Defibrinated blood is manufactured when whole blood from animals is aseptically drawn, without the use of an anticoagulant, into a bag or bottle containing marbles to promote clotting. The container is placed in a shaker to ensure that the clot is added to the marbles.
The clot is then removed, effectively removing clotting factors and eliminating the need for a blood thinner. A 48-hour microbial content test is performed to detect severe bacterial contamination; however, this test may not be applied if a cooler extraction is required.
Use 50 ml of horse/cattle/defibrinated blood.
LYSIS OF BLOOD
Procedure for lysis of horse blood by 3 x freeze-thaw (work in law bench with gloves)
NB: Blood is only kept 24 hours at room temperature and 3 weeks at 5 ° C.
2. Shake the bottle and pour 20 mL into each tube (50 mL conical plastic tubes).
3. Enter the lot number. of blood in the plastic tubes.
4. Freeze tubes at -20 ° C overnight.
5. The next morning, remove the blood from the freezer and thaw it at room temperature.
6. Repeat steps 4 + 5 until the blood has been frozen and thawed three times.
7. You can choose to check blood lysis by microscopy: use 100x magnification if all/almost all erythrocytes are lysed.
8. Add 20 ml of sterile MilliQ water to all plastic tubes containing blood. Invert the tubes a few times.
9. Seal the tubes and centrifuge at 5000 rpm for 30 minutes.
10. Carefully decant the supernatant into a new tube, leaving the pellet in the tube. Alternatively,
use a 20 ml disposable syringe/pipette to remove the supernatant. Discard the pill.
11. Write the lot number on the tube. of the blood along with the date and initials
12. Store lysed blood in a freezer (–20 ° C)
50% lysed horse blood is kept for four months at -20 ° C
50% lysed horse blood is kept for 24 hours at 5 ° C
1. Thaw the lysed blood (40 ml makes 40 tubes of 10 ml of MH + blood)
2. Use one batch of MH broth in 10 ml test tubes, preferably one that does not expire. up to four months later
3. Record the lot number. of the test tubes with MH together with lot no. of the blood. Determine the expiration date of the MH + blood as the shortest of
- Four months from the date of preparation (MH + BLOOD is kept for four months at 5 ° C)
- The expiration date of the MH broth
- The expiration date of blood
4. Invert the tube with the blood several times and transfer 1000 µl to each test tube. Invest the
test tube a few times.
5. Incubate the batch at 35-37 ° C overnight.
6. Next day: Examine the batch for contamination (towards the light, look for the pellet at the bottom)
7. Store the new batch at 5 ° C.
8. Measure the pH in two tubes. The temperature when measuring: 25º C. Record the pH in a logbook along with no. of contaminated pipes