g. Cultivation of Trypanosoma brucei. Weinman's medium is the medium of
choice for cultivating from both blood and spinal fluid.
(1)
Preparation of media.
(a) Agar base preparation.
Nutrient agar, 1.5 percent, pH 7.3 ..................... 31.0 gm
Agar ............................................................... 5.0 gm
Distilled water .................................................... 1,000.0 ml
Dissolve the agar and nutrient agar in distilled water by heating. Distribute solution in
250 milliliter Erlenmeyer flasks. Plug the top of the flasks with gauze-covered rolled
cotton. Autoclave the flasks at 12 pounds pressure for 30 minutes and store in
refrigerator at 4-10.
(b) Blood collection and preparation.
Inactivated human plasma ................................ 200.0 ml
Saline washed human red cells......................... 200.0 ml
STEP 1: Collect 450 milliliters of human blood in a plastic donor bag containing 50
milliliters of sodium citrate anticoagulant. The blood must be collected
aseptically and with great care to the donor as well as to the container of
blood. Outdated blood from blood banks may also be used, providing it still
retains its sterility.
STEP 2: Refrigerate the blood at 4-10 C. After the plasma separates from the red
cells by gravity, aseptically draw off the major portion of the plasma into a
separate bag. Centrifuge the red cell pack. Draw off the supernatant plasma
and add it to the first portion of plasma.
STEP 3: Inactivate the sample by heating it for 30 minutes in a 56 C waterbath.
STEP 4: Wash the red cells (as aseptically as possible) three times in three or more
volumes of sterile 0.9 percent sodium chloride solution.
STEP 5: Reconstitute the whole blood by mixing equal volumes of inactivated plasma
and washed red cells. Note the total volume.
(c)
Final media preparation.
STEP 1: Melt sufficient quantities of nutrient agar base (previously prepared) so as to
give an approximate three to one ratio of agar base to reconstituted blood
(e.g., 250 milliliters of base to about 83 milliliters of blood).
MD0841
2-24
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