Aquatic Animal Health - Sample Preparation
Sample Preparation - Shrimp
PCR or Dot blots
Non-lethal sampling (Broodstock):
• 0.2 ml of frozen hemolymph collected in a 1% sodium citrate coated syringe
• Frozen pleopods
• Pleopods placed in 95% ethanol
Lethal sampling (Larvae):
• Frozen larvae
• Larvae fixed in 95% ethanol
Histology or In situ gene probes
• Fix tissue in Davidsons fixative or RF fixative (use RF fixative if looking for the presence of an RNA virus, such as Taura).
• For postlarvae, immerse live shrimp into fixative for 24 hours
• For larger shrimp (> 1 g) inject fixative into live animals. Inject the hepatopancreas first, then the region anterior to the hepatopancreas, and lastly make two injections into the abdomen, one anterior and the other posterior.
• Inject fixative at 10% shrimps body weight (e.g. 1 ml for a 10 g shrimp)
• Using scissors, slit the cuticle open on both sides of the shrimp, from the 6th abdominal segment to the rostrum
• Immerse shrimp in 10x volume of fixative for each gram weight of shrimp (e.g. 100 ml of fixative for a 10 g shrimp)
• After 48 - 72 hours, remove shrimp from fixative and place in 50% or 70% ethanol for a minimum of a few hours before processing for shipment. Samples can be left in this solution indefineately.
• To ship specimens, remove them from the alcohol solution and wrap in paper towels which have been soaked in the solution. Place wrapped specimens in a sealable plastic bag. Seal, and then place in a second sealable bag to ensure samples will not dry out.
Sample Preparation - Fish
Histology
• Fix tissue in 10% neutral buffered formalin.
• For small fry, immerse in fixative for 24 hours
• For larger fish (fingerlings, small ornamentals) inject at 10% the body weight of the fish (e.g. 1 ml for a 10 g fish)
• For very large fish remove organs and tissue with suspected lesions and place in fixative
• After 48 - 72 hours, remove fish/organs and place in 50% or 70% ethanol for a minimum of a few hours before processing for shipment.
• To ship specimens, remove them from the alcohol solution and wrap in paper towels which have been soaked in the solution. Place wrapped specimens in a sealable plastic bag. Seal the bag and then place inside a second sealable bag to ensure that samples will not dry out.
Sample Preparation - Bivalves
Histology
• It is best to ship samples live and on ice for fixation and processing in the lab.
• To fix samples before shipment, remove the clam or oyster from its shell before placement in fixative. Seed can be placed directly in the fixative.
• Fix tissue or seed in Davidson's fixative for 24-48 hours.
• After samples have been fixed place in a 50% or 70% ethanol solution.
Fixatives Used
Davidsons AFA Fixative - 1 liter of solution
100% formalin (37% formaldehyde) - 220 ml
95% ethanol - 330 ml
Distilled water - 335 ml
Glacial acetic acid - 115 ml
RNA-friendly (R-F) fixative - 1 liter of solution
100% formalin - 349 ml
95% ethanol - 407 ml
Distilled water - 222 ml
Ammonium hydroxide - 22 ml
10% Neutral Buffered formalin - 1 liter of solution
100% formalin - 100 ml
Distilled water - 900 ml
Sodium phosphate, monobasic monohydrate - 4 g
Sodium phosphate, dibasic anhydrous - 6.5 g
Collecting Hemolymph
Shrimp
• Aseptically puncture a hemolymph sinus (pericardial or ventral) with a tuberculin syringe. If hemolymph is to be used for PCR or dot blot analysis, coat syringe with sodium citrate. Place collected hemolymph in a 1.5 ml microfuge tube. If hemolymph is to be used for bacterial sampling do not coat syringe. Place blood in a sterile tube or ship syringe intact
Fish
• Aseptically puncture the mid-tail region vessels just under the scales, below the lateral line with a syringe. If blood is to be used for PCR, coat syringe with 50 - 100 USP units/ml of heparin. If hemolymph is to be used for bacterial sampling do not coat syringe.
Pleopod collection
• Do not cut the pleopod where it is attached to the body. Cut it from the middle of the baspodite. Use scissors dipped in 70% ethanol or in an iodine solution. Place pleopod in 1.5 ml microfuge tube. Freeze and ship frozen or place in 95% alcohol
Bacterial sampling
• Ideally, samples should be shipped live. If not, ship samples at 4C (on ice) for processing. Alternatively samples can be frozen at -20C.
• Sterile swabs of organs/lesions can also be collected and shipped.
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