1. Ear punch identification

Ear punch codes have been developed as a quick and inexpensive method to label individual animals. There are three styles of ear punches: the scissor punch, the thumb punch, and the lever punch. The animal's right ear is used for single digits, the left ear for tens, and the middle of the ears for hundreds.

1. Restrain the animal so that the ears are easily accessible. Most mice are ear tagged for individual identification at weaning. This requires gentle handling due to their smaller, more fragile size. Some mice are less likely to struggle if the hind limbs can rest on a surface such as a cage top or a counter top.
2. Determine the ear punches needed to correspond to the animal's experimental number or to identify it within a cage.
   1. Punches will be notches or holes depending on the code used.
   2. The last number on the ear tag is often used as the ear punch identifier when it is the secondary method of identification.
   3. If animals are on a short-term study in stable cage groups, they can be given sequential numbers. This is often done when there are no ear tags present.
3. Make the ear punch.
   1. To make a notch, the punch is placed on the edge of the ear pinna.
   2. To make a hole, place the ear in the punch so that the hole of the punch is positioned away from the pinna margin in the desired position.
   3. Apply pressure to the thumb or lever punch, or close the scissor punch quickly and firmly to cut through the skin of the ear.
   4. Gently lift the punch away from the ear when using the thumb or lever punches. Open the scissor punch to remove it from the ear.
   5. Avoid pulling or twisting the punch to prevent tearing the ear.
   6. Punches do dull after time. Should a punch fail to cut through the ear, release the punch and select a new ear punch. Reposition the new punch in the same spot.
4. Clean the punches after each use.
   1. All punch types will dull if autoclaved. It is recommended that they be cleaned with a disinfectant and rinsed with alcohol. Dry them thoroughly to prevent rusting.
   2. Ear punches to be used for PCR are routinely soaked for 3 minutes in a solution to remove residual DNA or RNA. Before use they are rinsed in alcohol and wiped dry.

Figure 1. Ear punching in mice using scissor punch.

2. Ear tag identification

Customized ear tags can be ordered from manufacturers with alphabetical and/or numerical codes, which allows for the differentiation of mouse strains or investigators. Ear tags can be made of metal or plastic.

1. Select the ear tags appropriate for the animals to be identified. There are several styles of appliers available from ear tag manufacturers. Be sure to order the applier that is for the specific size and style of ear tags being used.
2. Gently remove a tag from the cardboard holder, and orient it into the applier so that the end with the hole is positioned over the notched area of the applier. The pointed end of the tag should be opposite the notch.
3. Restrain the animal so that the ears are easily accessible. Animals may be less likely to struggle if the hind limbs can rest on a surface, such as a cage top or a counter top.
4. Properly position the ear tag for placement.
   1. Position the point of the ear tag as deeply in the concha of the ear as possible, so the tag numbers face the back of the animal.
   2. To have the ear tag numbers facing the nose of the animal, place the point of the tag at the base of the pinna on the back of the ear.
5. Apply the tag.
   1. Firmly squeeze the applier closed. The ear tag will pierce the ear and lock together.
   2. Release the applier, and the tagged ear will fall out of the applier.

Figure 2. A mouse with an ear tag that is properly positioned on the pinna.

6. Remove the metal ear tags.
   NOTE: On occasion, metal ear tags can cause irritation and swelling of the ear. The swelling and irritation often leads to infections of the pinna. Treatment should include the removal of the tag; however, in cases of severe irritation or infection, the animal may need to be anesthetized.
   1. Remove debris from around the tag with a cotton-tipped applicator moistened with an antiseptic solution.
   2. Cut across the top of the tag loop from edge-to-edge with wire cutters. Do not cut across the flat surface, as this will compress the tag, pinch the ear, and cause further damage.
   3. Avoid applying torque while cutting to prevent tearing the ear.
7. Use hemostats to spread the cut ends of the ear tag apart.
   1. Gently guide the ear tag through-and out of-the ear using hemostats. Reduce trauma by avoiding the enlargement of the piercing hole and slipping the tag out so that the crimped end does not pass through the ear.
   2. Once the tag is gone, wipe the ear with an antiseptic-soaked cotton applicator stick.
   3. If there is a purulent discharge from the ear, follow-up treatment may be necessary and consultation with the veterinary staff for assessment is recommended.