Vol. 26, No. 12
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1988, p. 2667-2668
Copyright © 1988, American Society for Microbiology
Microtiter Method for MIC Testing with Spherule-Endospore-Phase
RICHARD F. HECTOR,,* BARBARA L. ZIMMER,2 AND DEMOSTHENES PAPPAGIANIS2
Cutter Biologicals, Berkeley, California 94701,1 and Department ofMedical Microbiology,
University ofCaliforniaat Davis, Davis, California 956162
Received 17 June 1988/Accepted 8 September 1988
A method was developed for susceptibility testing with spherule-endospore-phase Coccidioides immitis by
using a microtiter format. Isolated endospores were used to inoculate wells containing modified Converse
medium with various concentrations of azole or nikkomycin antifungal substances which then were sealed with
an acetate film. The plate was incubated at 37°C with shaking for 96 h, after which the control wells had visible
turbidity and endpoints were discernible. Microscopic examination revealed that both control and treatment
wells maintained cells predominantly in the spherule-endospore phase of growth.
Although a reasonable level of standardization exists for
antibacterial susceptibility testing, methods for antifungal
testing are numerous and have resulted in divergent test
results (16). Adding difficulty to this situation are the com-
plex environmental requirements of some fungi, such as the
dimorphic pathogenic fungi, and certain yeasts, such as the
lipophilic Malassezia furfur. Among the former group, the
parasitic phase of Coccidioides immitis has perhaps the most
stringent requirements for maintaining the parasitic cycle.
Such factors as pH, appropriate nutrients, temperature,
presence of a detergent, and cell density must be rigidly
controlled for liquid-based spherule-endospore (SE) cultures
to be successfully propagated. Under the conditions estab-
lished for in vitro growth (3-6, 13), liquid-phase MIC testing
would not be practicable without nephelometric measure-
ment because the culture is typically seeded to a density
already detectable with the unaided eye. At least three
previous reports made use ofthe SE phase in determinations
of MICs (2, 11, 17). Other reports of susceptibility testing
with C. immitis have been concerned either with the sa-
prophytic phase (7, 10, 17) or with an endospore inoculum
which was allowed to convert to the filamentous stage during
the incubation period (12, 14). We report herein a practical
microtiter method for susceptibility testing which uses and
maintains the SE cycle for the duration of the test procedure
and yields results consistent with previously reported data.
The compounds tested were the azoles miconazole, keto-
conazole, and itraconazole from Janssen Pharmaceutica,
Beese, Belgium; fluconazole from Pfizer Inc., Groton,
Conn.; and R 3783, N 7133, U 7476, clotrimazole, and
bifonazole from Bayer AG, Wuppertal, Federal Republic of
Germany; and the chitin synthase inhibitors nikkomycin X
and nikkomycin Z, also from Bayer. Ketoconazole was first
dissolved in 0.2 N HCl, fluconazole and the nikkomycins
were dissolved in modified Converse medium (4, 5, 13), and
all other compounds were first dissolved in 95% ethanol. All
drugs were subsequently diluted in the growth medium in
96-well microtiter plates (Costar Plastics) in 100-pul volumes.
A fresh endospore inoculum of C. immitis Silveira was
prepared by differential centrifugation of a liquid culture of
the parasitic phase inoculated 72 h earlier by following the
conditions described by Levine et al. (13). Endospores were
separated by spinning at 500 x g for 5 min, followed by a
high-speed spin of the upper portion of the supernatant at
800 x g for 10 min. The pellet was suspended to an optical
density of approximately 0.01 at 440 nm in modified Con-
verse medium, and 100-pli portions were added to all drug
and control wells. At this density, a 100-ml portion has the
usual turbidity used for culture maintenance, but the 100-pil
volumes added to test wells appear transparent. The final
volume in the wells was 200 pi. The wells were sealed with
an adhesive acetate film (Dynatech Laboratories, Inc., Alex-
andria, Va.), and the plates were incubated with shaking (80
rpm) at 37°C. Plates were read at 96 h, with the MIC
determined as the lowest concentration with no macroscop-
ically visible growth. At 7 days, minimum lethal concentra-
tions were determined by transferring 2.5 pul from wells
showing no visible growth to tubes containing 5 ml of
glucose-yeast extract broth and incubating at 37°C for 7
days. The minimum lethal concentration was defined as the
lowest concentration showing a complete absence ofgrowth.
The MIC results (Table 1) for miconazole, fluconazole,
and clotrimazole were consistent with previously reported
values (7, 8, 10, 14, 17), although the present method
resulted in values for ketoconazole considerably higher than
those ofother reports (7, 12). This latter finding cannot likely
be blamed on a pH effect such as is found with fungi tested
in yeast nitrogen base medium, since the present growth
medium has a pH much closer to neutral (6.4). The results
for the nikkomycins were consistent with the calculatedK,
values (15) and were considerably lower than MICs reported
for this class of agent versus Candida albicans (1). Micro-
scopic examination of control and several test wells at 96 h
revealed that the fungus was maintained in the SE phase.
Although the importance oftesting the parasitic versus the
saprophytic phase of dimorphic fungi has yet to be estab-
lished, using the former has an inherent appeal. In tests of
the susceptibility of C. immitis to amphotericin B, the MICs
for the SE phase were approximately 8- to 10-fold lower than
those for the mycelial phase (2, 11). Similarly, although the
SE phase of this fungus was found to be very sensitive to the
chitin synthase inhibitor polyoxin D, the mycelial phase
appeared to be refractory (9).
The method described above proved to be simple to
conduct; it conserved inocula, media, and antifungal agents;
and it resulted in data deemed consistent with other pub-
lished data. Additionally, determining MICs with the SE
phase instead of an arthroconidial inoculum offers the obvi-
J. CLIN. MICROBIOL. Download full-text
TABLE 1. MICs for C. immitis Silveira
aThe MICs were read at 96 h.
bThe MLCs (minimal lethal concentrations) were read at 7 days.
C1.56 ,uM=0.7 ,ug/ml.
ous advantage of safety for laboratories which may not have
the containment facilities to handle the saprophytic phase of
the fungus. However, since this requires the conversion of
the isolate from the saprophytic to the SE phase, the method
may be restricted to those reference centers with the capa-
bility and the experience to effect such a conversion.
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