Jürgen Fröhlich

Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator.

In order to facilitate the isolation of pure cultures from natural habitats we have developed a method for the isolation of single microbial cell clones from a mixed population, e.g. the flora of the termite gut, with the aid of a modern micromanipulator. The separated single prokaryotic or eukaryotic cells were grown after transfer in culture media or they were used for single cell PCR. The micromanipulator was also applied for the removal of nuclei from protozoa, of which the SSU rDNA was directly amplified.

Spirochaeta coccoides sp. nov., a Novel Coccoid Spirochete from the Hindgut of the Termite Neotermes castaneus

ABSTRACT A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus. The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta, and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta. Therefore, it represents a novel species, which was named Spirochaeta coccoides. In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosaccharides by forming formate, acetate, and ethanol as the main fermentation end products. Furthermore, strain SPN1 is able to grow anaerobically with yeast extract as the sole carbon and energy source. The fastest growth was obtained at 30°C, the temperature at which the termites were also grown. The cells possess different enzymatic activities that are involved in the degradation of lignocellulose in the termite hindgut, such as β-d-glucosidase, α-l-arabinosidase, and β-d-xylosidase. Therefore, they may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut.

Termite Gut Symbiotic Archaezoa Are Becoming Living Metabolic Fossils

ABSTRACT Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termites cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.

Lactic Acid Bacteria

A typical lactic acid bacterium grown under standard conditions is aerotolerant, acid tolerant, organotrophic, and a strictly fermentative rod or coccus, producing lactic acid as a major end product. It lacks cytochromes and is unable to synthesize porphyrins. Its features can vary under certain conditions. Catalase and cytochromes may be formed in the presence of hemes and lactic acid can be further metabolized, resulting in lower lactic acid concentrations. Cell division occurs in one plane, except pediococci. The cells are usually nonmotile. They have a requirement for complex growth factors such as vitamins and amino acids. An unequivocal definition of LAB is not possible (Axelsson, Lactic acid bacteria microbiological and functional aspects. Marcel Dekker, 2004). Lactic acid bacteria are characterized by the production of lactic acid as a major catabolic end product from glucose. Some bacilli, such as Actinomyces israeli and bifidobacteria, can form lactic acid as a major end product, but these bacteria have rarely or never been isolated from must and wine. The DNA of LAB has a G+C content below 55 mol%. LAB are grouped into the Clostridium branch of gram-positive bacteria possessing a relationship to the bacilli, while Bifidobacterium belongs to the Actinomycetes. They are grouped in one order and six families. From the 33 described genera, only 26 species belonging to six genera have been isolated from must and wine.

Use of a species-specific multiplex PCR for the identification of pediococci

In this study, the 23S rRNA genes of nine different Pediococcus type strains were sequenced. By using a multiple sequence alignment with 23S rDNA sequences of related lactic acid bacteria two primer pairs were constructed, one for the general identification of the genus Pediococcus and one for the identification of the atypical species, P. dextrinicus. Furthermore, a primer set for a rapid multiplex PCR identification of the eight typical Pediococcus species was developed. With this technique, the species P. damnosus, P. parvulus, P. inopinatus, P. cellicola, P. pentosaceus, P. acidilactici, P. claussenii, and P. stilesii could be discriminated simultaneously in a single PCR. Experiments with inoculated grape musts showed that the detection limit was 10 cells ml(-1). The multiplex PCR assay was tested by the usage of 62 Pediococcus strains from different culture collections and 47 strains recently isolated from German wines and musts. In addition, contaminations with P. parvulus and P. damnosus could be detected after purification of DNA from spoilt wine samples. The method demonstrates a rapid and easy to handle tool for the species affiliation of pediococci in beverages and food samples.

Rapid Detection and Identification with Molecular Methods

The only absolute criterion of purity for a bacterial culture is that it has been derived from the progeny of a single cell. Failure to apply this criterion may lead to much effort in proving the purity of a culture. All strains upon which research is to be based should therefore be rigorously purified before starting to investigate the properties of individual organisms (Johnstone 1969). Ecologically oriented wine microbiologists are especially faced with the problem of how to obtain a pure culture of certain microbial strains from their densely populated natural habitats. The used methods comprise thereby a range from simple devices up to very complex machines. Most approaches to identify and enumerate microbes in wine use enrichment techniques (Fugelsang and Edwards 2007). Such indirect methods do not enumerate the original cell number in the sample, but their progeny, as enriched in a specific medium. Fugelsang and Edwards (2007) describe both general and selective growth media for plating yeasts and bacteria from wine. Unfortunately, plating and enrichment procedures are time consuming as colonies for some wine-related microbes take up to a week or more to appear on a Petri dish. Additionally, once colonies appear on a plate, the identification of the microbes requires further testing. Moreover, sublethally injured or viable but nonculturable cells, common in wine, may fail to grow on plates but are metabolically active. As a rule, culturebased techniques typically underestimate the size and diversity of a population (Kell et al. 1998; Millet and Lonvaud-Funel 2000). For monitoring the succession of a microbiota, cultivation-free molecular biological approaches were applied which give a more realistic view of a population. These spatiotemporal “snapshots” are often presented in the form of gel electrophoretic pattern of PCR amplicons or

Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing

In the present study, a culture medium for qualitative detection of osmotolerant yeasts, named OM, was developed. For the development, culture media with different concentrations of glucose, fructose, potassium chloride and glycerin were analyzed in a Biolumix™ test incubator. Selectivity for osmotolerant yeasts was guaranteed by a water activity (aw)-value of 0.91. The best results regarding fast growth of Zygosaccharomyces rouxii (WH 1002) were achieved in a culture medium consisting of 45% glucose, 5% fructose and 0.5% yeast extract and in a medium with 30% glucose, 10% glycerin, 5% potassium chloride and 0.5% yeast extract. Substances to stimulate yeast fermentation rates were analyzed in a RAMOS® parallel fermenter system, enabling online measurement of the carbon dioxide transfer rate (CTR) in shaking flasks. Significant increases of the CTR was achieved by adding especially 0.1-0.2% ammonium salts ((NH4)2HPO4, (NH4)2SO4 or NH4NO3), 0.5% meat peptone and 1% malt extract. Detection times and the CTR of 23 food-borne yeast strains of the genera Zygosaccharomyces, Torulaspora, Schizosaccharomyces, Candida and Wickerhamomyces were analyzed in OM bouillon in comparison to the selective culture media YEG50, MYG50 and DG18 in the parallel fermenter system. The OM culture medium enabled the detection of 102CFU/g within a time period of 2-3days, depending on the analyzed yeast species. Compared with YEG50 and MYG50 the detection times could be reduced. As an example, W. anomalus (WH 1021) was detected after 124h in YEG50, 95.5h in MYG50 and 55h in OM bouillon. Compared to YEG50 the maximum CO2 transfer rates for Z. rouxii (WH 1001), T. delbrueckii (DSM 70526), S. pombe (DSM 70576) and W. anomalus (WH 1016) increased by a factor ≥2.6. Furthermore, enrichment cultures of inoculated high-sugar products in OM culture medium were analyzed in the Biolumix™ system. The results proved that detection times of 3days for Z. rouxii and T. delbrueckii can be realized by using OM in combination with the automated test system even if low initial counts (101CFU/g) are present in the products. In conclusion, the presented data suggest that the OM culture medium is appropriate for the enrichment of osmotolerant yeasts from high-sugar food products.

Micromanipulation Techniques for the Isolation of Single Microorganisms

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. The only absolute criterion of purity for a bacterial culture is that it has been derived from the progeny of a single cell. Failure to apply this criterion may lead to much effort in proving the purity of a culture. All strains upon which research is to be based should therefore be rigorously purified before starting to investigate the properties of individual organisms (Johnstone 1969). Ecologically oriented microbiologists are faced especially with the problem of how to obtain a pure culture of certain microbial strains from their densely populated natural habitats. The methods used range from simple devices up to very complex machines. The principal procedures for obtaining pure cultures of bacterial strains have not been greatly improved since Robert Koch (Koch 1881). The isolation according to conventional methods, like the separation of microorganisms by agar plates (Koch 1881) and agar shake tubes (cf. Pfennig and Truper 1981), cannot avoid the fact that individual colonies are formed by cell aggregates. This occurs particularly with filamentous bacteria, which could easily agglomerate. More sophisticated electronic enumeration and sampling systems such as Coulter counter or flow cytometry cannot prevent the formation of cell aggregates. In addition to the isolation of single cells with optical tweezers (Huber 1999), there are alternative approaches, e.g. the “Bactotip” and “Membrane” methods for the micromanipulation of individual microorganisms. With the aid of these methods single cells can be picked out of a mixed population under direct visual control. The isolated aerobic or anaerobic species can be grown in pure culture or can be subjected to single cell PCR (Frohlich andKonig 1999a, 2000; cf. Prescott et al. 2002; Frohlich et al. 2002).

Bacterial Ectosymbionts which Confer Motility: Mixotricha paradoxa from the Intestine of the Australian Termite Mastotermes darwiniensis

Springer-Verlag Berlin Heidelberg 2005

Molecular Methods for Identification of Wine Microorganisms and Yeast Development

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. Nevertheless, most of the environmental microorganisms are graded as “yet not cultivable” because the nutritional requirements are unknown or they could not be isolated due to the fact that fast-growing strains overgrow other microorganisms of a microbiota. In addition to plating techniques, isolation without cultivation and analysis of microbes could be performed by micromanipulation techniques or the application of optical tweezers followed by the utilization of PCR-based technologies.