Candida tropicalis filamentation assay with fluconazole

MEDIA PREPARATION

Medium to inhibit filamentation (RPMI+NAC)

RPMI1640: 10g

3-[N-morpholino]propane sulfonic acid buffer (MOPS): 35 g

N-acetyl glucosamine: 20 g

Distilled water 500 ml

Adjust pH at 7.0

Adjust volume at 1000 ml

Esterilize by filtration

MEDIUM TO PROMOTE THE FILAMENTATION (RPMI+SFB)

RPMI1640: 10g

3-[N-morpholino] propane sulfonic acid buffer (MOPS): 35g

Glucose: 20 g

Distilled water: 500 ml

Adjust pH at 7.0

Adjust volume at 1000 ml

Add 10% of Fetal bovine serum

Fluconazole preparation

Weigh 6.4 mg of Fluconazole (Cat Sigma: F8929)

Dissolve in 1 ml of Dimethyl sulfoxide (DMSO)

aliquot and freeze at -80°C

FLUCONAZOLE (FLU) DILUTION

USE A V or U bottom 96 wells plate

A	B	C	D	E	F	G	H	I	J	K
DMSO		50	75	175
FLU 6400 µg/mL		50	25	25
DMSO					50	75	175
FLU 800 µg/mL					50	25	25
DMSO								50	75	175
FLU 100 µg/mL								50	25	25

INOCOLUM DILUTION

Cultivate Candida tropicalis in Potato dextrosa agar o in Liquid Sabouraud

Incubate for 24 h at 35°C

Dilute one or two colonies in sterile saline solution

Vortex until homogeinity

Adjust the turbidity according to the 0.5 MacFarland Standard (1x10⁶-5x 10⁶CFU)

Make a 1:100 dilution of the inocolum using each media (filamentation and No fiamentation)

Vortex for 15 seg

Make a 1:20 dilution using the 1:100 dilution, using the same media (Final yeast concentration: 5x10² to 3x10³ FCU

MIC ASSAY USING MEDIA TO FILAMENTATION (SFB) AND MEDIUM TO INHIBIT THE FILAMENTATION (NAC)

USE A STERILE 24 WELLS PLATE

RPMI + NAC

A	B	C	D	E	F	G
RPMI+NAC	90 µL	90 µL	90 µL	90 µL	90 µL	90 µL
FLU (dil)	10 µL (6400 µg/mL)	10 µL (3200 µg/mL)	10 µL (1600 µg/mL)	10 µL (800 µg/mL)	10 µL (400 µg/mL)	10 µL (200 µg/mL)
INOCULUM (dil 1:20)	900 µL	900 µL	900 µL	900 µL	900 µL	900 µL
REAGENTS	1 µg/ml	0.5 µg/ml	0.250 µg/ml	0.125 µg/ml	C+	C-
RPMI+NAC	90 µL	90 µL	90 µL	90 µL	90 µL	1000 µL
FLU (dil)	10 µL (100 µg/mL)	10 µL (50 µg/mL)	10 µL (25 µg/mL)	10 µL (12.5 µg/mL)
DMSO					10 µL
INOCULUM (dil 1:20)	900 µL	900 µL	900 µL	900 µL	900 µL

C+: Positive control; C-: negative control

RPMI+SFB| A | B | C | D | E | F | G | | --- | --- | --- | --- | --- | --- | --- | | RPMI +SFB | 90 µL | 90 µL | 90 µL | 90 µL | 90 µL | 90 µL | | FLU (dil) | 10 µL (6400 µg/mL) | 10 µL (3200 µg/mL) | 10 µL (1600 µg/mL) | 10 µL (800 µg/mL) | 10 µL (400 µg/mL) | 10 µL (200 µg/mL) | | INOCULUM (dil 1:20) | 900 µL | 900 µL | 900 µL | 900 µL | 900 µL | 900 µL | | REAGENTS | 1 µg/mL | 0.5 µg/mL | 0.25 µg/mL | 0.125 µg/mL | C+ | C- | | RPMI+SFB | 90 µL | 90 µL | 90 µL | 90 µL | 90 µL | 1000 µL | | FLU (dil) | 10 µL (100 µg/mL) | 10 µL (50 µg/mL) | 10 µL (25 µg/mL) | 10 µL (12.5 µg/mL) | | | | DMSO | | | | | | | | INOCULUM (dil 1:20) | 900 µL | 900 µL | 900 µL | 900 µL | 900 µL | |

C+: Positive control; C-: Negative Control Incubate at 35°C for 24 hrs with constant agitation (200 rpm approx.)

Read using an inverted microscope, comparing controls with each well

The Minimal inhibitory concentration will be where was observed 50% or more of growth

Also, register the filamentation grade in each well

CULTURE OF C. tropicalis TO HARVEST YEAST FOR TRANSCRIPTOMIC ANALYSIS

Obtain a Fluconazole Strain and a certificate strain (ATCC750)

Establish the susceptibility grade of both strains

Culture both strains with filamentation (RPMI+SFB) and Non-filamentation (RPMI+NAC) media, using Fluconazole at subinhibitory concentrations

Incubate at 35°C by 24 hrs with constant agitation (200 rpm)

Check the growth degree and filamentation degree

Harvest the colonies with a sterile Pasteur pipet

Centrifuge at high speed

Extract the supernatant

Add RNA later (Sigma Aldrich Ref0901)

Freeze at -80°C until the RNA extraction