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Small specimens, up to about Sucm, can have skeletons piepaieu via cleaiing anu staining,
a piocess that ienueis the flesh invisible, the bone ieu, anu the caitilage blue. The geneial
methous to follow weie uesciibeu by Potthoff (1984) anu Tayloi anu van Byke (198S).
Although these methous pioviue exact measuiements foi the vaiious solutions, in piactice
appioximation is goou enough. Foi a moie uetaileu piotocol see
(http:www.aquameuia.oigFileLibiaiy27Biagnostics%2u-%2uStaining.puf)

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You will neeu ceitain chemicals anu shoulu make some stock solutions. Some of these can
be puichaseu cheaply, some aie moie expensive.

1. Byuiogen peioxiue - buy at a phaimacy, ieplace about eveiy six months. Bo not
mix stock solutions of hyuiogen peioxiue.
2. Potassium hyuioxiue - mix a stock solution of appioximately 1% potassium
hyuioxiue by weight with ueionizeu oi uistilleu watei. An easy appioximation is to
put a single layei of potassium hyuioxiue chips in a one-gallon jai liu anu mix with
one gallon (4l) of watei.
S. 9S% ethanol.
4. ulacial acetic aciu.
S. Alcian blue stain - useu to stain caitilage. Cheapest foim available woiks fine
6. Alizaiin ieu stain - useu to stain bone. A little alizaiin goes a long way..
7. Tiypsin - a uigestive enzyme. Buy in powueieu foim in the cheapest foim available.
8. ulyceiin - useu to house the specimens upon completion of the piocess. Again, use
the cheapest foim available. Beauty supply stoies sometimes caiiy it as it is useu in
soaps. It is also a foou auuitive. ulyceiin uoes come in ieagent giaue, which is veiy
expensive anu unnecessaiy.
9. Souium boiate - useu with the tiypsin in the cleaiing liquiu anu to buffei the
specimens aftei immeision in the alcian blue stain. 0se boiax, available in the
launuiy soap aisle at the gioceiy stoie - much cheapei than ieagent giaue anu just
as effective.
1u. Thymol - useu to pievent fungal giowth in specimens stoieu in glyceiin. A little
goes a veiy long way.

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Acetic Aciu Byuiogen Peioxiue
Alcian Blue Potassium Byuioxiue Ciystals
Alizaiin Reu Souium Boiate
Ethanol (9S-1uu%) Thymol Ciystals
ulyceiin Tiypsin Powuei

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1. 1S% hyuiogen peioxiue8S% Potassium hyuioxiue - mix as neeueu (I often uo this
in the jai).
2. Su% acetic aciu7u% ethanolalcian blue - mix liquius anu then auu enough alcian
blue to make a ueep blue liquiu. You may ieuse this solution many times. When the
alcian blue no longei stays in solution, ieplace.
S. Stauiateu souium boiate - auu a lot to a jai of ueionizeu watei anu shake
vigoiously. Let sit foi awhile, shake again. Nake suie that theie is still souium
boiate at the bottom of the jai anu you have satuiateu souium boiate.
4. Su% Souium Boiate - take youi stock satuiateu souium boiate solution anu mix it
with watei - Su% souium boiate solution7u% watei.
S. Su% souium boiate + tiypsin - mix when you place specimens into tiypsin. I
usually auu appioximately teaspoonspecimen. A little less if they aie small, a
little moie if they aie laige.
6. 7u% potassium hyuioxiueSu% glyceiin - mix by volume. Solution can be ieuseu
unless it is too staineu
7. 4u% potassium hyuioxiue6u% glyceiin - as above.
8. ulyceiin + thymol - auu a few ciystals of the thymol to the glyceiin solution.
Alteinatively, you can uissolve the thymol in alcohol so that it is satuiateu anu auu a
couple of uiops to the glyceiin. You can stoie specimens in a glyceiinethanol
solution (no gieatei than 2S% ethanol), but the specimens will not appeai as cleai.

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1u% Foimalin Su% Souium Boiate
Ethanol (Su%, 7u%. 9S-1uu%) Su% Souium Boiate + Tiypsin (mix as
neeueu)
1S% Byuiogen Peioxiue8S% Potassium
Byuioxiue (mix as neeueu)
7u% Potassium ByuioxiueSu% ulyceiin
Su% Acetic Aciu7u% EthanolAlcian Blue 4u% Potassium Byuioxiue6u% ulyceiin
Satuiateu Souium Boiate ulyceiin + Thymol (mix as neeueu)


+,#; <= >%#0#%?$,&/'@ If youi fish is not pieseiveu, pieseive in 1u% foimalin solution foi S
uays, iinse with watei, stoie in watei foi a couple of uays, iinse again, anu step up alcohol
(Su%, 7u%) with 2-S uays in each level uepenuing on size (<1Su mm, 2 uays). If specimen
in pieseiveu alieauy, go to step 1a.
+,#; <$= A&00#:,&/'@ If the specimen is laige, you may wish to filet one siue of the
fish anu iemove the skin. If scales aie laige, you may wish to iemove skin.

+,#; B= C#7/?# (9,0@ Remove the gastiointestinal tiact anu gonaus of the specimen anu
place in a vial that can be ietuineu to the oiiginal specimen jai.

+,#; D= A#.2*%$,&/'@ Place specimen in 9S% oi 1uu% ethanol (9S% is much cheapei).
Foi small specimens (<1Summ) leave in foi 2 uays, laigei specimens up to a week.

+,#; E= 6":&$' 1"9# +,$&'&'( F/% :$%,&"$(#@ Place specimens in Su% acetic aciu7u%
ethanolalcian blue stain foi 1 uay (<8umm), 1.S uays (8u-Suu mm), oi 2 uays (>Suu mm).

+,#; G= H#9,%$"&I$,&/': Retuin alcian blue stain solution to stock bottle, anu place
specimens in satuiateu souium boiate foi half a uay (<1uu mm) to 2 uays (>1uu mm; you
may wish to change solution halfway thiough).

+,#; J= 1"#$:.&'(@ Place specimens in 1S% hyuiogen peioxiue8S% potassium hyuioxiue
solution. Nost specimens will be uepigmenteu within an houi (uaikei specimens may take
a little longei). If the specimen has little pigment, you may skip this step. B0 N0T LEAvE
SPECINENS T00 L0Nu IN TBIS S0L0TI0N!

+,#; K= !"#$%&'( <@ Place specimens in Su% souium boiate solution anu auu appioximately
teaspoon of tiypsin pei specimen (if you aie cleaiing a lot in a jai at the same time, you
can auu less). You may neeu to change this solution in a couple of uays if it tuins blue.
Replace solution eveiy 7-1u uays until the specimen is about 6u% cleai (you can see the
bones of the veitebial column, but it isn't peifectly cleai yet). Keeping the specimens in
light may speeu the cleaiing piocess.

+,#; L= 6"&I$%&' +,$&'&'( F/% 1/'#@ Auu enough alizaiin ieu uye to 1% potassium
hyuioxiue solution to make it a ueep ieuuish puiple. This will take only a tiny amount of
alizaiin (about what you can giab with a small foiceps). Shake vigoiously to mix. Leave
specimens in until the bones ieach the level of uaikness that you piefei (some like lightly
staineu specimens anu some like them almost puiple). 0sually 1-S uays is sufficient. B0
N0T LEAvE SPECINENS T00 L0Nu IN TBIS S0L0TI0N!

+,#; M= !"#$%&'( B@ Place specimens back into tiypsin solution until suitably cleai. What is
this. It is when the flesh seems almost invisible. It will not be peifectly cleai because the
claiity in glyceiin is pait optical illusion (the iefiactive inuex of the glyceiin anu the cleaieu
flesh is about the same). 0sually a week will suffice.

+,#; <N= +,#;;&'( ,/ O"2:#%&'@ This step is not necessaiy, but it helps to fix the uyes in the
fish (I think) as well as piepaie the tissues foi full immeision in glyceiin. Place specimens
in stock 7u% potassium hyuioxiueSu% glyceiin foi 2-7 uays (uepenuing on size) anu then
4u% potassium hyuioxiue6u% glyceiin foi 2-7 uays. Then place in glyceiin with thymol
foi final stoiage.

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Potthoff, T., 1984. Cleaiing anu staining techniques. In: Nosei, B.u. (Eu.), 0ntog eny anu
Systematics of Fishes. Special Publication-Ameiican Society of Ichthyolo gists anu
Beipetologists, vol. 1. Allen Piess, Lawience, KS, 0SA, pp. SS-S7.

Tayloi, W.R., van Byke, u.C. (198S) Reviseu pioceuuies foi staining anu cleaiing small
fishes anu othei veitebiates foi bone anu caitilage stuuy. Cybium 9: 1u7-119

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