Tea tree oil reduces Candido adhesion and biofilm 365 Biofilm prevention assay tray containing serial dilutions of TTO but no microorgan- isms was incubated for 90 min at 35C.TTO was then Ten isolates including reference strains C.albicans ATCC removed and wells were washed twice with PBS with 10231.90028 and 90029 were examined for biofilm for- mation in the presence of TTO.Inocula were prepared by 0.001%Tween 80.After air drying.100 ul of a standardized collecting cells from overnight cultures in Sabouraud dex- suspension of C.albicans ATCC 10231 or C.albicans trose broth (SDB).washing.then resuspending in 0.85% 137548L was aliquoted into rows of the tray which was saline to~2X 10 cfu/ml.Doubling dilutions of TTO were then incubated for 90 min with shaking(75 rpm)at 35C. Well contents were removed.wells were washed and stained prepared in double-strength RPMI 1640 medium in 100 ul volumes in a 96-well flat-bottomed polystyrene microtitre with CV and optical densities determined as described tray (Nunc,Roskilde,Denmark).A final concentration of above.To assess whether TTO exposure alone reduced 0.001%Tween 80 was included throughout to aid oil sol- viability,suspensions of C.albicans ATCC 10231,137548L and 4047 prepared as described above were inoculated into ubility.Wells with no TTO served as positive growth con- solutions of TTO in PBS at final concentrations of 0.0.062 trols.Two dilution series were inoculated with 100 ul and 0.125%(v/v).Treatments were incubated with shaking volumes of inoculum per isolate to result in final concen- trations of~1X 106 cfu/ml and 0.25-0.016%(v/v)TTO. at 35C for 90 min and then viable counts were performed Corresponding TTO control wells each contained only using the Miles-Misra drop count technique. TTO solution and sterile growth medium.Microtitre trays were then incubated at 35C for 24 h,after which the trays Adhesion to mammalian cells were inverted to remove well contents and wells were Flow cytometry with FUN-1 stain was used to identify washed three times with PBS to remove any residual planktonic cells.Biofilm was then quantified by XTT(2.3- C.albicans bound to mammalian cells.Inocula were pre- bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- pared by collecting cells of C.albicans ATCC 10231,4047 and 137548L from overnight cultures in SDB,washing,then carboxanilide)reduction or crystal violet (CV)staining. XTT stain was prepared as described previously [15]. resuspending them in HEPES buffer(10 mM HEPES with with the exception that the XTT was prepared at 1 mg/ml 2%glucose)to a concentration of 10?cfu/ml.One ml vol- in PBS and menadione was prepared at 0.4 mM in ethanol umes of suspensions of each C.albicans strain,BEC,HeLa or A549 cells (10 cells/ml in 2X RPMI 1640).TTO solution (final concentration of 1 uM).Volumes of 200 uL of XTT/ and FUN-1 stain(2-chloro-4-(2.3-dihydro-3-methyl-(benzo- menadione were added to each well and trays were incu- 1,3-thiazol-2-yl)-methylidene)-1-phenylquinoliniumiodide, bated at 35C for 2 h in the dark.Then.100 ul volumes 0.5 uM,Invitrogen)were combined and incubated in the were transferred to new 96-well trays and the absorbance was determined at 490 nm using a microplate reader dark for 90 min at 35C with shaking at 50 rpm.Final TTO (Molecular Devices,Sunnydale,CA).Averages of the concentrations were 0,0.016.0.031 and 0.062%(v/v). Adhesion was then assessed by flow cytometry using a duplicate rows were calculated and the appropriate TTO blank value was subtracted. FACSCaliburTM flow cytometer.Mammalian cells (BEC, HeLa or A549)were gated based on forward/side scatter and For CV staining,biofilms were air dried and fixed with a total of 10,000 events were collected within the gate. methanol and then stained for 5 min with 1%(w/v)CV FUN-1 was excited with a 488 nm laser and the emission (225 ul per microtitre tray well).Excess stain was removed by rinsing under running tap water.After air drying,CV was collected with a 670 nm long pass filter(red live cells). was eluted by adding 225 ul of 33%(v/v)glacial acetic acid Mammalian cells positive for FUN-1 stain were indicative of binding of C.albicans.The following controls were to each well and leaving for 15 min.The absorbance of each included:(i)mammalian cells only.(ii)unstained C.albicans well was then determined at 540 nm as described above. and (iii)stained C.albicans.The percentage of mammalian cells with adherent C.albicans was calculated for each sample using CellQuest software (BD Biosciences,San Jose. Adhesion to polystyrene CA).Proportions of mammalian cells with adherent C.albi- Seven isolates including C.albicans ATCC 10231 were cans were calculated by dividing all test and control values examined for adhesion to polystyrene.The assay was per- by the relevant control.To examine the effects of TTO on formed as described above for the biofilm prevention assay pre-adhered C.albicans,equal volumes of C.albicans and with the exception that incubation was for 90 min at 35C HeLa cells prepared as described above were combined in a with shaking at 75 rpm [16]and final TTO concentrations glass McCartney bottle and incubated for 1 h at 35C with ranged from 0.25-0.008%(v/v).To eliminate the possibil- shaking at 50 rpm.TTO was then added to final concentra- ity that reductions in adhesion were an artefact of interac- tions of 0,0.031 or 0.062%and cells were incubated for a tions between TTO and the polystyrene tray,a microtitre further 90 min under the same conditions.Cells were then 2012 ISHAM,Medical Mycology,50,863-870© 2012 ISHAM, Medical Mycology, 50, 863–870 Tea tree oil reduces Candida adhesion and biofi lm 865 tray containing serial dilutions of TTO but no microorgan￾isms was incubated for 90 min at 35 ° C. TTO was then removed and wells were washed twice with PBS with 0.001% Tween 80. After air drying, 100 μ l of a standardized suspension of C. albicans ATCC 10231 or C. albicans 137548L was aliquoted into rows of the tray which was then incubated for 90 min with shaking (75 rpm) at 35 ° C. Well contents were removed, wells were washed and stained with CV and optical densities determined as described above. To assess whether TTO exposure alone reduced viability, suspensions of C. albicans ATCC 10231, 137548L and 4047 prepared as described above were inoculated into solutions of TTO in PBS at fi nal concentrations of 0, 0.062 and 0.125% (v/v). Treatments were incubated with shaking at 35 ° C for 90 min and then viable counts were performed using the Miles-Misra drop count technique. Adhesion to mammalian cells Flow cytometry with FUN-1 stain was used to identify C. albicans bound to mammalian cells. Inocula were pre￾pared by collecting cells of C. albicans ATCC 10231, 4047 and 137548L from overnight cultures in SDB, washing, then resuspending them in HEPES buffer (10 mM HEPES with 2% glucose) to a concentration of 10 7 cfu/ml. One ml vol￾umes of suspensions of each C. albicans strain, BEC, HeLa or A549 cells (10 6 cells/ml in 2  RPMI 1640), TTO solution and FUN-1 stain (2-chloro-4-(2,3- dihydro-3-methyl-(benzo- 1,3-thiazol-2-yl)- methylidene)-1- phenylquinolinium iodide, 0.5 μ M, Invitrogen) were combined and incubated in the dark for 90 min at 35 ° C with shaking at 50 rpm. Final TTO concentrations were 0, 0.016, 0.031 and 0.062% (v/v). Adhesion was then assessed by fl ow cytometry using a FACSCalibur ™ fl ow cytometer. Mammalian cells (BEC, HeLa or A549) were gated based on forward/side scatter and a total of 10,000 events were collected within the gate. FUN-1 was excited with a 488 nm laser and the emission was collected with a 670 nm long pass fi lter (red live cells). Mammalian cells positive for FUN-1 stain were indicative of binding of C. albicans . The following controls were included; (i) mammalian cells only, (ii) unstained C. albicans and (iii) stained C. albicans . The percentage of mammalian cells with adherent C. albicans was calculated for each sample using CellQuest software (BD Biosciences, San Jose, CA). Proportions of mammalian cells with adherent C. albi￾cans were calculated by dividing all test and control values by the relevant control. To examine the effects of TTO on pre-adhered C. albicans , equal volumes of C. albicans and HeLa cells prepared as described above were combined in a glass McCartney bottle and incubated for 1 h at 35 ° C with shaking at 50 rpm. TTO was then added to fi nal concentra￾tions of 0, 0.031 or 0.062% and cells were incubated for a further 90 min under the same conditions. Cells were then Biofi lm prevention assay Ten isolates including reference strains C. albicans ATCC 10231, 90028 and 90029 were examined for biofi lm for￾mation in the presence of TTO. Inocula were prepared by collecting cells from overnight cultures in Sabouraud dex￾trose broth (SDB), washing, then resuspending in 0.85% saline to ∼ 2  10 6 cfu/ml. Doubling dilutions of TTO were prepared in double-strength RPMI 1640 medium in 100 μ l volumes in a 96-well fl at-bottomed polystyrene microtitre tray (Nunc, Roskilde, Denmark). A fi nal concentration of 0.001% Tween 80 was included throughout to aid oil sol￾ubility. Wells with no TTO served as positive growth con￾trols. Two dilution series were inoculated with 100 μ l volumes of inoculum per isolate to result in fi nal concen￾trations of ∼ 1  10 6 cfu/ml and 0.25 – 0.016% (v/v) TTO. Corresponding TTO control wells each contained only TTO solution and sterile growth medium. Microtitre trays were then incubated at 35 ° C for 24 h, after which the trays were inverted to remove well contents and wells were washed three times with PBS to remove any residual planktonic cells. Biofi lm was then quantifi ed by XTT (2,3- bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide) reduction or crystal violet (CV) staining. XTT stain was prepared as described previously [15], with the exception that the XTT was prepared at 1 mg/ml in PBS and menadione was prepared at 0.4 mM in ethanol (fi nal concentration of 1 μ M). Volumes of 200 μ L of XTT/ menadione were added to each well and trays were incu￾bated at 35 ° C for 2 h in the dark. Then, 100 μ l volumes were transferred to new 96-well trays and the absorbance was determined at 490 nm using a microplate reader (Molecular Devices, Sunnydale, CA). Averages of the duplicate rows were calculated and the appropriate TTO blank value was subtracted. For CV staining, biofi lms were air dried and fi xed with methanol and then stained for 5 min with 1% (w/v) CV (225 μ l per microtitre tray well). Excess stain was removed by rinsing under running tap water. After air drying, CV was eluted by adding 225 μ l of 33% (v/v) glacial acetic acid to each well and leaving for 15 min. The absorbance of each well was then determined at 540 nm as described above. Adhesion to polystyrene Seven isolates including C. albicans ATCC 10231 were examined for adhesion to polystyrene. The assay was per￾formed as described above for the biofi lm prevention assay with the exception that incubation was for 90 min at 35 ° C with shaking at 75 rpm [16] and fi nal TTO concentrations ranged from 0.25 – 0.008% (v/v). To eliminate the possibil￾ity that reductions in adhesion were an artefact of interac￾tions between TTO and the polystyrene tray, a microtitre