Clear Resin Casting of Arthropods for Use in Education, Outreach, and Research

Summary

Here we show key steps in the process of creating high-quality, resin-embedded arthropods for educational instruction and outreach.

Abstract

Our prior work published a protocol for creating resin-embedded arthropods of medical importance for use in educational and outreach activities. This protocol has been adopted by diverse initiatives aiming to create a mounting technique that minimizes damage to the arthropod, creates a safe product for handling, and preserves the ability to see morphological characteristics for identification. Our protocol has evolved and is currently creating resin-embedded arthropods with improved quality thanks to the purchase of additional equipment and process modifications. We present this improved protocol with steps to minimize bubbles, adapt to different types of arthropods, and create final polished blocks with visibility from six sides. We also outline biosafety concerns from fumes during the resin curing, use of power tools, and from dust generated while sanding and polishing resin blocks. This visualized protocol will facilitate the adoption of clear resin casting of arthropods to a broader community. The list of equipment and consumables we have adopted over several years of trial and error will allow other programs to judge if this protocol is a viable option for adoption.

Introduction

Undamaged, high-quality specimens are critical resources for teaching arthropod identification skills to trainees^1,2. Often, teaching collections contain delicate pinned or pointed specimens that may be missing key features from frequent handling or may be difficult to view from several angles to understand the spatial relationships of key identifying features³. These can be frustrating for students and teachers alike, and rare, difficult-to-collect, and non-local species may be restricted or altogether absent from teaching collections for fear of destruction of the samples.

In addition to formal institutionalized learning, educating the public is part of the mission for many academic and extension programs, medical and veterinary practices, parks and nature centers, museums, and mosquito/vector control districts^4,5. In these situations, durable preserved specimens are critical, and it is optimal to show specimens that can survive hands-on investigation by children, accidental damage, or dropping. Pinned or pointed insect specimens do not offer the durability needed for these educational contexts, and specimens in a liquid preservative such as ethanol are difficult to view through containers and difficult to remove and handle. Given that 3D models of insects have been shown to enhance student education⁶, providing students with high-quality arthropods preserved in resin should also provide benefits. A recent study by Bron et al. used ticks and other arthropods embedded in resin to assess the public's ability to recognize tick vector species⁷. Another project used resin-embedded exotic forest insects ('travel bugs') placed in geocache locations to raise awareness of nonnative forest pests⁸. This work builds on prior entomologists who have been advancing protocols to embed insects in resin for decades^9,10. While working with various arthropods in the development of our protocol, we found that delicate insects with scales (e.g., mosquitoes) are more challenging to embed in resin, as scales and limbs are often lost during the resin-pouring stages. Moreover, specimens stored in ethanol over extended periods of time are less ideal to use in resin embedding as ethanol fades the coloration important for identification (e.g., ornate patterns on scuta of ticks).

To expand our teaching collections of arthropods for both formal instruction in classrooms (e.g., medical and veterinary entomology), and outreach and extension programs educating the public, we developed a protocol for clear casting of arthropods¹¹. We published these methods since most commercial companies that produce resin-embedded insects keep their protocols confidential. Since our 2018 publication, we have purchased additional equipment and further optimized the protocol, which is now presented here. In the Table of Materials we present all equipment and consumables used in this protocol.

Protocol

CAUTION: The hazards associated with the resin production protocol include exposure to toxic fumes from uncured resin, contact with uncured resin and catalyst, use of power tools, and dust production during the cutting/sanding/polishing steps. A fume hood or well-ventilated area and nitrile gloves are needed when working with uncured resin. If resin or catalyst comes into contact with skin, wash area immediately with soap and water. When cutting/sanding/polishing cured resin blocks, a properly fitted half-face respirator needs to be worn.

1. Specimen and label preparation

NOTE: The preparation of specimens depends on the arthropod being used. Specimens and labels should be prepared and set aside for use prior to preparing resin layer 1.

1. Choose a specimen to be placed in resin.
   1. Ticks: Remove ticks that have been stored in either the freezer or 70% ethanol. Place adult and nymphal ticks removed from 70% ethanol between two microscope slides while applying a light amount of pressure on either side of the tick to remove excess ethanol that may be inside of the tick. Then, place the tick on a flat surface and allow it to dry for at least 5 min prior to being added to resin.
      ​NOTE: Ticks stored in the freezer do not need to be pressed between microscope slides but should be allowed to sit at room temperature as mentioned above.
   2. Mosquitoes: Freshly emerged adult mosquitoes are best for embedding in resin, as they are most likely to have all diagnostic features (scales, etc.). To obtain freshly emerged adult mosquitoes when a colony or natural populations from the field exist, place larvae or pupae in water in an emergence chamber, and after eclosion, the adults will enter the top compartment of the emergence cup. Place newly emerged adult mosquitoes in the freezer to kill, then remove and allow them to sit at room temperature for at least 5 min prior to being added to resin.
      NOTE: The time to kill mosquitoes in the freezer is dependent on the temperature of the freezer (e.g., mosquitoes are killed in ~10 min after being placed in a -80 °C freezer). Adult mosquitoes should be used within hours of killing, as mosquitoes become desiccated when stored in the freezer for prolonged periods.
   3. Mosquito and other Diptera larvae/maggots: Remove larvae from ethanol and soak in resin catalyst for at least 20 min prior to being added to resin.
      NOTE: Placing these specimens in the catalyst replaces ethanol within their body without drying the specimen out and causing it to shrivel.
   4. Other arthropods: For many arthropods, follow the guidance outlined above for other ticks or mosquitoes, depending on how delicate the external features are.
2. Create labels with desired information (e.g., scientific names and life cycle stages). Print labels on transparency sheets with a laser (not inkjet) printer. Cut labels to size.
   1. Use a size 8 font Times New Roman italicized. Use a size 12 font Times New Roman italicized for life cycles or larger-bodied specimens.
   2. For teaching collections containing lab practical exams, make sure that arthropods in resin are labeled in a way that would tell the instructors what species they are, but not the students being tested on their identification skills. In this context, use small QR codes, about the size of 1 cm², that can allow a smartphone to link to a website containing information about the species.
      NOTE: Care should be taken to cut labels squarely and evenly to improve quality.

2. Resin layer 1

CAUTION: All steps involving uncured resin are done inside a fume hood vented outdoors, given the production of harmful fumes. Conducting these steps in a well-ventilated setting, such as outdoors, is another option.

1. Don nitrile gloves prior to working with uncured resin or catalyst.
   NOTE: If excessive amounts of resin get on gloves at any point while preparing the resin layers, remove gloves, being careful to avoid resin-to-skin contact. If gloves contain uncured resin, leave them in the fume hood for 24 h to fully cure before disposing of them in the garbage. If resin does come in contact with skin, immediately wash the affected area with soap and water, per label instructions.
2. Measure approximately 60 mL (~2 oz) of resin in a plastic cup. Pour resin from the plastic cup into a plastic bowl. Place the plastic bowl containing resin in a 32 °C water bath.
   NOTE: The resin should not be warmed in a water bath for more than 15 min.
3. Add 12 drops of catalyst (~540 μL) to the plastic bowl containing resin. Mix resin and catalyst with a plastic spoon slowly and carefully for ~1 min.
   NOTE: Resin to catalyst ratio may change based on resin brand. If the ratio differs from above, follow the manufacture's recommendations.
4. Pour resin/catalyst mixture from the plastic bowl into a silicone mold placed on a wooden block in the pressure pot (refer to step 5.1).
   NOTE: The viscosity of the mixture should be similar to corn syrup (or warmed honey).
5. Wait approximately 5-20 min for the resin to begin to harden and be sufficiently tacky before adding the specimen.
   NOTE: Resin cure times are dependent on many factors such as temperature, humidity, and elevation.

3. Specimens in resin

1. Place the specimen in the center of the first resin layer to ensure diagnostic features (e.g., spiracles on fly larvae) are positioned such that they can be easily viewed.
2. Place the label on top of the first resin layer in the desired proximity to the specimen.

4. Resin layer 2

1. Measure out approximately 60 mL (~2 oz)of resin in a plastic cup. Pour resin from the plastic cup into a plastic bowl. Place the plastic bowl containing resin in a 32 °C water bath.
   NOTE: The resin should not be warmed in a water bath for more than 15 min.
2. Add 12 drops of catalyst (~540 μL) to the plastic bowl containing resin. Mix resin and catalyst with a plastic spoon slowly and carefully for ~1 min.
   NOTE: Vigorous stirring will create excessive bubbles that might remain in the final product and reduce the clarity of the specimen (Figure 1). The mixture may also appear slightly green once mixing is completed.
3. Slowly pour the second resin/catalyst mixture from the plastic bowl directly over the specimens and along the edges of the inside of the mold, filling the remaining space in the mold (Figure 2C).
   NOTE: The viscosity of the mixture should be similar to corn syrup (or warmed honey).
4. Make final adjustments to the specimen, such as repositioning appendages (legs, wings, antenna) as needed (Figure 2D).
5. Remove and dispose gloves per disposal protocol.

5. Pressure pot

1. Place a block of wood into the pot to raise the surface on which the molds are to be placed and managed.
   NOTE: Molds may be placed in the pot on the block of wood prior to pouring the first layer to avoid accidental spillage or movement of the specimen that could result if molds were transferred after resin is poured before the layers are set.
2. Securely close the pot and apply pressure to 60 psi (Figure 3). Leave molds in the pressure pot for approximately 24 h.
   NOTE: Molds can be left in the pressure pot for up to 72 h without any apparent differences in the final product.
3. Release the pressure and remove the molds from the pressure pot.
4. Remove resin blocks from the molds and let them sit at room temperature until the remaining steps are ready to be performed.
   NOTE: The surfaces of the resin may feel a bit tacky to the touch (e.g., touching the resin will leave a fingerprint mark), and the sides that touched the mold may have some surface whitening. These surface imperfections will be removed during the following procedures.

6. Final product preparation

CAUTION: The cutting, sanding, and polishing steps are all done in a separate building with a shop vacuum, given the generation of dust and the need for respirator protection. A respirator should be put on and adjusted accordingly to fit securely on the face of the person cutting, sanding, or polishing the cured resin blocks prior to performing any of the following steps. At Texas A&M University, our Environmental Health & Safety program requires each employee to pass a respirator fit-test using a half-face respirator with a National Institute for Occupational Safety and Health P100 particulate filter.

1. Cut resin blocks to size using a bandsaw.
   NOTE: Care is taken to create even/balanced blocks with the specimens centered (Figure 4A).
2. Sand all six sides of the resin block using the disc sander (Figure 4B). Repeat steps for all sides using the 4 x 30 in 600 grit belt sander (Figure 5A), followed by sanding all sides with the 1 x 30 in 2,000 grit belt sander (Figure 5B).
   NOTE: To achieve the most clarity in the final product, hold each side of the resin block in the same orientation for each belt type.
3. Gently hold a red, aluminum oxide, buffing wheel polish against the buffing wheel for a few seconds, followed by gently holding the resin block against the buffing wheel at a 45° angle (Figure 5C).
4. Use cloth/fabric to remove excess polish from all sides of the resin block. Repeat as many times as needed until the desired clarity is achieved.
5. Repeat the same steps using a second buffing wheel with a white, diamond polish. Repeat as many times as needed until the desired clarity is achieved and the colored overcast from the red polish is removed.

Results

Successfully embedded specimens will be surrounded by clear and colorless resin, with all sides smooth. The specimen will be clearly visible from any side of the resin block. Specimen features important for identification, such as color patterns, scale shape and size, and other anatomical features, will be visible with the use of a dissecting microscope. Less desirable features of the resulting arthropods in resin sometimes result when a layer of air bubbles covers the specimen, scales ar...

Discussion

Preparation of specimens

Preservation methods can depend on the specimen being worked with. Common specimen preservation methods include 70% ethanol or freezing, both of which are compatible with resin embedding. These methods are useful in preserving key features of the specimen, such as coloration, scales, hairs, and other physical characteristics.

Preparation of resin layers and placement of the specimen

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Air compressor	California Air Tools CAT-1P1060S Light & Quiet Portable Air Compressor, Silver	California Air Tools	https://a.co/d/07y4iGha
Air compressor hose	California Air Tools HFH25.25 Hybrider Flex 1/4in 25ft Hybrid Air Hose with Quick Connect Air Fittings , Blue	California Air Tools	https://a.co/d/0cboxu7J
Bandsaw	G0803 9” Benchtop Bandsaw		https://www.grizzly.com/products/grizzly-9-benchtop-bandsaw-w-laser-guide-quick-release/g0803z (Analog Machine)
Belt Sander	Bucktool 1 X 30 in Bench Belt Sander with 5 in Disc with Wrench Storage and Easy Belt Cover Off	Bucktool	https://a.co/d/017GmVQI
Buffing wheel	6 inch Benchtop Buffer Heavy Duty 1/2 HP; Includes two buffing wheels	Central Machinery	https://a.co/d/0iGsbSCu
Disc sander	BUCKTOOL 5.0A Belt Disc Sander 4 in x 36 in Belt and 6 in Disc Sander with 3/4HP Direct-drive Motor and Portable Al Base	Bucktool	https://a.co/d/08AsKXva
Polishing wheel pad x2	Replacements Pads for Buffing Wheel	Enkay	https://a.co/d/07WajxZ5
Pressure pot	CALIFORNIA AIR TOOLS 255C 2.5 Gallon Pressure Pot for Casting, Blue	California Air Tools	https://a.co/d/0j3vGi3P
Shop vacuum to collect dust	BUCKTOOL 1.2 HP Auto Start 750CFM Dust Collector with 3 Dust Collection Bag, 5.4 Cubic Bag Capacity and 5 PCS Reducer for Woodworking DC50	Bucktool	https://a.co/d/0cEA39ii
Water bath	JOANLAB digital thermostatic water bath	JoanLab	https://a.co/d/0gJLNyWG
Consumables
Buffing wheel polish, red	SCOTTCHEN Red Buffing Polishing Cutting Compound Coarse, Jewelry Rouge	Scottchen	https://a.co/d/caLtcNL
Buffing wheel polish, white	D2903 1-Pound Buffing Compound	Woodstock	https://a.co/d/061T4kWU
Half face respirator	3M Rugged Comfort Quick Latch Half Facepiece Reusable Respirator 6502QL, NIOSH	3M	https://a.co/d/0g3wMldi
Half face respirator filters	3M Particulate Filter P100 Respiratory Protection, 2097/071184 (AAD)	3M	https://a.co/d/05a6ydbM
Resin	Clear-Lite Casting Resin, Tap Plastic	Tap Plastic	https://www.tapplastics.com/product/mold_making_materials/casting_products/tap_clear_lite_casting_resin/75
Resin catalyst	MEKP Liquid Catalyst, Tap Plastic	Tap Plastic	https://www.tapplastics.com/product/fiberglass/polyester_resins/tap_mekp_liquid_catalyst/40
Resin & Resin catalyst (Alternative)	Clear Casting UV Resin, Solarez (MEKP Catalyst included)	Solarez	https://solarez.com/solarez-clear-casting-uv-resin.html
Sanding belts	Red Label Abrasives 4 X 36 in 600 Grit Silicon Carbide	Red Label Abrasives	https://a.co/d/0hkYmjI7
Sanding discs	1 x 30 2000 Grit 9 Micron Blue Polishing Belt with Cushioned Ultra Flexible Mylar Backing	Pro Sharpening Supply	https://a.co/d/01pCnNnN
Sanding discs	Norton Metalite R228 PSA Disc, Pressure Sensitive Adhesive, Aluminum Oxide, 5’’ Diameter, Grit 80	Norton	https://a.co/d/0cqqdpMq
Silicone molds	Square Silicone Molds for Resin, Large Resin Mold Glossy Deep Square Molds 6.6''x 2'' w (with 2 Small Square or Rectangular molds)		https://a.co/d/06Qasc1G
Transparency sheets	Ohp Clear Printable Transparency Film 8.5 x 11 Inches for Overhead Projectors, for Laser Printers	Craftiff	https://a.co/d/07L2g0AT