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PubMed2006

Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards.

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.

Bacteria↗

PubMed2005

Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

Biotechnology↗

PubMed1996

Measurement methods and strategies for non-infectious microbial components in bioaerosols at the workplace.

Exposure to micro-organisms can be measured by different methods. Traditionally, viable methods and light microscopy have been used for detection of micro-organisms. Most viable methods measure micro-organisms that are able to grow in culture, and these methods are also common for the identification of micro-organisms. More recently, non-viable methods have been developed for the measurement of bioaerosol components originating from micro-organisms that are based on microscopic techniques, bioassays, immunoassays and chemical methods. These methods are important for the assessment of exposure to bioaerosols in work environments as non-infectious micro-organisms and microbial components may cause allergic and toxic reactions independent of viability. It is not clear to what extent micro-organisms should be identified because exposure-response data are limited and many different micro-organisms and microbial components may cause similar health effects. Viable methods have also been used in indoor environments for the detection of specific organisms as markers of indoor growth of micro-organisms. At present, the validity of measurement methods can only be assessed by comparative laboratory and field studies because standard materials of microbial bioaerosol components are not available. Systematic errors may occur especially when results obtained by different methods are compared. Differences between laboratories that use the same methods may also occur as quality assurance schemes of analytical methods for bioaerosol components do not exist. Measurement methods may also have poor precision, especially the viable methods. It therefore seems difficult to meet the criteria for accuracy of measurement methods of workplace exposure that have recently been adopted by the CEN. Risk assessment is limited by the lack of generally accepted reference values or guidelines for microbial bioaerosol components. The cost of measurements of exposure to microbial bioaerosol components may be high owing to expensive analyses and highly variable exposure levels. The use of qualitative indicators of microbial growth, recording of health effects, specific immunoglobulin G antibody levels to prevalent species in serum of exposed workers and stratified sampling may help to reduce the costs of exposure assessment. An example of a combined strategy for assessment of health risks from handling mouldy timber is shown.

Aerosols↗

PubMed1999

Oxidative stress in microorganisms--I. Microbial vs. higher cells--damage and defenses in relation to cell aging and death.

Oxidative stress in microbial cells shares many similarities with other cell types but it has its specific features which may differ in prokaryotic and eukaryotic cells. We survey here the properties and actions of primary sources of oxidative stress, the role of transition metals in oxidative stress and cell protective machinery of microbial cells, and compare them with analogous features of other cell types. Other features to be compared are the action of Reactive Oxygen Species (ROS) on cell constituents, secondary lipid- or protein-based radicals and other stress products. Repair of oxidative injury by microorganisms and proteolytic removal of irreparable cell constituents are briefly described. Oxidative damage of aerobically growing microbial cells by endogenously formed ROS mostly does not induce changes similar to the aging of multiplying mammalian cells. Rapid growth of bacteria and yeast prevents accumulation of impaired macromolecules which are repaired, diluted or eliminated. During growth some simple fungi, such as yeast or Podospora spp., exhibit aging whose primary cause seems to be fragmentation of the nucleolus or impairment of mitochondrial DNA integrity. Yeast cell aging seems to be accelerated by endogenous oxidative stress. Unlike most growing microbial cells, stationary-phase cells gradually lose their viability because of a continuous oxidative stress, in spite of an increased synthesis of antioxidant enzymes. Unlike in most microorganisms, in plant and animal cells a severe oxidative stress induces a specific programmed death pathway--apoptosis. The scant data on the microbial death mechanisms induced by oxidative stress indicate that in bacteria cell death can result from activation of autolytic enzymes (similarly to the programmed mother-cell death at the end of bacillary sporulation). Yeast and other simple eukaryotes contain components of a proapoptotic pathway which are silent under normal conditions but can be activated by oxidative stress or by manifestation of mammalian death genes, such as bak or bax. Other aspects, such as regulation of oxidative-stress response, role of defense enzymes and their control, acquisition of stress tolerance, stress signaling and its role in stress response, as well as cross-talk between different stress factors, will be the subject of a subsequent review.

Animals↗

PubMed2003

Separation, identification, and characterization of microorganisms by capillary electrophoresis.

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.

Animals↗

PubMed1985

Trichostrongylus colubriformis: egg lethality due to Bacillus thuringiensis crystal toxin.

A toxin from crystals of Bacillus thuringiensis israelensis was lethal in vitro to eggs of the ruminant nematode Trichostrongylus colubriformis, with an LD50 of 1.8 ng/ml. Larval viability declined after a 2-hr exposure to B. t. israelensis and was dependent on the development period of eggs prior to exposure. Alkaline solubilization suggested that the insecticidal delta-endotoxin of B. t. israelensis was not responsible for nematicidal activity. Filtration of the toxin through 0.2- or 0.45-micron-pore filters revealed that the nematicidal activity was retained on the filter. Toxicity for nematode eggs was decreased by the enzyme inhibitor L-1-tosylamide 2-phenylethylchloromethyl ketone (10(-4) M) or ethylenediaminetetraacetic acid (10(-5) M) and phenylmethylsulfonyl fluoride (10(-6) M). Ethylenediaminetetraacetic acid from 10(-9) to 10(-5) M had no effect on the toxicity while phenylmethylsulfonyl fluoride from 10(-9) to 10(-5) M inhibited toxicity. Fourteen mammalian and microbial enzymes had no significant effect on larval viability while 12 sugars and lipids failed to reduce the toxicity. Addition of 5 mM calcium to the eggs' medium decreased the B. t. israelensis toxicity by 20-fold. The calcium-dependent inhibition of toxicity was reversed by ethylenediaminetetraacetic acid (10(-5) M) and lanthanum chloride (100 microM). The ionophore A-23187 decreased the LD50 by 18-fold to 33.5 ng/ml. Addition of 5 mM calcium chloride to the ionophore and toxin yielded an LD50 of 9.2 ng/ml. Treatment of nematode eggs with B. t. israelensis toxin for 2 or 24 hr had no effect on subsequent binding of selected fluoresceinated lectins to the eggshell.

Animals↗

PubMed1996

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity.

Bacteria↗

PubMed2001

An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations.

BACKGROUND: When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but nonculturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. METHODS: Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell's physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. RESULTS: It was possible to resolve an individual cell's physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. CONCLUSIONS: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganisms in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation; microbial fermentation is discussed in this article.

Cell Physiological Phenomena↗

PubMed2001

Characterization of the sat operon in Streptococcus mutans: evidence for a role of Ffh in acid tolerance.

An essential protein translocation pathway in Escherichia coli and Bacillus subtilis involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon of Streptococcus mutans resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in S. mutans after construction of bona fide sat operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of ylxM that was responsible for upregulating the coordinate expression of all five genes of the sat operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of satD and one just 3' to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the sat locus resulted in viable mutants. Most striking, however, was the finding that a mutation in ffh did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in S. mutans.

Bacterial Proteins↗

PubMed2004

Viability of amoebae, fungal conidia, and yeasts: rapid assessment by flow cytometry.

Conventional methods for the evaluation of antimicrobials and disinfecting solutions with microorganisms involve culture-based techniques, which are time-consuming and underestimate the number of viable organisms. Rapid detection and viability measurements of microorganisms in homogenous and heterogenous microbial populations have been greatly enhanced by recent advances in the use of fluorescent stains in flow cytometry (FCM). FCM has been applied to enumerate, differentiate, and identify microorganisms, determine protein and DNA content of cells, analyze the physiological state of individual cells, and analyze the interaction of drugs, antibiotics, and antimicrobials with microbial cells. Four physiological states of cells can be distinguished by FCM: (1) reproductively viable, (2) metabolically active, (3) intact, and (4) permeabilized.FCM permits a rapid and quantitative measurement of the optical characteristics of cells as they pass through, in a single file, a focused beam of light. As cells are carried within a fast-flowing fluid stream and through the focus of exciting light, three parameters are measured: forward angle light scatter, side angle light scatter, and fluorescence emitted by dyes that have specific interaction with intracellular components of individual cells. FCM data that are presented in histogram and dot plots can be generated to give information on a variety of properties of interest among cells in the population as a whole.FCM offers major advantages in multiparameter data acquisition and multivariate data analysis, high-speed analysis, and cell-sorting capabilities. Disadvantages may be associated with the cost, which is usually over 100,000 (US Dollars) for a typical laser-based flow cytometer with just analyzing capabilities. Another disadvantage is that skilled personnel are usually required to operate these complex instruments so as to get optimum performance. A schematic overview of flow cytometry is presented in Fig. 1.

Amoeba↗

PubMed1986

Effects of Histoplasma capsulatum products on the functional activity of alveolar macrophages.

The possibility that Histoplasma capsulatum may affect the activities of alveolar macrophages through products of either secretion or lysis was examined. Varying amounts of histoplasmin (HP), the filtrate of cultures of H. capsulatum, were added to monolayer cultures of alveolar macrophages of Wistar rats, and the effects of the filtrate on viability, ingestion rate, random migration, adhesive properties and microbial killing capacity were evaluated. No clear cytotoxic or cytopathogenic effects were produced at any dose tested. The endocytic rate was enhanced when HP (0.2 micrograms) was added, the effect being due to the recruitment of non-phagocytosing cells. Alveolar macrophages that had remained nonadherent after 3 h of incubation adhered to glass slides after HP was added. Dose-dependent inhibition of random migration of macrophages was produced with the addition of HP. However, the microbial killing capacity was not modified significantly at any dose of the H. capsulatum product.

Animals↗

PubMed1983

[Yersinia enterocolitica survival in Bulgarian yogurt and Vita yogurt (experimental studies)].

Laboratory studies were carried out on the development and survival of Y. enterocolitica in Bulgarian sour milk and sour milk 'Vita'. The raw cow milk was contaminated with various amounts of Y. enterocolitica cells. Results showed that the development and survival of the organism in the sour milk was dependent on its count in the raw milk and the running of the process of lactic acid fermentation. Under refrigerator conditions (Bulgarian sour milk contaminated at the rate of 10(4) microbial cells per cm3) Y. enterocolitica retained its viability up to the 24 th hour. When contamination reached 10(5) and 10(5) cells per cm3 the viability of the organism lasted up to 72 hours. Sour milk 'Vita' with a starter of strain of Lb. bulgaricus, producing D (-) lactic acid, possessed stronger inhibitory action on Y. enterocolitica compared with milk with a starter of strain of Lb. bulgaricus, producing L (+) actic acid.

Animals↗

PubMed2000

Viability of microencapsulated bifidobacteria in set yogurt during refrigerated storage.

Bifidobacteria are probiotic organisms that improve the microbial balance in the human gut. They can be incorporated as live cultures in fermented dairy foods, including yogurt, for transmission to humans. Because bifidobacteria are sensitive to high acidity, their viability in yogurt is limited. The objective of the present study was to investigate the effect of microencapsulation on the viability of bifidobacteria in yogurt during refrigerated storage for 30 d. Live bifidobacterial cells were encapsulated in kappa-carrageenan. Cell enumeration, determination of titratable acidity and pH, quantitation of lactic and acetic acids, and sensory evaluation (consumer test) were carried out on the yogurt samples. Microbiological results showed a decline of 78 and 70.5% in the population of Bifidobacterium longum B6 and B. longum ATCC 15708, respectively, for the treatments containing nonencapsulated cells. No difference in bifidobacterial population was observed in the encapsulated treatments. The acetic acid content in the yogurt with nonencapsulated bifidobacteria was higher than that in the plain yogurt (control) and encapsulated treatments. The increase in lactic acid content during storage was not different among the various treatments for B. longum B6, but was greater for nonencapsulated than encapsulated B. longum 15708 and the control. Consumers judged the nonencapsulated treatment as the most sour, which was likely due to the higher acetic acid content. Consumers preferred the control and nonencapsulated treatments over the encapsulated treatment. Microencapsulation appears to increase the viability of bifidobacteria in yogurt. This technique can be used to transmit bifidobacteria via fermented products provided that sensory characteristics of the product are improved or maintained.

Acetic Acid↗

PubMed1990

The effect of bacterial products on human fibroblast and keratinocyte detachment and viability.

An in vitro model has been developed to study the effect of soluble bacterial products on the viability and detachment of skin cell types utilized cultured grafts. Microbial products prepared from clinical isolates of bacterial species which most commonly colonize burn lesions showed marked variation in their ability to detach and kill both keratinocytes and fibroblasts. All three isolates of Acinetobacter spp. tested were effective in causing detachment and death of keratinocytes and fibroblasts, whereas Escherichia coli, Proteus mirabilis and Enterobacter spp. tested had little, or no, effect on detachment or viability for either skin cell type. Four Staphylococcus aureus isolates elicited variable strain-dependent results with regard to detachment and viability. One isolate possessed activity specific for keratinocyte detachment and death. These results indicate the possible undesirable effects such bacterial species may have on graft success in colonized burn wounds.

Bacteria↗

PubMed2005

The microbiology of spacecraft hardware: lessons learned from the planetary protection activities on the Beagle 2 spacecraft.

We consider the aseptic assembly of the Beagle 2 Mars probe and how the requirements of COSPAR planetary protection category IVa were achieved. Several areas for future investigation became apparent. An ESA mission is outlined in which a microbial bioburden is recovered after Earth orbit to assess viability following re-entry through the atmosphere.

Environmental Microbiology↗

PubMed2004

Effect of commonly used enhancement solutions on the viability of Toxoplasma gondii tissue cysts in pork loin.

Retail meat cuts of pork are frequently enhanced with salt solutions to improve flavor and texture and to extend shelf life through reductions in microbial contamination. A study of the effect of commonly used meat enhancement solutions on the viability of Toxoplasma gondii tissue cysts was performed using tissues from experimentally infected mice and pigs. Brains of T. gondii-infected mice were injected to 110% of the original weight of the brain with solutions containing sodium chloride (1 and 2%), sodium diacetate (0.1 and 0.2%), sodium tripolyphosphate (0.25 and 0.5%), potassium lactate (1.4 and 1.96%), or sodium lactate (1.4, 1.5, and 2.0%) alone or in combination and stored at 4 degrees C for 7 days before feeding to T. gondii-seronegative cats. Loins were collected from pigs experimentally infected with T. gondii and injected as above and stored for 7, 28, or 45 days at 4 degrees C before feeding to T. gondii-seronegative cats. Cat feces were examined for 14 days to assess oocyst shedding. The present study demonstrated that injection of mouse brains or pork loins with solutions containing 2% sodium chloride or > or =1.4% potassium or sodium lactate, alone or in combination with other components, prevented transmission of T. gondii to cats.

Animals↗

PubMed2007

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study.

AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.

Alcanivoraceae↗

PubMed2002

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns.

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.

Chromatography, Affinity↗