Jun 18, 2026
Susceptibility testing of _Candida albicans_
- Marbio 1
- 1UiT The Arctic University of Norway
- Marbio
Protocol Citation: Marbio 2026. Susceptibility testing of _Candida albicans_. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldo3nog5b/v1
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: Working
We use this protocol and it's working
Created: June 11, 2026
Last Modified: June 18, 2026
Protocol Integer ID: 318911
Keywords: Susceptibility test, Candida albicans, growth inhibition, OD600nm, Amphotericin B (control), Natrual product, Bioactivity assay, Biodiscovery, Bioprospecting, lowest concentration of an antimicrobial compound, visible growth of microorganism, antimicrobial compound, susceptibility testing, assay, compounds for growth, microorganism, visible growth, inhibiting effect, activity in the initial screening, lowest concentration, turbid solution, turbid solution in the well, purpose of this method, defined concentration
Abstract
The purpose of this method is to test compounds for growth-inhibiting effects on C. albicans. The assay aims to determine the lowest concentration of an antimicrobial compound that inhibits visible growth of microorganisms. The culture is inoculated into microtiter plates together with a defined concentration of a compound or fraction. If the fraction inhibits C. albicans growth, the well will remain clear. If the fraction has no effect, continued growth will result in a turbid solution in the well. Fractions that show activity in the initial screening are retested in a dilution series. OD is measured in a plate reader after 24 hours and again after 48 hours.
Image Attribution
Figure 1. Layout of microtiter plate with samples and controls.
Materials
**Equipment:**
Microtiter plate
Inoculation loops
Pipettes
Incubator
Plate reader, Victor (PerkinElmer)
**Media/Reagents:**
Sterile MilliQ water
NaCl (Sigma-Aldrich, S5886)
Sterile RPMI with 0.165 mol/L MOPS (with phenol red, without bicarbonate) (Sigma-Aldrich, R7755)
MOPS (Sigma-Aldrich, M3183)
L-glutamine (Biowest, Cat no:X0551-100)
Remel 0.5 McFarland Equivalence Turbidity Standard (Fisher Scientific, 10026732)
Amphotericin B (AMP-B) (Sigma, A2942)
Potato dextrose broth (Sigma-Aldrich, P6685)
Agar-agar (Sigma-Aldrich, A1296)
**Microorganism:**
Candida albicans ATCC 90028
Safety warnings
C. albicans belongs to risk group 1. The strain is not known to cause disease in healthy individuals, but to minimize the risk of contamination, a biosafety cabinet, gloves, and a lab coat should be used. Additionally, any waste that has been in contact with the fungus should be autoclaved immediately after screening.
C. albicans can form spores when allowed to dry, which can easily spread through the air.
Preparation of Media
Assay medium: Add 10.4 g RPMI powder (containing phenol red, without bicarbonate) and 34.53 g MOPS to 900 ml distilled water. Adjust the pH to 7 and add water up to 1 liter. Autoclave the medium, then add 10.25 ml sterile L-glutamine (L-glutamine cannot withstand autoclaving).
0.9% NaCl: Add 9 g NaCl to 1 liter of distilled water.
Potato dextrose agar (PDA): Add 24 g Potato Dextrose Broth and 15 g agar to 1 L MQ-H2O. Autoclave and pour into agar plates.
Procedure
Day 1: Inoculation of Candida albicans (ATCC 90028) from a freeze stock onto a fresh PDA plate and incubated at 37°C overnight.
Day 2: From the overnight culture, pick 5–8 colonies and resuspend them in 5 ml sterile 0.9% NaCl.
Vortex the suspension for 15 seconds, then adjust the cell density to 1–5 × 10^6 cells/ml by adding sufficient 0.9% NaCl. Cell density is assessed using a 0.5 McFarland standard.
Further dilute the C. albicans suspension 1:50, and then 1:20 (resulting in 1–5 × 10^3 CFU/ml) in assay medium.
Add 100 µl of sample and controls to each wells first (as shown in Figure 1), then add 100 µl of the fungal suspension. The final concentration of fungal cells will be 0.5–2.5 × 10^3 CFU/ml. To avoid edge effect, the outer wells along the edges are only filled with medium.
Figure 1. Layout of microtiter plate with samples and controls.
Day 3: Measure OD at 600nm after 24 hours.
Day 4: Measure OD at 600nm after 48 hours.
Controls: Amphotericin B (AMP-B) control: 100 µl of 16 µg/ml AMP-B (diluted in MQ-H2O) + 100 µl fungal suspension in column 10 of the plate (final concentration: 8 µg/ml AMP-B).
Growth control: 100 µl autoclaved MQ-H2O + 100 µl fungal suspension in column 11 of the plate.
Medium control: 100 µl assay medium + 100 µl autoclaved MQ-H2O in column 12 of the plate.
Reading the Results
Gently shake the plates and perform a visual inspection. Note any inhibition of growth.
Measure the optical density using a Victor plate reader (PerkinElmer).
Interpretation of Results
Visual: Visible turbidity indicates fungal growth and therefore inactive fractions (I). Wells without turbidity indicate inhibition of fungal growth and are classified as active fractions (A). If growth appears reduced compared to the growth control but the well is not completely clear, the fraction is classified as questionable (Q).
Photometric: Fractions are categorized as active, questionable, or inactive based on absorbance values: A (Active): < 0.05, Q (Questionable): 0.05–0.09, I (Inactive): > 0.09