Although the best way to learn is by practice and from being trained by an experienced dissector, here are some general guidelines:

Sample #: Each fish should have its own unique sample number written. This number is obtained from the online dissection number program. Thus a tube may have 9497-101 written on it. The first fish euthanized within a pedigree will get the number "-101" as the first 100 numbers are reserved for fish keeping records. The next fish of the pedigree will be 9497-102 etc. If a fish already has been assigned a number by the XGSC or the SPRD facilities (typically under the number "xxxx-101") you MUST also enter this assigned number in the online dissection number program. Great care must be taken to avoid the accidental use of the same number for two fish. This sounds trivial and logical, but if individuals do not follow the rules, a sample number designation may be used for two different fish, with possibly devastating consequences.

Strain: Use only strain codes that are approved by the nomenclature committee. Each dissection station should be provided with a protocol book that contains these general guidelines, in addition to pages corresponding to strain codes, phenotype designations etc.

Treatment: Record the treatment of the fish individual (UV-A, UV-B, C (for control), MNU, DBP, None).

Sex: Note that within some crosses (such as H001BC1, for example) the animals are difficult to score even when internal anatomy is examined.

Sex confirmed: Upon dissecting, are there eggs, embryos or testis to confirm sex? If so write in "Yes". One can also write "Yes, has embryos", for example.

Albinism: Write "I" if the fish is not an albino. Write "i" as a phenotype if the fish is an albino (with the characteristic pink eyes and light body).

Macro Pattern: Record the macromelanophore patterns according to the accepted strain phenotype codes. Such codes are also available for each hybrid type.

P Locus: The P locus is the classically defined "tailspot" locus. Record these micromelanophore patterns according to the accepted strain phenotype codes. Such codes are also available for each hybrid type.

Gs/PGs: These micromelanophore patterns should be recorded. Note that Gs (Gravidity spot) and PGs (Pseudogravidity spot) are the same pattern, with the former reserved for females and the latter for males.

Xanth/Pter: Record the appropriate xanthophore/pterinophore patterns. Refer to the phenotype code section of the dissection manual.

Rank: "Rank" includes heavy, light, intermediate etc. but this varies depending on the cross type. As an example, a heavily pigmented H004BC1 may have less pigmentary coverage on the body than a lightly pigmented H007BC1. For certain crosses, a numeric designation for rank can be employed. Refer to the phenotype code section of the dissection manual.

Other Pheno: Other phenotypic information can be recorded here. Refer to the phenotype code section of the dissection manual.

Std Length: Measurement from the mouth to the base of the tail (hypural plate; in mm). This is taken with calipers.

DOB: Date of the fish's birth.

DOS: Date of sacrifice.

Tissue Codes: Fill in the boxed region and indicate if the tissue is intended for DNA, RNA or protein.

B	Brain
BE	Brain and Eye
DF	Dorsal Fin
EY	Eye
G	Gill
GO	Gonopodium
HD	Head
HR	Heart
K	Kidney
L	Liver
ML	Melanoma
MU	Muscle
P	Pancreas
S	Skin (normal)
SML	Melanotic Skin
SP	Spleen
SPKG	Spleen, Kidney, Gill together
TE	Testis
TF	Tail Fin
WF	Whole Fish

Other notes: Information such as the degree of pigmentation in a particular tissue should be recorded here.

Box: Record the box number where the samples will be stored.

Photo: Was the animal photographed? If it is a digital image write "DP" (for digital photograph) or "Film" for conventional photography.

Other notes: Any other information that the dissector feels is pertinent can be included here.

Optional: To make things easier to be traced later in time, we highlight certain entries with markers:

Yellow = Tumor
Orange = MNU
Pink =UVB
Blue = UVA
Green = Histology

This can, for example, make it easier to find all animals that were MNU treated etc.

An ice bucket and dry ice/ethanol bath should be prepared, and all dissection instruments should be handy and clean. The tubes should be adequately labeled. Each tube should have the individual fish sample number written (such as 9497-101). For the SPKG and TF tubes, scribe the outside with the sample number as well. In addition, the tubes should have the appropriate additional abbreviations:

BE (Brain and Eye)
L (Liver)
MU (MUscle)
SPKG (SPleen, Kidney, Gill)
TF (TailFin) (with 1.2 ml 95% ethanol, on ice)

and variably (depending if there is a need)

TE (Testis) (with no buffer in the dry-ice/ethanol bath)

In general we prefer use of 1.5 ml tubes because our teflon pestles for motorized grinding fit tightly in them. Screwcap (Nunc-type) sample tubes are used for the SPKG samples. Note that fish should be euthanized and tissues collected as rapidly as possible.

1. Place a fish in a 0.06% 3-Aminobenzoic Acid Ethyl Ester (methanesulfonate salt; C₉H₁₁NO₂* CH₄SO₄; also known as MS-222, Sigma A-5040) solution to euthanize it (~1 minute or until the fish is immobile). This solution is derived from a 0.4 % stock which is stored in a dark bottle.

2. Place the fish on a white plastic surface and photograph using a digital camera such as the Agfa 1680 (using macro mode). The photography information is recorded in the photo log so that this information can accompany the archived images.

3. Place the fish on a clean glass plate and measure the standard length (preferably with calipers).

4. With a #11 scalpel, remove the top of the cranium starting at the junction with the "neck" and working forward to expose the brain and eyes under the superorbital ridges. Break the spinal cord with a fine forceps after exposing it posteriously as far as desired (by breaking away the dorsal spinal column with the forceps) and tease the cord anteriorly to free it from the spine. Next, from the nasal area reach under the brain with the forceps to grasp the optic chiasma and lift upwards. With a little practice, both eyes and the brain and spinal cord can be lifted in one piece. Insert tissues into a 1.5 ml sample "BE" tube and tap so that the sample becomes immersed in the homogenizing solution. Place tube deep in wet ice. The brain and eye samples serve for various extractions of isozymes.

5. Use a scraper (a #21 scalpel blade works well for Xiphophorus) to scale the fish from the caudal peduncle to the operculum. Usually, it is easy to scale the fish rapidly while pressing the caudal fin down onto a glass plate. Clean the scalpel blade on a paper towel. NOTE: do not do this if you are dissecting a fish with an external tumor.

6. Cut the tail along with 2-3 mm of anterior musculature and place in the "TF" sample tube (on normal ice). Tap to insure that the sample is immersed in ethanol. This tissue serves for DNA extraction.

7. With iris scissors or equivalent, make a small incision just below the operculum, extend the incision posteriously to just in front of the anus, cut dorsally at both ends of the incision to above the lateral line, and cut off the resulting skin flap to make a window exposing the viscera.

8. With fine forceps, carefully lift the gall bladder from the liver and discard. Grasp the liver at an attachment to the intestine (after breaking other attachments) and lift the liver out. Though the liver is a very soft tissue, the entire liver can be lifted in one step after practice; place liver in 1.5 ml "L" tube in wet ice. The liver samples are used to extract isozymes.

9. With coarse and fine forceps as needed, remove spleen (bright red), kidney (pale colored), and gills and place in the "SPKG" sample tube which should be placed in a dry ice ethanol bath. This tissue also serves for DNA extraction.

10. Cut muscle away from remaining tissues and place in 1.5 ml sample "MU" tube. For a large fish the caudal peduncle area (carcass posterior to the cloaca of a small fish) is large enough. For a small fish, additional muscle must be dissected from the dorsal area underneath the dorsal fin. Place tube deep in wet ice.

11. Pipette 100 ul of tissue homogenizing solution (40 °C) into the BE and L tubes and spin on the microcentrifuge momentarily ("zip-spin") so that the tissue is immersed. This amount of volume is normally good enough for animal 25-40 mm Standard length. Adjust the volume of homogenizing solution added when smaller or larger animals are dissected (60 ml or less for a fish 20 mm SL).

12. Place the BE, L, MU, SPKG samples within a box at -800 °C. The TF sample should be stored at 40 °C. It is highly recommended that samples be placed in boxes which house only 1 cross type (such as H007BC1).

13. Note: At times, abnormalities such as internal tumors can occur. In such cases, adjust the notes "on the fly" and collect the material in histology cassettes (HistoPrep by Securline). These cassettes need to be labeled using specialty markers with the sample number, and initials of the P.I. The cassettes can be stored in a container with buffered formalin.

14. Be sure the dissection instruments and surfaces are clean before euthanizing another individual.

SOLUTIONS:

10 mM Tris-HCL, 1 mM EDTA, and 1mM 2-mercaptoethanol (pH=7.0)

1 L of buffered formalin = 900ml water/100ml formaldehyde/4gm Sodium Phosphate, monobasic-monohydrate/6.5gm Sodium Phosphate, dibasic-anhydrous

Contributed by Steven Kazianis.

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