A high-throughput microcultivation protocol for FTIR spectroscopic characterization and identification of fungi.

Characterization and identification of fungi in food industry is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for fungal characterization suffer from a low throughput and are time consuming. In this study we present a protocol for high-throughput microcultivation and spectral characterization of fungi by Fourier transform infrared spectroscopy. For the study 11 species of in total five different fungal genera (Alternaria, Aspergillus, Mucor, Paecilomyces, and Phoma) were analyzed by FTIR spectroscopy. All the strains were isolated from trouble-shooting incidents in the production of low and high acid beverages. The cultivation was performed in malt extract broth (liquid medium) in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Mycelium was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Four spectral regions, fatty acids + lipid (3200-2800 cm(-1), 1300-1000 cm(-1)), protein-lipid (1800-1200 cm(-1)), carbohydrates (1200-700 cm(-1)) and "finger print" (900-700 cm(-1)) were evaluated for reproducibility and discrimination ability. The results show that all spectral regions evaluated can be used as spectroscopic biomarkers for differentiation of fungi by FTIR. The influence of different growth times on the ability of species discrimination by FTIR spectroscopy was investigated, and an optimal separation of all five genera was observed after five days of growth. This work presents a novel concept for high-throughput cultivation of fungi for FTIR spectroscopy that enables characterization or identification of hundreds of strains per day.

Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci.

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage phi LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six phi LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30 degrees C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the phi LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the phi LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.