Davidson's AFA Fixative and How to Use it to Preserve Samples for Histology and/or In-situ Hybridizations

Source:  Lightner (1996, A Handbook of Pathology and Diagnostic Proceedures for Diseases of Penaeid Shrimp, World Aquaculture Society)

Davidson's Alcohol Formalin Acetic acid Fixative (Davidson's AFA)
Davidson's AFA fixative should be prepared as follows (makes 1 liter of fixative)

1. 330 mL 95% ethyl alcohol
2. 220 mL 100% formalin (saturated aqueous solution of formaldehyde gas, 37-39% solution)
3. 115 mL glacial acetic acid
4. 335 mL tap water (preferably distilled water, if available)
5. Store at room temperature, No refrigeration necessary.

Fixation Proceedures with Davidson's AFA

NOTE: Precautions should be taken to avoid skin and eye contact with the fixative. In the case of accidental exposure, flush immediately with large amounts of water, then seek medical advice.

Note: Prepare an adequate supply of fixative; a rule of thumb is that a minimum of approximately 10X their volume of fixative should be used for each specimen (e.g. a shrimp of 10 mL volume (10g) would require 100 mL of fixative).

Larvae and Early Postlarvae:

1. Immerse shrimp selected for sampling directly into fixative.

2. Fix for 12 to 24 hours in Davidson's, then transfer to 50 to 70% ethyl alcohol for storage.

Larger Postlarvae, Juveniles, and Adults:

1. Inject fixative (0.1 to 10 mL depending on size of shrimp), via needle and syringe (needle gauge dependant upon shrimp size; i.e. 27 guage needle for PLs and small juveniles) into the living shrimp.

2. The site of injection should be laterally in the hepatopancreas proper, in the region anterior to the hepatopancreas, in the anterior abdominal region, and in the posterior abdominal region.

3. The fixative should be divided between the different regions, with the cephalothoracic region, specifically the hepatopancreas, receiving a larger share than the abdominal region.

4. A good rule of thumb: "Inject an equivalent of 5-10% of the shrimp's body weight; all signs of life should cease, and visible color change should occur in injected areas.

5. Immediately following injection, slit the cuticle, with dissecting scissors, from the sixth abdominal segment to the base of the rostrum, paying particular attention not to cut deeply into the underlying tissue. The incision in the cephalothoracic region should be just lateral to the dorsal midline, while that in the abdominal region should be approximately mid-lateral.

6. Shrimp larger than ~12 grams should then be transversely bisected at least once just posterior to the abdomen/cephalothorax junction, and (optional) again mid abdominally.

7. Following injection, incisions, and bisection/trisection, immerse the specimen in the remainder of the fixative.

8. Allow the shrimp to remain in the fixative at room temperature for 24 to 48 hours depending on the size of the shrimp (larger shrimp stay in the fixative longer).

9. Following proper fixation, the specimens should be transferred to 50 to 70% ethanol, where they can be stored for up to a week.

10. Record a complete history of the specimen at the time of collection: gross observations on the condition of the shrimp, species, age, weight, source (pond, tank, raceway identifying number), source of parent stock, and any other pertinent historical information that may at a later time provide clues to the source and cause of the problem. Use soft-lead pencil on paper (plastic paper is recommended as it will not fall apart in the fixative).

Transportation or Shipment for Processing:

1. Remove the specimens from the 50 or 70% ethyl alcohol.

2. Wrap with paper towels to completely cover.

3. Place towel-wrapped specimen in a sealable plastic bag and saturate with 50% ethyl alcohol.

4. Label: Include the history, as recorded above, with the shipment. Use soft-lead pencil on paper (plastic paper is recommended).

5. Place bag within a second sealable bag.

6. Multiple small sealable bags can again be placed within a large sealable bag.

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