STEP 2: Cool (or warm) the agar base to 45 C and add one part of reconstituted
blood (warmed to 45 C) to three parts base. Quickly swirl until well mixed.
Do not shake the mixture since this causes bubbles and partial hemolysis of
STEP 3: Aseptically dispense the still liquid media into 16 by 150 millimeter test tubes
(5 milliliters of media per tube) and stopper the tubes with vaccine-type
rubber stoppers. Place the tubes on a slanting rack at an angle that will
result in the media's being in a long slant.
STEP 4: After the medium has set (which may require several days), incubate the
tubes at 37 C and observe after 24 hours for sterility (lack of microbial
growth). Store them in the refrigerator at 4-10 C until they are needed.
500 milliliters of blood should yield about 300 tubes of final medium. The
medium, if kept under refrigeration at 4-10 C will remain satisfactory for
about six months.
Preparation of anticoagulant and innoculation of specimen.
(a) Anticoagulant preparation (PVSA).
Polyvinyl sulfuric acid, potassium salt ............... 0.5 gm
0.9 percent sodium chloride solution ................. 100.0 ml
STEP 1: Prepare a 0.5 percent solution of polyvinyl sulfuric acid in 0.9 percent sodium
STEP 2: Adjust the pH to 7.5 and sterilize by filtration through a sintered glass filter,
"UF" porosity (pyrex), into sterile vaccine-stoppered test tubes. Refrigerate at
4-10 C until needed.
STEP 3: Use 0.1 ml. of anticoagulant for every milliliter of patient's blood.
(b) Venipuncture and inoculation.
STEP 1: Use a sterile one milliliter serological pipet to deliver 0.4 milliliter of
anticoagulant into a sterile nine milliliter screwcap bottle.
STEP 2: Perform a venipuncture on the patient using a sterile syringe. Draw about
four milliliters of blood and mix it thoroughly with the anticoagulant in the
STEP 3: Use a sterile five-milliliter syringe to withdraw the thoroughly mixed
anticoagulated blood from the bottle and inoculate two tubes of medium
(warmed to room temperature) with two milliliters each of the mixture.