Rodent injection techniques
Guidelines: The procedures described in this SOP should be followed when liquids will be
administered to mice, rats, or hamsters by intraperitoneal, subcutaneous, intramuscular,
intravenous or intradermal injection. All substances must be sterile and administered using sterile
instruments to minimize the risk of infection. Maximum injection volumes and needle sizes must
be followed to minimize trauma to tissues and discomfort for the animals.
Maximum injection volumes
Route
Intraperitoneal
(IP)
Subcutaneous
(SC)
Intramuscular
(IM)
Intravenous (IV)
Intradermal (ID)
Mice
Volume
Needle
(mL)
size
2.0
25G
Rats
Volume
Needle
(mL)
size
10ml/kg
23G
Hamsters
Volume
Needle
(mL)
size
3-4
23G
2.0
25G
5ml/kg
23G
3-4
23G
0.05
25G
0.2
23G
0.1
23G
0.2
25G
5
23G
0.05
27G
0.05
27G
Very difficult, requires
anesthesia
0.05
27G
Materials:
Sterile needles- 23G, 25G, 27G
Sterile syringes- ½ CC, 1CC, 3CC, 5CC, 10CC
Clippers
70% ethanol
Gauze
Rodent restrainer
Heat lamp
Rodent EZ anesthesia machine (located in the B16 suite of ARF)
Methods:
Procedures in the mouse can generally be performed by one person. For awake procedures in rats
and hamsters it may be easier to have two people, one to restrain the animal and one to
administer the injection.
Intraperitoneal injection:
1. Prepare the syringe and needle with the inoculum, removing the cap from the needle and
expelling any air bubbles.
2. Manually restrain the animal by grasping the lose skin at the scruff of the neck with the
thumb and forefinger to immobilize the head and torso. Pick the animal up and turn it so the
abdomen is facing up, supporting the back and hind end.
3. Wipe the lower left quadrant of the abdomen with 70% alcohol and allow to dry.
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Rodent injection techniques
4. Tip the animal so the nose is pointing down slightly then insert the needle at a 30º angle in
the lower left quadrant of the abdomen.
5. Retract the plunger slightly and if any fluid is aspirated remove the needle and prepare a new
inoculum before repeating the injection.
6. When the needle is correctly positioned, slowly inject the inoculum then remove the needle.
7. Once the injection is complete, release the animal into the cage.
Subcutaneous injection
1. Prepare the syringe and needle with the inoculum, removing the cap from the needle and
expelling any air bubbles.
2. Manually restrain the animal by grasping the lose skin at the scruff of the neck with the
thumb and forefinger to immobilize the head and torso. Place the animal on a flat surface to
stabilize the body.
3. Gently pull up the skin over the desired injection site, i.e. nape of neck, flank, or rump, to
create a small pocket.
4. Insert the needle into the pocket and retract the plunger slightly. If any fluid or blood is seen,
remove the needle and reposition it.
5. Depress the plunger slightly and if any resistance is felt remove the needle and reposition it.
6. Once the needle is correctly positioned, slowly inject the inoculum then remove the needle.
7. Once the injection is complete release the animal into the cage.
Intramuscular injections:
Intramuscular injections can be painful and should be performed on anesthetized animals to
minimize discomfort. Isoflurane inhalant anesthesia in the rodent EZ anesthesia chamber is
recommended. It is recommended that the smallest needle gauge possible is used to minimize
discomfort at the injection site. The thigh muscles on the front or back of the leg are most
commonly used, although small volumes can be injected into the lower leg muscles. Care must
be taken when injecting into the caudal thigh muscles to avoid damage to the sciatic nerve.
1. Prepare the syringe and needle with the inoculum, removing the cap from the needle and
expelling any air bubbles.
2. Place the animal in the induction chamber and observe the animal until it stops moving and
its breathing slows.
3. After the animal is immobile, firmly pinch one of the paws to test the depth of anesthesia. If
there is no purposeful movement the depth of anesthesia is adequate.
4. Remove the animal from the induction chamber and place the animal on the heated stage
with its nose positioned in the nose cone.
5. Turn the animal to access the desired muscle group.
6. Wipe the injection site with ethanol and allow todry. Gently pinch the leg to stabilize the
muscle group then insert the needle into the muscle until the bevel of the needle is embedded
in the muscle, but not deep enough to touch the bone.
7. Retract the plunger slightly and if any blood is seen, remove the needle and reposition it.
Once the needle is correctly positioned slowly inject the inoculum.
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Rodent injection techniques
8. Gently remove the needle from the muscle and apply gently pressure to the injection site for
about 10 seconds to prevent leakage of the inoculum.
9. Place the animal in a recovery cage on its side or sternum and monitor it continuously until it
is awake and able to walk normally, then return the animal to its home cage.
Intravenous injection:
Intravenous injections in rodents are most easily administered into the tail veins. Due to the short
tail of hamsters, IV injections are commonly administered through the cephalic or jugular veins
under anesthesia. These procedures should only be performed by highly experienced individuals
under supervision of a veterinarian, and are therefore not included in this SOP.
1. Prepare the syringe and needle with the inoculum, removing the cap from the needle and
expelling any air bubbles.
2. Place a heat lamp over the cage of animals for no more than 5 minutes to warm mice and
dilate vessels.
3. Once the animals are warmed, place them in an appropriately sized restraint device with the
tail extended. (An alternative method to using the heat lamp is to place the animal in a
restraint device, then rub the tail for several seconds with a gauze pad soaked in very warm,
but not hot, water to dilate the vessels.)
4. Wipe the tail with 70% alcohol and allow to dry.
5. Insert the needle into one of the lateral tail veins at a shallow angle, starting close to the end
of the tail. Advance the needle approximately 1 mm, then slowly depress the plunger. If the
inoculum flows with no resistance continue to inject the full amount. If there is resistance,
remove the needle, apply pressure to the site to stop any bleeding, and reinsert it a few
millimeters above the first injection site.
6. Once the injection is complete, remove the needle and apply gentle pressure to the site for 10
seconds to prevent leakage of the inoculum and stop any bleeding.
7. Once the bleeding and leakage is controlled release the animal from the restraint device and
return to it to its cage.
Intradermal injections:
Anesthesia may be required to immobilize the animal long enough to achieve proper positioning
of the needle in the dermal layer. Isoflurane inhalant anesthesia in the rodent EZ anesthesia
chamber is recommended. If the animal cannot be properly positioned for injection with inhalant
anesthesia, injectable anesthesia can be used. A 27G or smaller needle is recommended to
facilitate entry into the dermal layer. Commonly used intradermal injection sites include the base
of the tail, flank, footpad, and pinnae of the ears.
1. Prepare the syringe and needle with the inoculum, removing the cap from the needle and
expelling any air bubbles.
KMJ/ Drafted March 2010
Rodent injection techniques
2. Place the animal in the induction chamber and observe the animal until it stops moving and
its breathing slows. If injectable anesthesia is being used, administer the appropriate dose
following the injection methods described above.
3. After the animal is immobile, firmly pinch one of the paws to test the depth of anesthesia. If
there is no purposeful movement the depth of anesthesia is adequate.
4. Remove the animal from the induction chamber and place the animal on the heated stage
with its nose positioned in the nose cone.
5. Turn the animal on its side, sternum or back to access the desired injection site. It may be
necessary to shave the area to visualize the skin at the injection site.
6. Wipe the injection site with ethanol and allow to dry. Positioning the needle parallel to the
skin, insert the needle into the skin just until the bevel of the needle is within the dermal
layer.
7. Gently depress the plunger to inject the inoculum. Resistance should be felt and a firm bleb
will rise at the injection site.
8. Gently remove the needle from the skin. A small amount of leakage of the inoculum may be
seen, but using a smaller gauge needle will minimize leakage. The smallest volume possible
should be used, as larger volumes are more likely to leak.
9. Place the animal in a recovery cage on its side or sternum and monitor it continuously until it
is awake and able to walk normally, then return the animal to its home cage.
Post-procedural monitoring:
1. Anesthetized animals must be monitored continuously until they are awake and able to walk
normally.
2. Awake animals should be monitored at a minimum 1 hour and 24 hours after the procedure .
3. Observe the animals for the following signs:
a. Bleeding from the injection site
b. Labored breathing
c. Discharge from the nose or mouth
d. Ruffled fur
e. Hunched posture
f. Lethargy
g. Minimal responsiveness
4. Lethargy, labored breathing, minimal responsiveness and uncontrolled bleeding or discharge
are criteria for immediate euthanasia.
5. Document all observations on the experimental health monitoring sheet. Alert the Attending
Veterinarian of any ill or injured animals.
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