研究目的
To test if visible light can inactivate fungi and to elucidate the mechanisms involved, specifically for Saccharomyces cerevisiae irradiated with violet (405 nm) and blue (450 nm) light.
研究成果
The irradiation doses for one log reduction are 182 J/cm2 for 405 nm and 526 J/cm2 for 450 nm light. Trypan blue staining indicates intact cell membranes, suggesting photoinactivation may lead to a viable but nonculturable state rather than membrane damage. Spectral analysis supports protoporphyrin IX and zinc protoporphyrin as key photosensitizers for 405 nm, and flavins for 450 nm irradiation.
研究不足
The study is based on a single strain of Saccharomyces cerevisiae and specific culture conditions; results may not generalize to other strains or conditions. Variations in photosensitizer concentrations and experimental setups could affect outcomes.
1:Experimental Design and Method Selection:
The study involved irradiating Saccharomyces cerevisiae with 405 nm and 450 nm light from LEDs to assess photoinactivation effects and mechanisms, including colony quantification, trypan blue staining, and spectroscopic measurements of cell lysates.
2:Sample Selection and Data Sources:
The yeast strain Saccharomyces cerevisiae (DSM no. 70449) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and cultivated in YEPG medium.
3:List of Experimental Equipment and Materials:
Equipment included LEDs (LZ4-OUUB
4:00U8 and LZ4-00B208 from LED Engin), optical power meter (OPM 150 from Qioptiq), magnetic stirrer, water bath, vibrating tube mill (B. Braun Biotech), mill beads (Sartorius), cuvette spectrometer (Specord Plus from Analytik Jena), lasers (Flexpoint 405 nm from Laser Components and LDMC-470-2600 from Lasertack), fluorescence light probe (Ocean Optics), and spectrometer (SensLine AvaSpec-2048 XL from Avantes). Materials included YEPG medium, PBS, trypan blue (Merck), and various chemicals for lysate preparation. Experimental Procedures and Operational Workflow:
Yeast cells were cultivated, washed, irradiated with specified doses, and then subjected to CFU quantification on agar plates, trypan blue staining with microscopic examination, and cell lysate preparation for absorption and fluorescence spectroscopy.
5:Data Analysis Methods:
Data were analyzed using linear regression for CFU log reduction, manual counting for staining, and spectral analysis for identifying photosensitizers.
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Cuvette Spectrometer
Specord Plus
Analytik Jena
Used to measure absorption spectra of cell lysates.
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Spectrometer
SensLine AvaSpec-2048 XL
Avantes
Used to detect fluorescence emissions from cell lysates.
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LED
LZ4-OUUB00.00U8
LED Engin
Used for irradiation with violet light at 407 nm peak emission for photoinactivation experiments.
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LED
LZ4-00B208
LED Engin
Used for irradiation with blue light at 453 nm peak emission for photoinactivation experiments.
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Optical Power Meter
OPM 150
Qioptiq
Used to measure irradiation intensity during experiments.
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Vibrating Tube Mill
B. Braun Biotech
Used for homogenizing yeast cells to prepare cell lysates.
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Mill Beads
Sartorius
Used with the vibrating tube mill for cell homogenization.
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Laser
Flexpoint
Laser Components
Used for excitation at 405 nm in fluorescence measurements of cell lysates.
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Laser
LDMC-470-2600
Lasertack
Used for excitation at 465 nm in fluorescence measurements of cell lysates.
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Fluorescence Light Probe
Ocean Optics
Used to couple laser radiation into the sample for fluorescence detection.
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Trypan Blue
Merck
Used as a dye for staining to assess cell membrane integrity.
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