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Real-time monitoring of fungal inhibition and morphological changes
S D Aunsbjerg1, K R Andersen2, S Knøchel1
1Department of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
Abstract:
Mold growth constitutes a problem in many food and clinical environments and there is therefore focus on studying antifungal activity. Methods for determining growth inhibition by measuring colony growth or biomass are, however, time-taking and rapid methods for evaluation of antifungal effects are needed. Propionic acid and diacetyl are antifungal compounds produced by a range of dairy-associated bacteria. Their activity against Penicillium spp. was monitored real-time using an optical detection system with tilted focus plane to assess growth and morphological changes of Penicillium spp. by image recording inside a 96 well microplate. Images were used for generation of growth curves by using a segmentation and extraction of surface areas (SESA) algorithm and for quantifying morphology changes. Using image analysis growth could be detected within 15 h compared with more than 30 h when using standard optical density measurements. Induced morphological changes of fungi could furthermore be visualized and quantified using morphological descriptors such as circularity, branch points, perimeter and area of spores and growing hyphae. Propionic acid inhibited two out of two Penicillium spp. while morphological changes were strain dependent at the concentrations tested. Diacetyl inhibited six out of six Penicillium spp. strains and increased spore size and number of germination sites in two out of six of the strains prior to germination.
Insights
Rapid optical detection of antifungal activity against Penicillium spp. using image analysis significantly reduces detection time compared to standard methods. This approach visualizes and quantifies fungal growth and morphological changes, aiding in the evaluation of compounds like propionic acid and diacetyl.
Area of Science:
- Food science and technology
- Mycology
- Antimicrobial agent development
Background:
- Mold growth in food and clinical settings necessitates effective antifungal strategies.
- Current methods for assessing antifungal activity are time-consuming.
- Rapid evaluation methods are crucial for antifungal research.
Purpose of the Study:
- To develop and validate a rapid, real-time method for assessing antifungal activity against Penicillium spp.
- To monitor fungal growth and morphological changes using an optical detection system and image analysis.
- To evaluate the efficacy of propionic acid and diacetyl against various Penicillium strains.
Main Methods:
- Utilized an optical detection system with a tilted focus plane for real-time image recording in 96-well microplates.
- Employed a segmentation and extraction of surface areas (SESA) algorithm for growth curve generation and morphological quantification.
- Analyzed fungal morphology using descriptors like circularity, branch points, perimeter, and spore area.
Main Results:
- Image analysis enabled growth detection within 15 hours, significantly faster than standard optical density measurements (over 30 hours).
- Propionic acid demonstrated inhibitory effects on tested Penicillium spp., with strain-dependent morphological changes observed.
- Diacetyl inhibited all tested Penicillium spp. strains and induced pre-germination morphological alterations in some strains, including increased spore size and germination sites.
Conclusions:
- The developed image analysis method offers a rapid and efficient alternative for evaluating antifungal effects on Penicillium spp.
- Propionic acid and diacetyl show promise as antifungal agents, with distinct mechanisms of action and strain-specific responses.
- Real-time monitoring of fungal morphology provides valuable insights into antifungal compound interactions.
