Post-extraction cleaning of entomological specimens (Microhymenoptera)

Generously shared with the CCG by Bruna Cortat Simoneli, CAS Lakeside Fellow, Laboratório de Biodiversidade de Insetos (Insect Biodiversity Laboratory), Universidade Federal do Espírito Santo (UFES), Brazil. Some modifications have been made to reflect CCG equipment and materials.

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Background information

Following DNA extraction, crystals may form on the specimen from the buffers used during cell and tissue lysis. To avoid this, the specimen should be cleaned after extraction.

Crystal formation on a chalcid wasp following DNA extraction without proper cleaning. Image credit: Bruna Cortat Simoneli. Crystal formation on a chalcid wasp following DNA extraction without proper cleaning. Image credit: Bruna Cortat Simoneli.

Ethanol is used to dehydrate the specimen, preventing decay and fungal growth. However, the exoskeletons of very small and delicate samples may buckle under osmotic pressure if transferred directly to 100% ethanol after rinsing in water. To prevent this, the specimen is transferred to gradually increasing ethanol concentrations until 100% is reached. Finally, ethyl acetate is used to further dehydrate the specimen and remove any oily remaining oily residues.

This procedure is optimized for small, delicate specimens (microhymenoptera). For more robust specimens (e.g., beetles), fewer wash steps may be acceptable. See notes below, and consult with your PI or collection manager to determine appropriate modifications.

Materials/Reagents

  • Molecular-grade ethanol
  • Molecular-grade water
  • Ethyl acetate (if the specimen is to be mounted)
  • Squeeze or micropipettes (ethyl acetate compatible, if the specimen is to be mounted)
  • Eppendorf tubes (ethyl acetate compatible, if the specimen is to be mounted)

Ethyl acetate is highly volatile and can cause drowsiness, dizziness, and narcosis. It should only be handled in a fume hood. Final specimen drying should also be accomplished in a hood if possible. If your specimens are so small/delicate that a fume hood’s airflow could cause damage or total loss, ensure that the final drying step takes place in a well-ventilated area.

Ethyl acetate (EA) will cloud or dissolve some plastics, including polystyrene and polycarbonate. Do not use plastic Petri dishes (typically polystyrene), and do not place EA-soaked specimens on unknown foam or plastic surfaces. LDPE, HDPE, polypropylene, and glass are all compatible with EA and can be safely used for short periods at room temperature.

Preparation

  • Prepare aliquots of ethanol (diluted with water) at the following concentrations: 70–75%, 80%, 90%, 100%.

Protocol

Graduated ethanol wash

  1. After removing the insect from the extraction solution, transfer to a new tube and fill with water. Pipette off the water and discard.
  2. Repeat step 1 twice for a total of three washes. The specimen can also be left soaking in water for up to 30 minutes.
  3. Add sufficient 70%–75% ethanol to completely immerse the specimen. Pipette off the ethanol and discard. This helps remove residual water, which would reduce ethanol concentration in following washes.
  4. Add sufficient 70%–75% ethanol to fully immerse the specimen. Allow the specimen to soak for at least 30 minutes to maximize water displacement.
  5. Repeat steps 3 and 4 using 80% ethanol.
  6. Repeat steps 3 and 4 using 90% ethanol.
  7. Repeat steps 3 and 4 using 100% ethanol.
  8. At this stage, the specimen is ready for long-term preservation in an appropriate storage tube and 100% ethanol. If mounting the specimen, proceed to mounting prep.

Mounting prep: Final dehydration and oil removal

  1. Prepare a container, tray, etc, for drying specimens after soaking with ethyl acetate.

Ethyl acetate will cloud or dissolve some plastics. See details above.

  1. Remove the ~100% ethanol from the tube and discard.
  2. Add sufficient ethyl acetate to completely immerse the specimen. Pipette off the ethyl acetate and discard.
  3. Add sufficient ethyl acetate to completely immerse the specimen. Allow the specimen to soak for approximately 60 seconds.
  4. Remove the specimen and place it on a paper towel to absorb excess ethyl acetate and allow residual solvent to evaporate. Discard liquid acetate and tube.
  5. After these steps, the specimen is ready for mounting.
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Abbreviated protocol for robust specimens (e.g., Coleoptera)

Specimens with heavily sclerotized bodies may not require as many steps as more delicate species (e.g., microhymenopterans). In these cases, the use of ethyl acetate may not be necessary. Some coleopterists even mount their specimens immediately after the 75% ethanol wash (skipping steps 5-6 below), then allow them to dry in a drying chamber or inside a Schmitt box.

Protocol

Materials/Reagents

  • Molecular-grade ethanol
  • Molecular-grade water
  • Squeeze or micropipettes
  • Eppendorf tubes

Preparation

  • Prepare aliquots of ethanol (diluted with water) at the following concentrations: 70–75%, 100%.

Protocol

  1. After removing the insect from the extraction solution, transfer to a new tube and fill with water. Pipette off the water and discard.
  2. Repeat step 1 twice for a total of three washes. The specimen can also be left soaking in water for up to 30 minutes.
  3. Add sufficient 70%–75% ethanol to completely immerse the specimen. Pipette off the ethanol and discard. This helps remove residual water, which would reduce ethanol concentration in following washes.
  4. Add sufficient 70%–75% ethanol to fully immerse the specimen. Allow the specimen to soak for at least 30 minutes to maximize water displacement.
  5. Repeat steps 3 and 4 using 100% ethanol.
  6. At this stage, the specimen is ready for long-term preservation in an appropriate storage tube and 100% ethanol.
  7. As soon as the specimen is removed from the ethanol, mount it.
  8. The mounted specimen can be dried in a drying chamber or Schmitt box for approximately two days.

GHS Irritant GHS Flammable All ethyl acetate waste must be disposed of in its designated waste bottle. This must read: “Ethyl acetate – Danger! Flammable.”