents that are of sufficient importance to be preserved, but which for some reason are not aopropriate for scientific publication. No restriction is olaced on subject matter and the series should reflect the broad research interests of FRS. These Reports can be cited in publications, but care should be taken to indicate their manuscript status. Some of the material in these Reports will eventually appear in scientific publication. Inquiries concerning any particular Report should be directed to the issuing PRS establishment which is indicated on the title page.

3 FISHERIES RESEARCH BOARD OF CANADA TECHNICAL REPORT NO. 123 CLEARING AND STAINING JUVENILE PACIFIC SAI1ION FOR SKELETAL STUDIES by Linda D. Hungar FISHERIES RESEARCH BOARD OF CANADA Biological Station, Nanaimo, B. C. July 1969

4 By modifying Alizarin Red techniques described by Hollister (1934), a method for clearing and staining juvenile Pacific salmon was do?veloped for the examination of skeletal parts in toto. To simplify the i!l toto examination of bony parts, a method of clearing and staining juvenile Pacific salmon (Oncorhynchus spp.) was desired. The method described below was developed from that of Hollister (1934) for various non-salmonids, and produced specimens from which accurate counts of vertebrae, branchiostegals, gill rakers and fin rays could be obtained. Specimens to be treated ranged from 30 to 110 mm fork length. Best results were obtained from fish killed in MS222 and hardened for a 2-weck period in 10% formalin (3.7% formaldehyde). The addition of borax to the formalin did not improve the results. Samples preserved in 7~ alcohol were tried but cleared slowly in KOH, became wrinkled and limp in the early stages of staining and ultimately became unsuitable for vertebral counts. Unhardened fish di sintegrated during pre-stain clearing in KOH. Prior to staining, the specimens were rinsed for 5-24 hours in running water to remove most of the formalin. Evisceration was not found to be essential but tended to facilitate staining of the vertebrae. Scales stained readily and obscured the vertebrae, and hence were removed by lightly scraping the sides of the fish prior to treatment. The samples were then divided into two groups according to length. The larger specimens (say, 70 to 110 nvn), which required longer staining and clearing periods, could then be transferred through the solutions independently of the smaller ones. Both size groups were placed in 500 ml of 1% hydrogen peroxide solution for 24 hours to eliminate natural pigmentation and then moved to a 2% KOH solution for 2 to 4 days (depending upon the size of the fish)

5 - 2 - for clearing. As noted by Lipman (1935), successful staining was found to depend upon correct initial clearing in KOH; excellent post-staining results were obtained if the entire dorsal area and the caudal peduncle became transparent before staining was attempted. The staining and clearing solutions were prepared as follows: L Prepare Alizarin dye solution using distilled water: Glacial acetic acid, c.p., 50% solution Glycerin, c.p. Chloral hydrate crystals, c.p., 1% solution Alizarin Red S, added until solution is medium brown in colour..qq. not saturate. 5 ml 10 ml 60 ml 2. Add 0.5 ml of the above dye solution to 500 ml of 2% KOH Additional Alizarin powder may be added to this KOH staining solution to obtain a medium-purple colour. However, solutions containing too much Alizarin will result in over-staining of the sample which no amount of clearing will successfully correct. Until the right amount of dye to be sprinkled in (approx. 4 mg) can be determined, it is better to prepare a solution lighter in colour and immerse the samples for a longer period. Staining took 1 to 3 days depending upon the strength of the dye solution and the size of fish. After staining, the samples were transferred to a 1% KOH solution to remove excess colour from the flesh. Specimens were cleared for examination by replacement of KOH solution with glycerin in the following steps: parts 2% KOH, 40 parts glycerin: hr parts 4% KOH, 30 parts glycerin: 24 hr parts 4% KOH, 60 parts glycerin: 24 hr parts 4% KOH, 90 parts glycerin: 24 hr 5. Storage in pure glycerin, plus a crystal of thymol to prevent mold. Replace glycerin when it becomes discoloured.

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7 - 3 - Exposure of the specimens to ultraviolet light or sunlight during the post-staining stages was recoltlllended by Hollister as a method to eliminate excessive pigmentation. However, we found that young salmon (with less pigment than many of the deep-sea fish she processed) sufficiently bleached by the pre-staining bath in hydrogen peroxide. The entire procedure, from hydrogen peroxide to pure glycerin, required 7 to 9 days for smaller fish and 10 to 15 days for the larger. 1. Harden specimens in 10% formalin for at least 2 weeks. 2. Rinse in running water (2-24 hr). Eviscerate and des::ale if desired. 3. Bleach in 1% hydrogen peroxide solution (24 hr). 4. Clear in 2% KOH (2-4 days). 5. Stain in KOH dye solution (1-3 days). 6. Pla:e in 1% KOH solution to remove excess Alizarin (1-6 hr). 7. Clear as follows: 60 parts 2% KOH, 40 parts glycerin: hr. 70 parts 4% KOH, 30 parts glycerin: 24 hr. 40 parts 4% KOH, 60 parts glycerin: 24 hr. 10 parts 4% KOH, 90 parts glycerin: 24 hr. 8. Store in glycerin with crystal of thymol. Acknowledgements Suggestions by Mr. P. J. McCart regarding staining methods were most useful The author also wishes to thank Mr. F. C. Withler and Dr. R. R. Parker for their guidance in preparing this report.

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9 - 4 - Hollister, G Clearing and dyeing fish for bone study. Zoologica, 12: Lipman, H. J Staining the skeleton of cleared embryos with Alizarin Red S. Stain Techno!.,