I would like to plate whole blood taken from mice via cardiac puncture for CFU counts. Does anyone have a protocol for this, specifically that addresses the use of heparin? I want to save most of the blood collected for serum separation but some of the blood will be plated on agar plates for CFU. I can't treat all of the blood with heparin because of the serum separation but what should I do with the aliquot of whole blood that I will be plating, in regards to heparin use?
Blood clots very quickly, but if you're quick you can put part of the blood into a heparin containing eppie and part into a normal eppie. When I did CFU counts on blood collected by cardiac punctures I collected the blood into 1.5 mL eppies containing 2uL of concentrated heparin. The recommended working concentration of heparin is between 10-30 units/mL (found on the internet by searching for heparin coated tubes, Sigma recommends 20-50 units/mL). I prepared a 6000U/mL stock from powder and used 2 uL of that per eppie which should be enough for about 1 mL of blood according to the recommendations. A colleague of mine used to coat syringes with a ready-to-use heparin solution (not sure of the concentration) just prior to drawing blood - you could do the same with the eppies if you prefer this method. Attached is a data sheet for Sigma heparin I used. Hope this helps.
Alicja
Hi
I agree with Alicja. You get Heparin coated eppeies now a days. The other way is to add 2 uL of heparin into the eppies you are using for aliquots.
regards
Amarnath Pisipati
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