Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine

Abstract

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling researchers to model human disease. Recording and tracking individual animals allows researchers to reduce the number of animals required for study and refine practices to improve animal wellbeing. Several well-documented methods exist for marking and tracking mammals, including ear punching and ear tags. However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly. In this manuscript, we outline methods and best practices for marking Xenopus laevis with a rotary tattoo machine. Proper tattooing results in high-quality tattoos, making individuals easily distinguishable for researchers and posing minimal risk to animals' health. We also highlight the causes of poor-quality tattoos, which can result in tattoos that fade quickly and cause unnecessary harm to animals. This approach allows researchers and veterinarians to mark amphibians, enabling them to track biological replicates and transgenic lines and to keep accurate records of animal health.

Figure 1:. Protocol workflow and bench setup.

(A) Workflow and timing. (1) Frogs spend 7-8 min being anesthetized in a solution of 1.5 g/L tricaine and 3.5 g/L sodium bicarbonate, pH 7.15, dissolved in frog-safe water. While one frog is being tattooed, a second frog can be placed on deck in the anesthesia tank. Only one frog can be in tricaine solution at a time. (2) Frogs spend about 10 min out of water being tattooed. Time will vary depending on the size of the tattoo and the lab personnel’s experience. (3) Unconscious frogs are placed on Styrofoam for 30-45 min to prevent drowning and covered with a wet paper towel to prevent them from drying out. (4) After tattooing, frogs spend 24 h recovering in fresh frog-safe standing water. Once they have recovered, frogs can be returned to their colony. (B) Bench setup for an example workspace. The tattooing workspace, an unbleached paper towel, is centered on the bench. The tattoo machine and voltage supply are to the left of the paper towel. These are placed on the personnel’s dominant side (the personnel is left-handed). An inkpot and tattoo ink are placed near the voltage supply and out of the way of the tattooing workspace. The foot pedal is on the same side as the voltage supply. Pressing this foot pedal controls rotary motion. Lint-free wipes and a squeeze bottle with frog-safe water are to the right of the paper towel. These are used to clean excess ink from the frog’s skin during tattooing. An Allen wrench is used to tighten and loosen screws in the tattoo machine. This is used to assemble and disassemble the tattoo machine. The anesthesia tank and extra paper towels are also kept on the bench during tattooing.

Figure 2:. Tattoo machine assembly.

(A) Image of the tattooing needle, grip, and related components disassembled. The screws on the metal grip are circled in red. (B) Image of the tattooing needle appropriately assembled into the grip. (C) Image of the tattooing needle and grip assembled into the tattooing gun.

Figure 3:. Positioning and reference images for frog tattooing.

(A) Image of a frog laying face-up on a paper towel. The arrow points to the frog’s sternum. (B) Image of lab personnel inking frog. (C) Images of numbers during inking, before (C), and after (C’) excess ink has been washed away. (D) Image of frog’s tattoo immediately after inking, before recovery.

Figure 4:. Post-tattoo recovery.

(A) Styrofoam island. The Styrofoam ice box lid is placed inside of a zip-top bag. (B) Frogs in recovery position post-tattooing. Styrofoam islands can typically fit 1 or 2 frogs.

Figure 5:. Examples of representative results.

(A) A fresh tattoo 24 h after being tattooed. (B) Poor quality techniques. In (B), the tattoo machine was not properly assembled but was still capable of rotary motion. This photo was taken 4 months after tattooing. In (B’), the tattoo machine was not assembled properly and was not capable of rotary motion. This photo was taken 6 months after tattooing. (C) Improperly assembled tattoo machine. The metal tube and plastic tip are not present, and the metal grip is not correctly secured to the rest of the tattoo machine. Rubber band impairs rotary motion (D) Before (D) and after (D’) of a frog tattooed with good technique and proper equipment setup. (D) was taken immediately after tattooing, and (D’) was taken 4 months after tattooing. (E) Quantitative data of frog tattooing results. Of the frogs tattooed, 100% had visible tattoos immediately after tattooing, 100% had them 24 h after tattooing, and 91% had them 4 months after tattooing. n = 58 frogs.