Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Summary

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J·cm^-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Abstract

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J·cm^-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Introduction

The increased incidence of diabetes, immunosuppressive therapy applications, HIV infection, AIDS epidemic, invasive clinical procedures and broad-spectrum antibiotic consumption in the past years have increased the incidence of Candida albicans related diseases^1,2. C. albicans infections are commonly related to biofilm development and may cause clinical manifestations, such as candidiasis, or systemic manifestations, such as candidemia^1,2. One of the most noteworthy virulence factors of biofilm growth is the extracellular polysacchari....

Protocol

1. Preparation of culture media

1. Prepare sabouraud dextrose agar (SDA). Suspend 65 g of SDA supplemented with chloramphenicol (50 mg/L) in 1,000 mL of distilled water. Boil to dissolve the medium completely. Sterilize by autoclaving at 15 PSI (121°C) for 30 min. Cool to 45-50 °C. Mix well and pour 20 mL of SDA into sterile Petri plates (size: 100 mm x 15 mm).
2. Prepare yeast nitrogen base (YNB) medium supplemented with 100 mM glucose by mixing 6.7 g of YNB and 18 g of dextrose to 1,000 mL of ultrapure water. Mix using a stirrer and sterilize the medium using a 0.22 µm vacuum filter system.
3. Prepare RPMI by mixing 10.4 g of RP....

Results

Figure 2 displays the outcomes of Log₁₀ CFU/mL of C. albicans after per diem treatments with red light for 1 min. Red light significantly reduced the Log₁₀ CFU/mL compared to the NC (p = 0.004). Figure 3 presents the outcomes of the biomass (mg) of C. albicans biofilms after daily treatments. All treated groups showed reduction of the biomass compared to the NC (p = 0.000) and the red .......

Discussion

The most critical steps for successful culturing of C. albicans biofilm are: 1) to do the pre-inoculum and the inoculum in YNB medium complemented with 100 mM glucose; 2) to wait 90 min for the adhesion phase and carefully wash twice the wells with 0.89% NaCl to remove non-adhered cells; and 3) to add RPMI medium to the adhered cells to start biofilm formation, since RPMI will stimulate hyphae growth. Aneuploidies can occur when culturing C. albicans. Consequently, it is important not to use coloni.......

Materials

Name	Company	Catalog Number	Comments
Clorhexidine 20%	Sigma-Aldrich	C9394
Dextrose (D-Glucose) Anhydroous	Fisher Chemical	D16-500
Ethanol 200 proof	Decon Laboratories	DSP-MD.43
LumaCare LC-122 A	LumaCare Medical Group, Newport Beach, CA, USA
NaCl	Fisher Chemical	S641-500
NaOH	Fisher Bioreagents	BP 359-500
Phenol 5%	Milipore Sigma	843984
RPMI 1640 buffered with 3-(N-morpholino)	Sigma	R7755
Sabouraud dextrose agar supplemented with chloramphenicol	Acumedia	7306A
Sulfuric acid	Fisher Chemical	SA200-1
Yeast nitrogen base	Difco	DF0392-15-9
3-(N-morpholino)propanesulfonic acid MOPS	Sigma-Aldrich	M1254
24-well polystyrene plate	Falcon	353935