Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system

The micro-colony method was used to enumerate viable bacteria in composts. Cells were vacuum-filtered onto polycarbonate filters and incubated for 18 h on LB medium at 37 degrees C. Bacteria on the filters were stained with SYBR Green II, and enumerated using a newly developed micro-colony auto counting system which can automatically count micro-colonies on half the area of the filter within 90 s. A large number of bacteria in samples retained physiological activity and formed micro-colonies within 18 h, whereas most could not form large colonies on conventional media within 1 week. The results showed that this convenient technique can enumerate viable bacteria in compost rapidly for its efficient quality control.     

细菌“活的不可培养状态”的生态意义及研究进展

"活的不可培养(VBNC)"状态是细菌在不良条件下的一种生存方式.VBNC状态作为细菌的一种生理状态,对传统微生物学产生了深远的影响.进入VBNC状态的细胞发生了一系列变化,无法继续用常规培养方法检测,在医学健康,环境科学等领域产生了巨大的影响,改进检测方法具有重要的意义.本文介绍了进入VBNC状态细菌在DNA、蛋白质组成等方面发生的变化,复苏过程.同时还介绍了VBNC状态的最新检测方法,最后对VBNC状态未来的研究方法进行了讨论.     

Solubilization of gellan gels by chelation of cations

Summary Chelation solubilization of gellan under mild conditions has been accomplished for the first time by exposure to either 10 mM sodium citrate buffer (pH 6.0) or to 1 mM sodium hexametaphosphate (pH 6.6). The citrate system was preferred for most applications since its is a ubiquitous cellular component, its solutions are autoclavable, and because viable plant tissues, fungi, and bacteria could be recovered from culture. Such recovery is not possible from more commonly used media such as agar.     

Adaptation of Rhodococcus erythropolis cells to high concentrations of toluene

Cells of Rhodococcus erythropolis DCL14 were adapted to increasing toluene concentrations in a mechanically stirred reactor. When the initial non-adapted cells were placed in contact with toluene, only 10.5% of cells remained viable after 1 h in the presence of 20% (v/v) toluene, while 8.6% of cells were viable after 28 h in the presence of an organic phase containing 80% (v/v) toluene in n-dodecane. Cell adaptation was studied by following the toluene consumption rate, the viability of the cell population, and the composition of the bacteria cellular membrane in the presence of increasing concentrations of toluene in the reactor. A maximum toluene concentration of 4.9 M, which corresponds to 52.4% (v/v) toluene in the organic phase, was achieved, toluene being consumed at 10.7 mg/(h mg protein). The adapted cells showed a substantially increased resistance to 50% ethanol and to concentrations of Betadine and Micropur tablets currently used in water purification, when compared to non-adapted cells.     

Recovery of Viable Bacteria from Probiotic Products that Target Oral Health

Probiotic therapy has predominantly been directed toward promoting and maintaining intestinal health. In recent years, however, probiotic regimens that target oral health have appeared on the market. These regimens are often delivered in the form of lozenges. Despite the oral health claims made by the manufacturers of these products, there is little independent evidence in the literature to support such claims. In theory, probiotic organisms can be beneficial by several different means including direct inhibition of pathogens and boosting of the host immune response, with the underlying assumption that these mechanisms require a critical number of viable organisms. In this study, five brands of probiotics marketed for oral health were tested for the recovery of viable bacteria. For only one brand could viable bacteria be recovered within one log of the manufacturer’s stated starting amount of bacteria. Nearly, a billion viable bacteria could be recovered from a lozenge of this brand. The other brands claimed similar starting amounts of bacteria at the time of manufacture but at least a three-log drop-off was observed in the amount of viable bacteria recovered from those products. Refrigeration of the probiotics significantly improved the recovery for one brand, but recoveries for all but one brand remained below the recommended daily dosage for probiotic regimens. It is concluded that probiotic brands differ significantly in the quantities of bacteria that remain viable with most failing to meet recommended dosage targets.     

Immobilization of bacteria and Saccharomyces cerevisiae in poly(tetrafluoroethylene) membranes.

A novel method for immobilization of bacteria and Saccharomyces cerevisiae cells is described. Microorganisms may be entrapped in a matrix of poly(tetrafluoroethylene) (PTFE) fibrils. Cells are blended with an aqueous emulsion of PTFE stabilized with Triton X-100 surfactant to form a thick paste. The paste is calendered biaxially in a standard rubber mill. This process causes fibrillation of the PTFE and formation of the fibril matrix, which serves only to impart physical integrity to the composite microporous membrane. The cells trapped in the membrane were shown to be viable by incubation of the membrane on solid media and in broth culture. This bioactive membrane represents a new means of immobilization of cells for bioprocessing.     

Immobilization of bacteria and Saccharomyces cerevisiae in poly(tetrafluoroethylene) membranes

A novel method for immobilization of bacteria and Saccharomyces cerevisiae cells is described. Microorganisms may be entrapped in a matrix of poly(tetrafluoroethylene) (PTFE) fibrils. Cells are blended with an aqueous emulsion of PTFE stabilized with Triton X-100 surfactant to form a thick paste. The paste is calendered biaxially in a standard rubber mill. This process causes fibrillation of the PTFE and formation of the fibril matrix, which serves only to impart physical integrity to the composite microporous membrane. The cells trapped in the membrane were shown to be viable by incubation of the membrane on solid media and in broth culture. This bioactive membrane represents a new means of immobilization of cells for bioprocessing.     

Phagocytosis of mycobacteria by U937 cells: a rapid method for monitoring uptake and separating phagocytosed and free bacteria by magnetic beads

A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.     

Short-Term Storage at 4 C of Trypsinized Tissues

Cells dispersed from tissues of certain animals remained viable for several days at 4 C although the per cent of viable cells varied among the different species.     

The viable but non-culturable state of wine micro-organisms during storage

Colony counting and DEFT did not give the same results when wine micro-organisms were enumerated. Both methods were used to monitor the population of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) during wine storage. Results suggest that part of the populations had reached a viable but non-culturable (VBNC) state. These cells were unable to produce colonies but could hydrolyse fluorescent esters and could be counted by DEFT. For AAB, O2 deprivation quickly induced this state. Recovery from this state was very rapid as soon as O2 was available. The response was not so clear for LAB during wine storage. However, a similar state was induced by sulfiting. Moreover, filtration of wine stored in barrels and contaminated by Brettanomyces, AAB and LAB demonstrated that cell size was not homogeneous. Cells which remained in wine after several weeks could pass through a 0.45-microm membrane. However, when they re-entered a growing phase, they were again retained by membrane filtration. During and after the decline phase, wine micro-organisms might survive as smaller cells in a VBNC state.     

Transfer of spores, bacteria and yeast into toluene containing phospholipids and low amounts of water: preservation of the bacterial respiratory chain

A method that allows the transfer of spores, bacteria and yeasts into a ternary system composed of toluene, phospholipids and low amounts of water is described. Initially an emulsion is formed by sonication of cells suspended in water in presence of toluene and phospholipids. The emulsion formed was subsequently clarified by blowing N2 on its surface and transparency was achieved when the water content of the system was reduced to 1-3 microliter/ml of organic solvent. The cells in ternary systems exhibit a reduction of cell volume, but the general structure is preserved. About 1/10,000 bacilli or yeast were viable, but spores were viable after 30 days in the ternary system. Yeast cells transferred to the ternary system, and back to an all water media failed to show oxygen uptake. In contrast bacilli that remained in the ternary system for 2 days respired to 80% of their maximal capacity when returned to an aqueous media.     

Effect of starch- and lipid-based encapsulation on the culturability of two Bifidobacterium longum strains

AIMS: To assess the applicability of starch- and lipid-based encapsulation methods for improving the viability and culturability of two Bifidobacterium longum strains stored in fermented and nonfermented foods. MATERIALS AND RESULTS: Cells were encapsulated with partially hydrolysed potato starch granules combined with amylose coating, or entrapped in cocoa butter matrix. The tested B. longum strains were not adherent to the starch granules, and the culturability of the cells stored in fermented and nonfermented foods was not improved by starch-based encapsulation. Encapsulation of the cells in cocoa butter was found to increase the plate counts during storage. In addition to plate counts, viability of the cells was measured by fluorescent microscopy using LIVE/DEAD BacLight viability assay. Microscopic counts of the viable cells did not change significantly during storage, suggesting that the cells remained alive despite becoming unable to grow on nutrient agar plates. CONCLUSIONS: Encapsulation with cocoa butter increased the culturability of the cells, but encapsulation with hydrolysed potato starch had no effect. Culture-independent viability assay suggested that cells remained viable despite being unable to grow on agar plates. SIGNIFICANCE AND THE IMPACT OF THE STUDY: This study indicates that encapsulation techniques may be useful in improving the culturability of bacteria, but the plate counts may yield insufficient data on the actual viability of the cells.     

A novel cytomedical vehicle capable of protecting cells against complement

We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.     

Rupture of the cell envelope by induced intracellular gas phase expansion in gas vacuolate bacteria.

Using a new approach, we estimated the physical strength of the cell envelopes of three species of gram-negative, gas vacuolate bacteria (Microcyclus aquaticus, Prosthecomicrobium pneumaticum, and Meniscus glaucopis). Populations of cells were slowly (0.5 to 2.9 h) saturated with argon, nitrogen, or helium to final pressures up to 100 atm (10, 132 kPa). The gas phases of the vesicles remained intact and, upon rapid (1 to 2 s) decompression to atmospheric pressure, expanded and ruptured the cells; loss of colony-forming units was used as an index of rupture. Because the cell envelope is the cellular component most likely to resist the expanding intracellular gas phase, its strength can be estimated from the minimum gas pressures that produce rupture. The viable counts indicated that these minimum pressures were between 25 and 50 atm; the majority of the cell envelopes were ruptured at pressures between 50 and 100 atm. Cells in which the gas vesicles were collapsed and the gas phases were effectively dissolved by rapid compression tolerated decompression from much higher gas saturations. Cells that do not normally possess gas vesicles (Escherichia coli) or that had been prevented from forming them by addition of L-lysine to the medium (M. aquaticus) were not harmed by decompression from gas saturation pressures up to 300 atm.     

Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed

AIMS: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. METHODS AND RESULTS: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>10(8) active fluorescent units). CONCLUSIONS: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. SIGNIFICANCE AND IMPACT OF THE STUDY: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.     

Growth and Starvation of a Strain of Klebsiella pneumoniae Isolated from a Brazilian Oil Formation

Summary The changes that occur in cell size and shape during experimental starvation and resuscitation of bacteria may find practical application in the biotechnological processes used in microbial enhanced oil-recovery (MEOR), in in situ bioremediation and in the creation of biobarriers. In the work described here, the aim was to observe certain aspects of the response of a strain of Klebsiella pneumoniae, isolated from oil producer wells belonging to PETROBRAS (National Oil Company of Brazil), to starvation and growth under various culture conditions. Cells of K. pneumoniae were taken from an exponentially-growing culture, washed and suspended in phosphate-buffered saline solution, without nutrients, where they remained for 91 days. Aliquots were withdrawn periodically, to perform viable and total cell counts and measure the optical density and cell dimensions. The starved cells were resuspended in sodium citrate medium, where they recuperated and exhibited a typical growth curve. The results indicate that this strain is a viable option for future trials on the transport and growth of microorganisms inside porous media, aimed at possible applications in MEOR.     

Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria

Aims:  To assess the stability of 16S rRNA of viable but nonculturable (VBNC) probiotics during storage when compared with different attributes of viability.
Methods and Results:  Levels of RNA of the probiotic strains Bifidobacterium longum 46, B. longum 2C and B. animalis subsp. lactis Bb-12 were monitored during storage in fermented and nonfermented foods. Cells which gradually lost their culturability in fermented products retained high level of rRNA, whereas rRNA of acid-killed control cells decreased at faster rate. Furthermore, the viability of B. longum 2C was monitored during storage by measuring changes in reductase activity, cytoplasmic membrane integrity and esterase activity using a flow cytometer. All of the culture-independent viability assays suggested that the cells remained viable during storage. In nonfermented media, the observed losses in culturability were smaller, and the changes in cell counts were comparable with the changes in rRNA levels.
Conclusions:  Viable but nonculturable probiotics maintain high levels of rRNA and retain properties of viable bacteria including reductase activity. Quantification of 16S rRNA complements culture-independent viability assays.
Significance and Impact of the Study:  Culture-independent viability assays allow the detection of VBNC probiotics, and can be used parallel to conventional culture-dependent methods to obtain accurate information on probiotic viability.     

Behaviour of the pharmaceutical probiotic preparation VSL#3 in human ileostomy effluent containing its own natural elements

The pharmaceutical probiotic VSL#3 (300 billion cfu/g lactic acid bacteria & bifidobacteria) was inoculated into human ileostomy effluent (HIE) to assess its behaviour vs the ileo-caecal tract. Separately, yogurt bacteria (yog) and bifidobacteria (Bif) present in VSL#3 were also inoculated into HIE. During 37 degrees C incubation (anaerobic condition) at zero, six and 24 hours, both cell growth in control HIE and indigenous Bif growth in HIE+yog were observed. Cells remained viable and metabolically active as shown by the increase in L(+) lactic acid in HIE+VSL#3 and HIE+yog and the pH decreased (approx. 5.5 compared with the 6.2 of control HIE). Total SCFA Short Chain Fatty Acids decreased in HIE+yog and HIE+VSL#3 at 6h and in all cultures at 24h; butyric acid decreased in HIE+Bif and HIE+VSL#3. Generally in vivo the bacteria remain in the ileo-caecal tract no longer than six h, therefore VSL#3 bacteria seem able to pass this barrier viably, colonizing the large bowel.     

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose

Abstract Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria ( Escherichia coli ). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.     

Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

AIMS: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. METHODS AND RESULTS: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant-depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLight LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. CONCLUSIONS: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. SIGNIFICANCE AND IMPACT OF STUDY: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.