ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2001, p. 2475–2479 Vol. 45, No.

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0066-4804/01/$04.00⫹0 DOI: 10.1128/AAC.45.9.2475–2479.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Standardized Method for In Vitro Antifungal Susceptibility

Testing of Candida albicans Biofilms
GORDON RAMAGE,1 KACY VANDE WALLE,2 BRIAN L. WICKES,1 AND JOSÉ L. LÓPEZ-RIBOT2*
Department of Microbiology and Department of Medicine, Division of Infectious Diseases,2
1

The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245
Received 29 March 2001/Returned for modification 9 May 2001/Accepted 5 June 2001

Candida albicans is implicated in many biomaterial-related infections. Typically, these infections are asso-
ciated with biofilm formation. Cells in biofilms display phenotypic traits that are dramatically different from
those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents.
Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal
treatment regimens. Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for
the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells
by using a formazan salt reduction assay. The assay was used for in vitro antifungal susceptibility testing of
several C. albicans strains grown as biofilms against amphotericin B and fluconazole and the increased
resistance of C. albicans biofilms against these antifungal agents was demonstrated. Because of its simplicity,
compatibility with a widely available 96-well microplate platform, high throughput, and automation potential,
we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility
testing of fungal biofilms.

Candida spp. are increasingly associated with biomaterial- haps related to the testing strategies that do not account for
related infections (11). Indeed, the majority of manifestations this alternative mode of growth. NCCLS guidelines use free
of candidiasis are associated in one way or another with the suspended planktonic cells for in vitro susceptibility testing.
formation of Candida biofilms on the surface of inert or bio- However, sessile cells from biofilms are phenotypically distinct
logical surfaces, and this phenotype is associated with infec- from their planktonic counterparts and are associated with
tions at both the mucosal and systemic sites (6, 8, 9, 11, 13). an increased-resistance phenotype (2–4, 22, 26, 27). Conse-
One of the main consequences of the biofilm mode of growth quently, for suspected biofilm-related infections, NCCLS stan-
is the increased resistance to antimicrobial therapy, which is dardized testing does not provide an accurate in vitro-in vivo
the main reason why biofilm-associated infections are fre- correlation. As a result, an alternative testing strategy should
quently refractory to conventional antibiotic therapy (2, 10, 22, be sought.
27). Decreased susceptibility of sessile cells to antimicrobial
Antifungal susceptibility testing represents a means of pre- agents compared to that of planktonic cells has been reported
dicting therapeutic concentrations of antifungal drugs used to extensively over the past decade (1, 2, 15–17, 22, 24, 30, 37).
treat a variety of Candida infections (14, 19, 20). It was not However, the comparatively new field of biofilm research has
until recently that the National Committee for Clinical Labo- progressed at such a rate that the development of assays to
ratory Standards (NCCLS) published its guidelines for a stan- measure sessile antimicrobial data, often ingenious, has re-
dardized broth macro- and microdilution assay for in vitro sulted in a plethora of different antimicrobial testing strategies.
testing of antifungal susceptibilities (35). Although these tests, Moreover, biofilms can be quantified by a variety of tech-
to some extent, have been shown to exhibit good in vitro-in niques, such as direct microscopic enumeration, total viable
vivo correlation, mainly in the setting of oropharyngeal candi- plate counts, metabolically active dyes, radiochemistry, and
diasis in human immunodeficiency virus-infected individuals luminometry (1, 2, 4, 7, 12, 15, 16, 18, 23, 25, 29, 37). Conse-
(19, 36), occasionally the antifungal susceptibility data do not quently, there are a myriad of potential techniques to measure
correlate with the desired clinical outcome. A variety of host biofilm antimicrobial susceptibilities. It is therefore imperative
factors could account for the lack of good correlations, partic- that a standardized antimicrobial susceptibility testing protocol
ularly in disseminated infections in individuals with various for biofilms be implemented, from both a clinical and research
degrees of immunosupression. Also, the lack of correlation is standpoint.
likely to be related to the fact that Candida infections can be Here we report on a rapid, inexpensive, easy to use, accu-
chronic and are most commonly associated with microbial bio- rate, and reproducible methodology for antifungal susceptibil-
films. Discrepancies in correlating susceptibility data are per- ity testing of Candida biofilms that benefits from the use of
conventional 96-well microtiter plates coupled to a colorimet-
ric method to assess the effects of antifungal agents against
* Corresponding author. Mailing address: Department of Medicine/ biofilm cells.
Division of Infectious Diseases, The University of Texas Health Sci-
ence Center at San Antonio, South Texas Centers for Biology in
Medicine, Texas Research Park, 15355 Lambda Dr., San Antonio, TX MATERIALS AND METHODS
78245. Phone: (210) 562-5017. Fax: (210) 562-5016. E-mail: RIBOT Isolates. Candida albicans isolates SC5314, 3153A, ATCC 64550, ATCC
@UTHSCSA.EDU. 64558, ATCC 76615, ATCC 90028, and ATCC 90029 were used in the course of

2475
2476 RAMAGE ET AL. ANTIMICROB. AGENTS CHEMOTHER.

filter, aliquoted, and stored at ⫺70°C. Prior to each assay, an aliquot of stock
XTT was thawed, and menadione (Sigma; 10 mM prepared in acetone) was
added to a final concentration of 1 ␮M. A 100-␮l aliquot of the XTT-menadione
solution was then added to each prewashed biofilm and to control wells (for the
measurement of background XTT-reduction levels). The plates were then incu-
bated in the dark for up to 2 h at 37°C. A colorimetric change in the XTT-
reduction assay, a direct correlation of the metabolic activity of the biofilm, was
then measured in a microtiter plate reader (Benchmark Microplate Reader;
Bio-Rad, Hercules, Calif.) at 490 nm.
Kinetics of biofilm formation on microtiter plates. C. albicans 3153A biofilm
formation was initiated in microtiter plates as described above. Biofilms were
formed over a series of time intervals (2, 4, 6, 8, 24 and 48 h). At each time
interval, biofilm formation was measured with the XTT assay and concurrently as-
sessed by light microscopy. For each time interval, 11 biofilm replicates were formed.
Total viable cell counts in control and fluconazole-treated biofilms. Biofilms
were preformed on 15-mm-diameter polystyrene discs within 24-well tissue cul-
FIG. 1. Colorimetric readings (OD490s) of biofilms formed in wells ture trays and challenged with antifungal agents, as described above. Following
of microtiter plates. Values represent the mean and standard devia- antifungal challenge and subsequent washing, sessile cells were removed from
tions of multiple independent biofilms formed in wells of each of 10 the surface of the disc by scraping with a sterile scalpel. The sessile cells were
different columns of the same microtiter plate. The variability between added to sterile PBS, sonicated for 5 min to disaggregate clumps, and then
the colorimetric readings was analyzed by statistical methods as de- vortexed for 30 s. Total viable counts were then estimated by the method
scribed in Materials and Methods. described by Miles and Misra (as cited in reference 5). Briefly, serial 10-fold
dilutions in sterile PBS were performed on each sample. Measured volumes of
this study. They were stored on Sabouraud dextrose slopes (BBL, Cockeysville, each dilution were dispensed onto YPD plates and then incubated for 18 to 24 h
Md.) at ⫺70°C. at 37°C. Colonies were counted the following day to estimate the total viable cell
Antifungal susceptibility testing. Fluconazole (Pfizer, Inc., New York, N.Y.) counts from each disc. SMICs were then assessed relative to those of the nondrug
and amphotericin B (Bristol-Myers Squibb, Princeton, N.J.) were used in the controls. XTT analysis of duplicate biofilms was performed in parallel to the total
course of this study. viable counts assay to demonstrate correlation between these two techniques.
Antifungal testing to determine the MICs of planktonic cells was performed by Statistical analysis. The optical density (OD) values from individual biofilms
the NCCLS M-27A broth microdilution method (35). We used the spectropho- were compared by one-way analysis of variance and by using the Bartlett’s test
tometric method of inoculum preparation corresponding to a concentration of for homogeneity of variances and the Bonferroni’s multiple comparison post-test.
0.5 ⫻ 103 to 2.5 ⫻ 103 cells per ml for each of the isolates prepared in the test P ⬍ 0.05 was considered significant. The analyses were performed with GraphPad
medium. Yeast inocula (100 ␮l) were added to each well of microdilution trays Prism version 3.00 for Windows (GraphPad Software, San Diego, Calif.).
containing 100 ␮l of antifungal drug solution (prepared at a 2⫻ final concentra-
tion). Antibiotic-free controls were also included. The microtiter plates were
RESULTS
then incubated at 35°C, and the endpoints were read visually at 48 h. Testing of
these isolates was performed in quadruplicate.
For antifungal susceptibility testing of sessile cells, isolates were propagated in
C. albicans biofilm formation in wells of microtiter plates.
yeast peptone dextrose (YPD) medium (1% [wt/vol] yeast extract, 2% [wt/vol] We performed a series of preliminary experiments to assess the
peptone, 2% [wt/vol] dextrose [U.S. Biological, Swampscott, Mass.]). Flasks variability between C. albicans 3153A biofilms formed in inde-
containing liquid medium (20 ml) were inoculated with a loopful of cells from pendent wells of the same microtiter plate. All biofilms formed
YPD agar plates containing freshly grown isolates and incubated overnight in an
on the microtiter plates over a 24-h time period displayed
orbital shaker (100 rpm) at 30°C. All strains grew in the budding yeast phase
under these conditions. Cells were harvested and washed in sterile phosphate- consistent XTT readings when the intensity of the colorimetric
buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 product was measured in a microtiter plate reader at 490 nm.
mM sodium chloride [pH 7.4] [Sigma, St. Louis, Mo.]). Cells were resuspended As seen in Fig. 1, no statistically significant difference was
in RPMI 1640 supplemented with L-glutamine and buffered with morpholinepro- noted between C. albicans 3153A biofilms formed on multiple
panesulfonic acid (Angus Buffers and Chemicals, Niagara Falls, N.Y.) to a
cellular density equivalent to 1.0 ⫻ 106 cells per ml with a Bright Line hemocy-
wells in each of 10 columns of the same microtiter plate (P ⬎
tometer (Hausser Scientific, Horsham, Pa). This density of cells was selected 0.05), a requisite for valid comparisons for susceptibility test-
because previous experiments in our laboratory demonstrated that optimal bio- ing. Furthermore, there were no significant differences in the
film formation occurs at this particular density (not shown). Biofilms were XTT absorbance readings from 70 independent biofilms formed
formed on commercially available presterilized, polystyrene, flat-bottom 96-well
on the same microtiter plate. Table 1 shows a description of
microtiter plates (Corning Inc., Corning, N.Y.). Biofilms were formed by pipet-
ting standardized cell suspensions (100 ␮l of the 106 cells/ml) into selected wells statistical measurements of multiple biofilms to demonstrate
of the microtiter plate and incubating them for 48 h at 37°C as described above. the validity of 70 independent biofilms. The remaining 26 wells
After biofilm formation, the medium was aspirated, and nonadherent cells were
removed by thoroughly washing the biofilms three times in sterile PBS. Residual
PBS was removed by blotting with paper towels before the addition of antifungal TABLE 1. C. albicans biofilm formation on 70 independent wells of
agents. Fluconazole and amphotericin B were then added to the biofilms in polystyrene microtiter plates grown for 24 h
serially double-diluted concentrations (1,024 to 1 ␮g/ml and 32 to 0.125 ␮g/ml,
respectively, from stock [concentrated] solutions of each antifungal agent pre- Parameter OD490a
pared in RPMI medium directly) and incubated for a further 48 h at 35°C. A
Mean ................................................................................................0.4466
series of antifungal agent-free wells and biofilm-free wells were also included to
Median.............................................................................................0.4435
serve as positive and negative controls, respectively. Sessile MICs (SMICs) were
Standard deviation .........................................................................0.0297
determined at 50 and 80% inhibition SMIC50 and SMIC80, respectively) by using
Standard error ................................................................................0.00355
the XTT reduction assay described below. Testing of these isolates was per-
Lower 95% confidence limit .........................................................0.4395
formed in quadruplicate.
Upper 95% confidence limit.........................................................0.4536
XTT-reduction assay. A semiquantitative measure of biofilm formation was
Maximum.........................................................................................0.5150
calculated by using an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetra-
Minimum .........................................................................................0.3860
zolium-5-carboxanilide]-reduction assay, adapted from previous reports (28, 39).
Briefly, XTT (Sigma) was prepared in a saturated solution at 0.5 g/liter in a
The variability between the colorimetric readings was analyzed by statistical
Ringer’s lactate. The solution was filter sterilized through a 0.22-␮m-pore-size methods as described in Materials and Methods.
VOL. 45, 2001 SUSCEPTIBILITY TESTING OF CANDIDA BIOFILMS 2477

deviate significantly from the positive non-drug-treated con-

trol. Likewise, the total viable cell counts from fluconazole-
treated discs (mean ⫽ 2.1 ⫻ 108 cells/ml) were only slightly
reduced compared to that of the drug-free control (mean ⫽
2.7 ⫻ 108 cells/ml). These data confirmed the resistance of
C. albicans biofilm cells to this azole derivative and the validity
of the XTT-reduction assay as an indicator of the efficacy of
antibiotic treatment.

DISCUSSION
In this study, we present a method for antifungal suscepti-
bility testing of sessile organisms. In this era of widespread
FIG. 2. Kinetics of C. albicans biofilm formation in wells of micro- increased antimicrobial resistance and increased use of in-
titer plates as determined by the colorimetric XTT-reduction assay. dwelling devices, it is crucial that we establish standard meth-
odologies that allow evaluation of current and new antifungal
served as negative controls. The results validate the equivalency agents against cells in biofilms (11). This painstaking work has
between each biofilm formed in multiple independent wells. been previously developed for many planktonic organisms;
The biofilm growth curve (Fig. 2) demonstrates how the however, the consideration of a sessile microbial lifestyle ap-
increased colorimetric reading obtained by the XTT-reduction pears to have been so far neglected. The increased resistance
assay correlates with increased cellular density in the biofilms, phenotype of sessile organisms emphasizes the need for a
as assessed by microscopy techniques. The biofilms were highly standardized assay to test biofilm antimicrobial susceptibilities
metabolically active in the first 8 h. However, as the biofilm (10, 17, 22, 30, 38). The microplate method described here is
matured and the complexity increased (24 to 48 h), the meta- fast, efficient, reliable, and reproducible, with high throughput
bolic activity reached a plateau, but remained high, reflecting potential. For semiquantitative analysis of the preformed bio-
the increased number of cells that constituted the mature bio- films exposed to antifungal drugs, a colorimetric assay was
film. Light microscopy observations demonstrated how the bio- developed, based on the studies by Tellier et al. and Hawser
film began with small microcolonies comprised mainly of yeast (26, 28, 39). A semiquantitative colorimetric technique was
cells (2 h). After 2 to 4 h, the yeast cells budded and started to chosen in preference to classical total viable cell counts pri-
filament, forming pseudohypha and eventually true hyphae. At marily because of the inherent problems associated with enu-
8 h, microcolonies then merged into an intricate network of merating highly variable morphological forms of C. albicans.
spatially dispersed filamentous forms that intertwined to form Colorimetric evaluation shows no bias in this respect. This
a coherent woven-like structure (24 to 48 h), with yeast cells assay was reliant upon the mitochondrial dehydrogenases of
forming aggregates along the hyphae. the live cells to convert an XTT tetrazolium salt into a reduced
Susceptibility of C. albicans biofilms to clinically used flu- formazan-colored product that could be measured spectropho-
conazole and amphotericin B. The antifungals tested in this tometrically (26, 28, 39). The colometric XTT metabolic assay
study showed decreased activity against sessile cells of all C. al- was shown to produce color changes that upon spectrophoto-
bicans strains tested (Table 2). The planktonic MICs reported metric determination of absorbance exhibited no statistically
for strain ATCC 90028, used for quality control purposes, fell significant differences between 70 independent biofilms formed
within the breakpoints quoted by experts and NCCLS guide- on 96-well microtiter plates. We clearly showed by using the
lines (35). Biofilms from all C. albicans strains tested were growth curve that the XTT assay absorbance readings were
intrinsically resistant to fluconazole. The resistance to ampho- proportional to the cellular density of the biofilm (Fig. 2).
tericin B was less pronounced and more variable between the These phenomena have been previously demonstrated by Tel-
isolates tested. Amphotericin B was up to 1 to 32 times less lier et al. and Hawser (26, 28, 39), which lends validity to this
active against sessile cells than planktonic MICs. This polyene alternative method of biofilm quantification. Because of its
antifungal still demonstrated some activity against C. albicans water solubility, the XTT-reduction assay can be easily quan-
biofilms, as indicated by SMIC50s. However, all SMIC80s al-
ready fell within the resistant range for this antifungal agent
TABLE 2. Antifungal susceptibilities of different C. albicans strains
(⬎1 ␮g/ml). Of note, even at the highest concentration of am- under planktonic or biofilm (SMIC data) growth conditions
photericin B used against biofilms (16 ␮g/ml) complete killing
was never achieved. Quadruplicate antifungal susceptibility MIC (␮g/ml) of:
testing of biofilms from the same strain displayed identical Strain Fluconazole Amphotericin B
MICs for both antifungal agents tested. Planktonic SMIC50 SMIC80 Planktonic SMIC50 SMIC80
Correlation between colorimetric readings with the XTT
assay and viable cell counts after exposure to fluconazole. SC5314 4 ⬎1,024 ⬎1,024 0.25 0.25 1.0
3153A 4 ⬎1,024 ⬎1,024 0.5 0.5 1.0
C. albicans 3153A biofilms were formed on plastic discs in a ATCC 64550 16 ⬎1,024 ⬎1,024 0.25 0.25 8.0
similar manner to those in microtiter plates. XTT absorbance ATCC 64558 0.5 ⬎1,024 ⬎1,024 0.25 0.5 1.0
measurements and total viable cell counts were performed ATCC 76615 0.25 ⬎1,024 ⬎1,024 0.25 0.5 2.0
with two triplicate sets of discs. XTT absorbance measure- ATCC 90028 0.5 ⬎1,024 ⬎1,024 0.5 0.5 1.0
ATCC 90029 0.5 ⬎1,024 ⬎1,024 0.5 0.5 2.0
ments of fluconazole-treated discs (up to 1,024 ␮g/ml) did not
2478 RAMAGE ET AL. ANTIMICROB. AGENTS CHEMOTHER.

tified without performing additional steps such as centrifuga- may partially explain the increased resistance of sessile cells
tion, addition of lysis buffer, solubilization, removal of me- compared to their planktonic counterparts (SMICs MICs).
dium, and sonication. It was our hypothesis that sessile cells However, this is also the situation that antifungal agents en-
that resisted the actions of antifungal agents would continue to counter in vivo against biofilms formed within the host. Over-
be metabolically active and therefore initiate color changes of all, the complexity of resistance associated with sessile cells is
XTT, whereas dead cells would not. in reality most likely to be multifactorial.
We have also successfully used this model to evaluate anti- In summary, we have developed a methodology that allows
fungal susceptibility testing against biofilms formed by a num- simple, inexpensive, rapid and accurate testing of the in vitro
ber of C. albicans clinical isolates and by other Candida spp., susceptibility of Candida biofilms to antifungal agents. Because
such as C. glabrata and C. dubliniensis (unpublished observa- of its compatibility with the 96-well microtiter platform and
tions). Other authors have described the ability of C. krusei, high throughput potential, this technique should prove impor-
C. tropicalis, C. guilliermondii, and C. parapsilosis to bind to the tant in the standardization of in vitro antifungal susceptibility
wells of microtiter plates (25). Overall, by using this method- testing of fungal biofilms, both as a research tool and in the
ology, multiple parameters can be easily investigated with rel- clinical laboratory. Use of this technology should be helpful for
ative ease, which is in contrast to other proposed techniques the selection of antifungal agents active against biofilms and
for the examination of antibiotic susceptibilities of biofilm for the screening of new effective antifungal agents to combat
cells. For example, Domingue et al. (12) proposed the use of biofilm-associated infections.
the Modified Robbin’s Device (MRD) technology to produce
multiple biofilms for antimicrobial testing. While this tech- ACKNOWLEDGMENTS
nique is a well-recognized model, it requires expert handling,
This work was supported by grant ATP 3659-0080 from the Texas
relatively few equivalent biofilms can be produced, requires Higher Education Coordinating Board (Advance Technology Pro-
longer processing times, and is more open to contamination gram, Biomedicine). J.L.L.-R. is the recipient of a New Investigator
than our method. Formation of biofilms by using other tech- Award in Molecular Pathogenic Mycology from the Burroughs Well-
nologies such as the perfused biofilm fermenter models or come Fund.
We thank W. Fonzi for C. albicans strain SC5314.
membrane-associated biofilm models (3, 18) are not amenable
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