Dec 24, 2021
Candida tropicalis filamentation assay with fluconazole
- 1Universidad de Caldas;
- 2Universidad de Manizales
- BIOSALUD
Protocol Citation: Laboratorio de Microbiología Mr JORGE ENRIQUE PEREZ CARDENAS, Sebastian Hernandez 2021. Candida tropicalis filamentation assay with fluconazole. protocols.io https://dx.doi.org/10.17504/protocols.io.bxh8pj9w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 19, 2021
Last Modified: December 24, 2021
Protocol Integer ID: 52512
Abstract
This protocol was used to obtain enough quantity of RNA of a nonsusceptible strain of Candida tropicalis, with the goal to do a transcriptomic analysis to demonstrate the degree of differential expression of genes under filamentation and non-filamentation conditions and against a non-filamenting strain, susceptible to fluconazole (Candida tropicalis ATCC750)
RPMI PREPARATION
RPMI PREPARATION
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
| REAGENTS | FLU 6400 µg/mL | FLU 3200 µg/mL | FLU 1600 µg/mL | FLU 800µg/mL | FLU 400 µg/mL | FLU 200 µg/mL | FLU 100 µg/mL | FLU 50 µg/mL | FLU 25 µg/mL | FLU 12,5 µg/mL | |
| 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 |
Susceptibility test
Susceptibility test
1d
1d
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 106 CFU)
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
| REAGENTS | 64 µg/ml | 32 µg/mL | 16µg/mL | 8 µg/mL | 4 µg/mL | 2 µg/mL | |
| 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+ | ||
| 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
| REAGENTS | 64 µg/mL | 32 µg/mL | 16 µg/mL | 8 µg/mL | 4 µg/mL | 2 µg/mL | |
| 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
1d
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