mouse by the scruff of the neck. Both of these restraining methods are only used for brief restraint; for example transferring animals from cage to cage. Scruff restraint
and mechanical restrainers are used for procedures that require more than momentary restraint, such as injection or blood withdrawal.
D.7 Proper Disposal
When performing euthanasia on the mice, the procedure must be approved by the Institutional Animal Care and Use Committee IACUC. Mice may be euthanized
using the CO
2
chamber method. Compressed CO2 gas is the only recommended source of CO2 for euthanasia. Carbon dioxide generated from dry ice is unacceptable.
With an animal in a chamber, an optimal flow rate should displace 10 – 20 of the
chamber volume per minute until the mouse is unconscious. This flow rate is associated with a rapid loss of consciousness and minimal distress to the animal.
Once the mouse is unconscious, the flow rate can be increased. Gas flow should be maintained for at least 1 minute following apparent clinical death. Death should be
verified by the absence of the heartbeat, performing cervical dislocation or by perforating the diaphragm prior to proper disposal of the animal.
E. Blood Collection
E.1 Extraction of Blood
A safe maximum for a single sample is 1.25 of body weight 1.25 ml100 grams of body weight taken every 2 weeks. Animals on chronic studies requiring multiple
blood samples may require hematocrit monitoring.
E.2 Handling, Storage and Transport of Blood
When blood is collected in a glass tube, it solidifies or coagulates within 5-10 minutes, forming a clot. Clotting can be prevented by adding a special anticoagulant
fluoride oxalate, trisodium citrate, EDTA dipotassium salt solution, Wintrobe mixture to the blood as soon as it is collected. Blood treated with an anticoagulant
separates into two liquid components: 1 the plasma, a yellow liquid, and 2 the blood cells.
Blood can be refrigerated and stored for up to three weeks. The temperature should not exceed 2
– 8 degrees Celsius. A temperature recording device is recommended to be included with the transported samples to ensure the appropriate
temperature has been maintained. It must not be stored with vaccines, reagents o medicines that require frequent use of the refrigerator.
The blood samples should be transported in a refrigerated state. No sample should be allowed to freeze, and should ideally be kept at a temperature of approximately 4
degrees. Transport of blood sample from site of collection to laboratory should be made in less than 48 hours.
E.3 Determination of the Number of Platelets
The blood sample is diluted with a 1 ammonium oxalate solution. The isotonic balance of the diluent is such that all erythrocytes are lysed while the leukocytes,
platelets, and reticulocytes remain intact. The standard dilution for platelet counts is
1:100. Platelet counts should be performed within three hours after the dilution has been prepared. If clumps of platelets are seen in the hemacytometer, the procedure
should be repeated. Clumps may be due to inadequate mixing of blood or to poor
technique in obtaining the blood specimen.
F. Related Studies
