The 96-well microtiter plate biofilm model and XTT assay have been invaluable tools for the quantitation of Candida
biofilms and the testing of their susceptibility to antifungal drugs (1
). However, our previous studies identified variability in assay results. Our experience has been informally confirmed via communication with numerous laboratories. We examined our experience with these assays over the past several years and attempted to identify experimental variables that might account for these inconsistencies. The primary goal of the current set of experiments was to investigate potential assay factors that might account for the observed variability in study accuracy and sensitivity in order to improve the assay for antifungal drug testing. The second objective of the present study was to develop a high-throughput XTT assay to be used for screening the susceptibility of biofilms to drugs. By systematically varying the assay conditions, we did identify factors that impact the results of the 96-well plate assay. These variables include (i) the duration of biofilm growth prior to antifungal administration, (ii) the antifungal dosing regimen, (iii) the XTT incubation time, (iv) transfer of the supernatant and washes prior to colorimetric measurement, and (v) the XTT manufacturer and lot. Experimental variables that did not appear to alter the XTT results significantly include (i) subculture medium and temperature conditions, (ii) washing of the subculture prior to the inoculation of wells, (iii) the XTT concentration, and (iv) transfer of the XTT supernatant to a new plate for colorimetric measurement.
Previous unpublished studies in our laboratory identified outcome variability with different subculture conditions prior to biofilm formation. We hypothesized that this may be due to the impact of the subculture medium and temperature on the presence or degree of filamentation, which has been shown to influence biofilm formation (16
). We found that the susceptibility patterns of C. albicans
, C. glabrata
, and C. parapsilosis
biofilms were not profoundly impacted by subculture conditions (). Small differences were observed, possibly related to changes in cellular morphology or biofilm architecture. In our experience, altering the subculture conditions can be helpful for certain strains ( and ). For example, C. albicans
K1 biofilms formed under RPMI-MOPS subculture conditions are more durable and easier to wash without disruption. We have found this phenotype for strains of C. parapsilosis
and C. glabrata
as well. However, for certain C. albicans
strains, such as DAY185, the formation of hyphae under subculture conditions at 37°C or in RPMI-MOPS complicates hemocytometer enumeration. In sum, propagation of Candida
spp. at 30°C in YPD medium plus uridine results primarily in yeast cells, which are easily enumerated with a hemocytometer. However, subculture in RPMI-MOPS may be useful for strains that are not forming consistent biofilms with YPD-plus-uridine subcultures.
Summary of Candida biofilm microtiter plate model and XTT assay variables for experimental design
Techniques for XTT assay troubleshooting
A number of studies have shown that the susceptibility of Candida
biofilms to drugs may be dependent on maturation or the biofilm growth phase. For example, the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p appear to play a role in early biofilm drug resistance (20
). Strains with disruption of these pumps are more susceptible to fluconazole during early biofilm formation but remain resistant if treated when biofilms are fully mature (20
). We hypothesized that testing C. albicans
earlier in biofilm formation (6 h) may increase XTT sensitivity for the detection of other resistance mechanisms or for some antifungal compounds. For a C. albicans
glucan synthase mutant, we observed increased XTT assay sensitivity for the detection of fluconazole antibiofilm activity when the biofilm growth period was reduced from 24 h to 6 h () (24
). The increased susceptibility of early-phase biofilms has been described previously and may be related to decreased matrix deposition, a smaller biofilm mass, or other biofilm phenotypic properties (19
). A shorter biofilm maturation time may be especially useful for the testing of azole drugs, such as fluconazole. C. albicans
biofilms are frequently resistant to azoles at concentrations near drug solubility, and testing of higher drug concentrations may not be possible (2
). We considered a decrease in the starting inoculum as an alternative method of reducing the biofilm mass. However, quorum sensing is critical for biofilm formation, and the use of a smaller inoculum may result in uneven or inconsistent biofilm formation (11
biofilms are more susceptible to amphotericin B and echinocandins than to azoles. We consistently find a dose-response curve with concentrations higher than the planktonic MIC. Therefore, we have found altering the duration of biofilm growth prior to drug treatment less helpful for optimizing resistance testing for these drugs.
Another factor that appeared to improve the sensitivity of the triazole antibiofilm activity assay was altering the drug dosing scheme. The application of additional antifungal drug or redosing improved the ability to discern differences in antifungal susceptibility between C. albicans
strains (). It is possible that the redosing replaces drug that has become inactive (unstable) over the treatment period (34
). Another possibility is that the mechanism of resistance is saturable and that additional antifungal dosing is capable of “overcoming” the biofilm resistance. Additional studies with the glucan synthase mutant suggest the presence of a saturable resistance mechanism for the strains examined in the current studies (24
). However, we cannot rule out the impact of drug stability on this study result. As described above, Candida
biofilms may be resistant to azole concentrations approaching solubility, and techniques to improve the sensitivity of the assay for the detection of susceptibility can be especially useful for this drug class (2
Another assay variable that impacted the biofilm susceptibility results was the XTT concentration. We were somewhat surprised to find that variation in the XTT concentration over a range from 0.25 to 1 mg/ml produced similar endpoint results (). Although absorbance values were lower for treatment with XTT at 0.25 mg/ml (optical density at 492 nm [OD492], approximately 1.5) than at 1 mg/ml (OD492, approximately 2.5), it appears that both XTT concentrations produce colorimetric products in the dynamic range. Therefore, lower XTT concentrations may be considered in the assay design; this change would decrease the XTT cost 4-fold for high-throughput assays, saving more than $10 per 96-well plate. Adjustments in the XTT concentration may also be helpful for Candida strains with absorbance values too low or too high to accommodate a full dose range effect.
We also tested the impact of the duration of the XTT incubation period. We found that the greatest assay sensitivity was observed with incubations of approximately 30 min for C. albicans
, a duration considerably shorter than that often described in previous publications () (10
). The shorter incubation period improved the dynamic range for the detection of susceptibility for the strains and drugs we studied over a wide concentration range. This assay change both improved sensitivity and reduced the time necessary for assay completion ( and ). In addition, we found that the use of XTT from different manufacturers and lots impacts the XTT endpoint, and we suggest that comparisons among antifungals and strains should be made using XTT from the same manufacturer and lot number. Pilot testing of new lots may be helpful for optimal study design and interpretation ().
An important consideration for interpretation of the activity of antifungal drugs against biofilm cells is the selection of the endpoint. The endpoint most commonly used is the antifungal concentration associated with a 50% reduction in optical density from that for an untreated control (EC50). The EC50 endpoint gives a single value that often reflects a steep part of the dose-response curve. It can be used for comparisons among strains or drugs in a manner similar to the use of planktonic MICs. Another method of comparison is determination of the percentage of reduction at a single drug concentration. This approach easily allows statistical analysis when experiments are performed in triplicate. However, the percentage of reduction at a single given concentration may not best represent the entire dose-response curve. Comparison of multiple points along the dose-response curve will provide a more complete reflection of drug impact. The trapezoid-AUC rule considers the percentage of reduction at each of the drug concentrations tested and estimates the area for the dose-response curve. This value can be used for comparisons of the entire drug-effect relationship.
In summary, the XTT assay and Candida
biofilm microtiter plate model have been useful tools for the study of drug activity against biofilms. However, assay inconsistency has been reported. The current experiments identified a number of variables that may be altered so as to optimize the assay for studying drug efficacy and comparing genetically modified strains for the investigation of drug resistance mechanisms. Although we considered many experimental variables, the assay variables investigated were by no means exhaustive. We focused on factors that we had hypothesized or observed to influence the XTT assay in a positive manner. We limited our study to include two antifungals with differing antibiofilm activities (fluconazole and amphotericin B) and one biocide (hydrogen peroxide). We believe the techniques for increasing assay sensitivity, troubleshooting, and saving time can be applied to the study of other antifungal drugs and biocides. Our experiments included C. glabrata
and C. parapsilosis
but focused on C. albicans
. The variables found to increase assay sensitivity for C. albicans
may not apply directly to the other strains. For example, a shorter biofilm growth period for C. glabrata
may result in poor biofilm formation, considering the lower growth rate of this species. The processing of XTT may differ among Candida
strains, and thus, direct correlations should be made with caution (14
). XTT requires uptake and processing by the Candida
cells. It is possible that strains with genetic alterations in the cell wall or biofilm matrix may limit uptake, but we have not observed this phenomenon.
These experiments identify factors for optimizing and troubleshooting the XTT assay for the assessment of drug activity against biofilms. The techniques have been useful for teaching new students and helping other labs adopt this method of susceptibility testing.