STEP 4: For spinal fluid inoculation, inoculate approximately one milliliter of spinal fluid
directly from its sterile container to the culture tube.
It is important to mix the blood and anticoagulant solution thoroughly and to
inoculate promptly, for coagulation is inhibited only temporarily and may take
place within 15 minutes.
STEP 5. Add one milligram of dihydrostreptomycin sulfate in 0.2 milliliter of 0.9 percent
sodium chloride solution or 4000 units of penicillin in 0.2 milliliter of 0.9
percent sodium chloride solution for two milliliters of blood. For spinal fluid,
add 0.5 milligrams dihydrostreptomycin sulfate in 0.1 milliliters of saline or
2,000 units of penicillin in 0.1 milliliter of 0.9 percent sodium chloride solution
for one milliliter of spinal fluid. Contamination with bacteria or fungi almost
invariably inhibits trypanosomal multiplication and is the greatest single cause
for failure of this procedure.
Do not add any antifungal agents or mycotics because they may inhibit or kill
STEP 6: Tilt the inoculated tubes back and forth several times to spread the blood or
spinal fluid over the surface of the medium.
STEP 7: Incubate the tubes in darkness at 25 C. Cultures rarely become positive
before five or after 30 days. To check for positives, withdraw samples from
the tubes with sterilized Pasteur pipets (not with bacteriological loops).
Aspirate fluid from the bottom of the tube and repeatedly wash over the
surface of the slant to dislodge colonies. The colonies are colorless,
translucent, round, with regular outline, and rarely exceed two millimeters in
diameter. They are not completely distinctive on the medium. Therefore, do
not identify them based on gross appearance alone.
STEP 8: Withdraw small amounts of fluid suspension. Make wet mounts or stained
smears and search under the microscope for the characteristic forms. These
are numerous and occur singly, or in dividing forms, or appear in large and
h. Xenodiagnosis for Trypanosoma cruzi. One method for diagnosing
Chagas' disease consists of feeding noninfected laboratory-bred reduviid bugs (the
insect vector) with the blood of the suspected person and demonstrating the cyclical
development of the trypanosome in the insects' intestinal tract. This procedure is known
(1) The most important criteria for this technique is to select only clean
noninfected reduviid bugs and rear them in the laboratory. Each generation must be
properly protected from outside infection.