Chapter 5

Penetration of Antifungal Agents Through Candida Biofilms

L. Julia Douglas
Abstract
A filter disk assay is described, which measures the penetration of antifungal agents through Candida biofilms.
The technique involves forming a colony biofilm on a polycarbonate membrane filter, and capping it with a
second, smaller membrane filter followed by a wetted paper disk of the type used in zone-of-inhibition assays.
The entire assembly is transferred to agar medium containing the antifungal agent of interest. During
subsequent incubation, the drug diffuses out of the agar and through the biofilm sandwich to the moistened
paper disk. The drug concentration in the disk can be determined by measuring the zone of growth inhibition
that it produces on medium seeded with an indicator strain of Candida albicans in standard bioassays.
Additional procedures are outlined for determining the viabilities of drug-treated biofilms and for examining
biofilm morphology by scanning electron microscopy.
Key words: Biofilm, Candida, antifungal agent, drug penetration, scanning electron microscopy.

1. Introduction
During recent years, Candida albicans, together with related Candida
species, has become one of the commonest agents of hospital-acquired
infections (1, 2). Many of these are implant-associated infections, in
which adherent fungal populations, or biofilms, are formed on the
surfaces of medical devices including catheters, prosthetic heart valves
and endotracheal tubes (3, 4). Biofilm cells are organized into structured communities enclosed within a matrix of extracellular material.
They are phenotypically distinct from planktonic (suspended) cells;
most notably, they are significantly less susceptible to antifungal agents
(515). This property makes implant infections difficult to treat and
often the implant must be removed.
The mechanisms of biofilm resistance to antimicrobial
agents are not fully understood. One long-standing hypothesis is
Ronald L. Cihlar, Richard A. Calderone (eds.), Candida albicans: Methods and Protocols, vol. 499
Humana Press, a part of Springer ScienceBusiness Media, LLC 2009
DOI 10.1007/978-1-60327-151-6_5 Springerprotocols.com

37

38

Douglas

that the biofilm matrix resists drug penetration by forming a

reactiondiffusion barrier (16), and that only the surface layers
are exposed to a lethal dose of the agent. The extent to which the
matrix acts as a barrier to drug diffusion would depend on the
chemical nature of both the antimicrobial agent and the matrix
material. This protocol allows the penetration of antifungal agents
through Candida biofilms to be investigated (17) using a filter
disk assay modified from one previously reported for bacterial
biofilms (18). The procedure is also suitable for measuring drug
penetration through mixed-species biofilms (17). In addition,
methods for determining the viabilities of drug-treated biofilm
cells, and for examining the biofilms by scanning electron microscopy, are described.

2. Materials
2.1. Preparation of
Biofilm Inoculum

1. Slope cultures of Candida species on Sabouraud dextrose agar

(Difco).
2. Yeast nitrogen base (YNB) medium (Difco) containing 50 mM
glucose: batches of 50 mL of medium in 250-mL Erlenmeyer
flasks.
3. Wash buffer: 0.15 M phosphate-buffered saline (PBS; pH 7.2).

2.2. Biofilm Formation

1. Polycarbonate membrane filters (Whatman): diameter 25 mm

and pore size 0.2 mm. Sterilize by exposure to UV radiation for
15 min on both sides.
2. YNB agar containing 50 mM glucose in Petri dishes.

2.3. Antifungal Agents

1. Flucytosine (Sigma), fluconazole (Pfizer) and caspofungin

(Merck) solutions in sterile distilled water, prepared immediately
before use.
2. Voriconazole (Pfizer) and amphotericin B (Sigma) solutions in
dimethyl sulfoxide, prepared immediately before use. Other
antifungal agents may be used, dissolved fresh in the appropriate
solvent.

2.4. Penetration of
Biofilms by Antifungal
Agents

1. Antifungal-agent-supplemented agar: using a sterile filtration

unit (Sartorius), filter the drug solution into culture medium
(YNB agar containing 50 mM glucose) buffered with 0.165 M
morpholinepropanesulfonic acid (Sigma) to pH 7, and kept
molten at 50C. High drug concentrations are used (see
Note 1).
2. Polycarbonate membrane filters (Whatman): diameter 13 mm
and pore size 0.2 mm. Sterilize by exposure to UV radiation for
15 min on both sides.

Drug Penetration of Candida Biofilms

39

3. Paper concentration disks (Becton Dickinson): diameter

6 mm. Sterilize by exposure to UV radiation for 15 min on
both sides.
4. Plates of YNB agar containing 200 mM glucose and seeded
with 150 mL of a standardized suspension of planktonic C.
albicans (used here as an indicator organism and adjusted to
an optical density at 520 nm of 1.0).
2.5. Viable Counts
of Biofilm Cells
Exposed to Antifungal
Agents

1. Plates of YNB agar containing 200 mM glucose.

2. Dilution buffer: 0.15 M PBS (pH 7.2).

2.6. Scanning
Electron Microscopy

1.
2.
3.
4.

2.5% (v/v) Glutaraldehyde in PBS.

1% (w/v) Osmium tetroxide.
1% (w/v) Uranyl acetate.
Series of ethanol solutions ranging, in 10% increments, from
30% (v/v) ethanol in distilled water to dried absolute ethanol.

3. Methods
3.1. Preparation
of Biofilm Inoculum

1. Batches of YNB medium containing 50 mM glucose (50 mL in

250-mL Erlenmeyer flasks) are inoculated from fresh Candida
slopes and incubated at 37C for 24 h in an orbital shaker
operating at 60 rpm.
2. Cells are harvested and washed twice in PBS.
3. Before use in biofilm experiments, all washed cell suspensions
are adjusted to an optical density at 600 nm of 0.2.
4. If mixed-species biofilms are to be tested (see Note 2), equal
volumes of the standardized suspension of each organism are
mixed immediately before use.

3.2. Biofilm Formation

1. Biofilms are grown on membrane filters resting an agar culture

medium in Petri dishes. Sterile polycarbonate membrane filters
(diameter 25 mm) are placed on the surface of YNB agar
containing 50 mM glucose (see Note 3).
2. A standardized cell suspension (50 mL) is applied to the surface
of each membrane and the plates are incubated at 37C.
3. After 24 h, the membrane-supported biofilms are transferred
to fresh agar for a further 24 h, giving a total incubation time of
48 h for biofilm formation.

3.3. Penetration
of Biofilms by
Antifungal Agents

1. After biofilm formation on membrane filters, smaller sterile

polycarbonate membrane filters (diameter 13 mm) are carefully
placed on top of the 48-h-old biofilms.

40

Douglas

2. Sterile paper concentration disks (diameter 6 mm) are moistened

with growth medium (normally 29 mL; see Note 4) and positioned
on top of the 13-mm-diameter membranes.
3. Biofilms sandwiched between the membranes and moistened
disks are transferred to plates of antifungal-agent-containing
medium using sterile forceps and incubated at 37C for time
periods ranging from 60 min to 6 h (see Fig. 5.1 and Note 5).
4. The amount of antifungal agent, which penetrates each biofilm
and reaches the concentration disk, is determined by using
the disk in a standard drug diffusion assay. After the appropriate
exposure time, concentration disks are removed from the biofilm
sandwiches and placed on plates of YNB agar containing
200 mM glucose (see Note 6), which have been seeded with
the indicator strain of C. albicans. The plates are incubated at
37C for 24 h.
5. Zones of growth inhibition are measured and used to
determine the concentration of active antifungal agent in the
disks by the reference to a standard curve prepared by using
drug solutions of different concentrations but fixed volumes
(see Note 7).
6. Control assays are included where concentration disks are
placed on the two-membrane system to which no cells have
been added, i.e., the unit without the biofilm.
7. The drug concentration that penetrates the biofilm (C) is
divided by the drug concentration determined for the control
(C0) to provide a normalized penetration curve (see Figs. 5.2
and 5.3, and Note 8).

C
D
B

Fig. 5.1. Experimental system used to determine the penetration of antifungal agents
through biofilms. The biofilm (B ) is formed on a 25-mm-diameter membrane filter (A )
resting on YNB agar. A second, smaller filter (C ) is placed on top of the biofilm, and a
moistened concentration disk (D ) is positioned on top of the second filter. The entire
assembly is then transferred to antifungal-containing agar (E ).

Drug Penetration of Candida Biofilms

b)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

C/Co

C/Co

a)

41

60

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0

120 180 240 300 360

60

Time (min)

120 180 240 300 360

Time (min)

Fig. 5.2. Penetration of flucytosine through biofilms of (a) C. albicans GDH 2346 (closed triangles ), C. albicans GDH 2023
(open squares ), and C. albicans GRI 682 (open circles ); (b) C. krusei (open squares ), C. glabrata (open diamonds ), C.
parapsilosis (closed triangles) and C. tropicalis (closed circles). Error bars indicate the standard errors of the means. C,
drug concentration on distal edge of biofilm; Co, drug concentration measured in control. Reproduced from Ref. (17 ) with
permission from the American Society of Microbiology.

b)
1.0

1.0
0.9
0.8
0.7
0.6

0.9
0.8
0.7
C/Co

C /Co

a)

0.5
0.4
0.3
0.2
0.1
0.0

0.6
0.5
0.4
0.3
0.2
0.1
0.0

60

120 180 240

Time (min)

300 360

60

120

180

240

300

360

Time (min)

Fig. 5.3. Penetration of fluconazole through biofilms of (a) C. albicans strain GDH 2346 (closed triangles ), C. albicans GDH
2023 (open squares), and C. albicans GRI 682 (open circles ); (b) C. krusei (open squares ), C. glabrata (open diamonds ),
C. parapsilosis (closed triangles) and C. tropicalis (closed circles ). Error bars indicate the standard errors of the means. C,
drug concentration on distal edge of biofilm; Co, drug concentration measured in control. Reproduced from Ref. (17 ) with
permission from the American Society of Microbiology.

3.4. Viable Counts of

Biofilm Cells Exposed
to Antifungal Agents

1. After incubation on antifungal-agent-containing agar, biofilm

cells are gently scraped from the membranes with a sterile
scalpel and resuspended in 10 mL of PBS.
2. Serial dilutions (101106) of each biofilm cell suspension
are prepared in PBS.
3. Triplicate samples (0.1 mL) of the 104, 105 and 106 dilutions
are spread on YNB agar containing 200 mM glucose and these
plates are incubated at 37C for 24 h.
4. In control assays, membranes are transferred to growth
medium containing no antifungal agent and viable counts
determined as above.

42

Douglas

Fig. 5.4. Scanning electron micrograph of a 48-h membrane-supported biofilm of C.

tropicalis. Arrows indicate extracellular matrix material. Bar, 10 mm. Reproduced from
Ref. (17 ) with permission from the American Society of Microbiology.

3.5. Scanning
Electron Microscopy

1. Biofilms formed on polycarbonate membranes are fixed in

2.5% (v/v) glutaraldehyde in PBS for 1 h at room temperature
and immersed in 1% (w/v) osmium tetroxide for 1 h. They are
then washed three times with distilled water (3 mL), treated
with 1% (w/v) uranyl acetate for 1 h and washed again with
distilled water (3 mL).
2. Dehydration of the samples is achieved by immersion in a series
of ethanol solutions ranging, in 10% increments, from 30% (v/v)
ethanol in distilled water to dried absolute ethanol.
3. The final step before gold coating is drying. Simple air-drying in
a desiccator will suffice for many purposes, although it can lead
to a significant loss of the biofilm extracellular matrix. A typical
electron micrograph of a membrane-supported biofilm is shown
in Fig. 5.4.

4. Notes
1. Drug concentrations are selected on the basis of their ability to
give large zones of growth inhibition in control assays for drug
penetration. Examples of recommended concentrations would

Drug Penetration of Candida Biofilms

2.

3.

4.

5.
6.
7.

8.

43

be flucytosine, 30 times the MIC for planktonic cells of the

same strain; fluconazole, 60 times the MIC; voriconazole,
220 times the MIC; and amphotericin B, 60 times the MIC.
The protocol can also be used to test drug penetration through
mixed-species biofilms. In such cases, a medium should be
selected that is best able to support the growth of both the species.
For example, tryptic soy broth (Difco) supports the growth of
both Candida species and Staphylococcus species, and is a suitable
choice for propagation of biofilms containing both the organisms.
If mixed-species biofilms are to be tested, a suitable solid medium
should be selected. Tryptic soy agar is an excellent medium for
Candida/Staphylococcus biofilms.
Because of an occasional variation in disk thickness, a slightly
lower or higher volume of medium is sometimes required to
saturate the disks. Wetting of the disks helps prevent the
capillary action of the antifungal medium through the biofilms.
Exposure times of 60, 90, 120, 180, 240 and 360 min are
convenient for most purposes.
YNB agar containing 200 mM glucose, rather than 50 mM,
gives optimal lawn production by the indicator strain.
A standard curve is obtained by plotting the log of the drug
concentration used against the diameter of the zone of growth
inhibition.
Colony biofilms formed on membrane filters appear to lack
the water channels that typically surround matrix-enclosed
microcolonies in many other biofilms. The possibility that
drugs simply move through water channels without reaching
cells deep within the microcolonies is, therefore, largely eliminated by using this model system.

References
1. Calderone, R. A. (ed.) (2002) Candida and
Candidiasis. ASM Press, Washington, D.C.
2. Kojic, E. M., and Darouiche, R. O. (2004)
Candida infections of medical devices. Clin.
Microbiol. Rev. 17, 255267.
3. Douglas, L. J. (2003) Candida biofilms
and their role in infection. Trends Microbiol.
11, 3036.
4. Donlan, R. M. (2001) Biofilms and deviceassociated infections. Emerg. Infect. Dis. 7,
277281.
5. Hawser, S. P., and Douglas, L. J. (1995)
Resistance of Candida albicans biofilms to
antifungal agents in vitro. Antimicrob. Agents
Chemother. 39, 21282131.
6. Baillie, G. S., and Douglas, L. J. (1998)
Effect of growth rate on resistance of

7.

8.

9.

10.

Candida albicans biofilms to antifungal

agents. Antimicrob. Agents Chemother.
42, 19001905.
Baillie, G. S., and Douglas, L. J. (1998) Ironlimited biofilms of Candida albicans and their
susceptibility to amphotericin B. Antimicrob.
Agents Chemother. 42, 21462149.
Baillie, G. S., and Douglas, L .J. (1999)
Candida biofilms and their susceptibility to
antifungal agents. Meth. Enzymol. 310,
644656.
Baillie, G. S., and Douglas, L. J. (1999) Role
of dimorphism in the development of
Candida albicans biofilms. J. Med. Microbiol.
48, 671679.
Chandra, J., Mukherjee, P. K., Leidich, S. D.,
Faddoul, F. F., Hoyer, L. L., Douglas, L. J.,

44

Douglas

and Ghannoum, M. A. (2001) Antifungal

resistance of candidal biofilms formed on denture acrylic in vitro. J. Dent. Res. 80,
903908.
11. Chandra J., Kuhn, D. M., Mukherjee, P. K.,
Hoyer, L. L., McCormick, T., and Ghannoum,
M. A. ( 2001) Biofilm formation by the fungal
pathogen Candida albicans: development,
architecture, and drug resistance. J. Bacteriol.
183, 53855394.
12. Lewis, R. E., Kontoyiannis, D. P., Darouiche,
R. O., Raad, I. I., and Prince, R. A. (2002)
Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model
of Candida catheter-related bloodstream
infection. Antimicrob. Agents Chemother.
46, 34993505.
13. Ramage, G., VandeWalle, K., Wickes, B.
L., and Lopez-Ribot, J. L. (2001) Standardized method for in vitro antifungal
susceptibility testing of Candida albicans
biofilms. Antimicrob. Agents Chemother.
45, 24752479.

14. Ramage, G., VandeWalle, K., Wickes, B. L., and

Lopez-Ribot, J. L. (2001) Biofilm formation by
Candida dubliniensis. J. Clin. Microbiol. 39,
32343240.
15. Andes, D., Nett, J., Oschel, P., Albrecht, R.,
Marchillo, K., and Pitula, A. (2004) Development and characterization of an in vivo central
venous catheter Candida albicans biofilm
model. Infect. Immun. 72, 60236031.
16. Gilbert, P., Maira-Litran, T., McBain, A. J.,
Rickard, A. H., and Whyte, F. W. (2002) The
physiology and collective recalcitrance of
microbial biofilm communities. Adv. Microb.
Physiol. 46, 203256.
17. Al-Fattani, M. A., and Douglas, L. J. (2004)
Penetration of Candida biofilms by antifungal
agents. Antimicrob. Agents Chemother. 48,
32913297.
18. Anderl, J. N., Franklin, M. J., and Stewart, P. S.
(2000) Role of antibiotic penetration limitation
in Klebsiella pneumoniae biofilm resistance to
ampicillin and ciprofloxacin. Antimicrob.
Agents Chemother. 44, 18181824.