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The still poorly explored world of microbial functioning is about to be uncovered by a combined application of old and new technologies. Bacteria, especially, are still in the dark with respect to their phylogenetic affiliations as well as their metabolic capabilities and functions. However, with the advent of sophisticated flow cytometric and cell sorting technologies in microbiological labs, there is now the possibility to gain this knowledge at the single-cell level without cumbersome cultivation approaches. Cytometry also facilitates the understanding of physiological diversity in seemingly likewise acting populations. Both individuality and diversity lead to the complex and concerted actions of microbial consortia. This review provides an overview of the state of the art in the field. It deals with the handling of microorganisms from the very beginning (i.e. sampling, and detachment and fixation procedures) and goes on to discuss the pitfalls and problems in analysing cells without any further treatment. If information cannot be gained by specific staining procedures, phylogenetic technologies, transcriptomic and proteomic approaches may be options for achieving advanced insights. All in all, flow cytometry will be a mediator technology to gain a deeper insight into the heterogeneity of populations and the functioning of microbial communities.

Greatly increasing the amounts of flaxseed oil [rich in alpha-linolenic acid (ALNA)] or fish oil (FO); [rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in the diet can decrease inflammatory cell functions and so might impair host defense. The objective of this study was to determine the effect of dietary supplementation with moderate levels of ALNA, gamma-linolenic acid (GLA), arachidonic acid (ARA), DHA, or FO on inflammatory cell numbers and functions and on circulating levels of soluble adhesion molecules. Healthy subjects aged 55 to 75 yr consumed nine capsules per day for 12 wk. The capsules contained placebo oil (an 80:20 mix of palm and sunflowerseed oils) or blends of placebo oil with oils rich in ALNA, GLA, ARA, or DHA or FO. Subjects in these groups consumed 2 g ALNA; approximately 700 mg GLA, ARA, or DHA; or 1 g EPA plus DHA (720 mg EPA + 280 mg DHA) daily from the capsules. Total fat intake from the capsules was 4 g per day. None of the treatments affected inflammatory cell numbers in the bloodstream; neutrophil and monocyte phagocytosis or respiratory burst in response to E. coli; production of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 in response to bacterial lipopolysaccharide; or plasma concentrations of soluble intercellular adhesion molecule-1. In contrast, the ALNA and FO treatments decreased the plasma concentrations of soluble vascular cell adhesion molecule-1 (16 and 28% decrease, respectively) and soluble E-selectin (23 and 17% decrease, respectively). It is concluded that, in contrast to previous reports using higher amounts of these fatty acids, a moderate increase in consumption of long-chain n-6 or n-3 polyunsaturated fatty acids does not significantly affect inflammatory cell numbers or neutrophil and monocyte responses in humans and so would not be expected to cause immune impairment. Furthermore, we conclude that moderate levels of ALNA and FO, which could be incorporated into the diet, can decrease some markers of endothelial activation and that this mechanism of action may contribute to the reported health benefits of n-3 fatty acids.

Animal and human studies have shown that greatly increasing the amounts of flax seed oil [rich in the (n-3) polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALNA)] or fish oil [FO; rich in the long chain (n-3) PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in the diet can decrease mitogen-stimulated lymphocyte proliferation. The objective of this study was to determine the effect of dietary supplementation with moderate levels of ALNA, gamma-linolenic acid (GLA), arachidonic acid (ARA), DHA or FO on the proliferation of mitogen-stimulated human peripheral blood mononuclear cells (PBMC) and on the production of cytokines by those cells. The study was randomized, placebo-controlled, double-blinded and parallel. Healthy subjects ages 55-75 y consumed nine capsules/d for 12 wk; the capsules contained placebo oil (an 80:20 mix of palm and sunflower seed oils) or blends of placebo oil with oils rich in ALNA, GLA, ARA or DHA or FO. Subjects in these groups consumed 2 g of ALNA or 770 mg of GLA or 680 mg of ARA or 720 mg of DHA or 1 g of EPA plus DHA (720 mg of EPA + 280 mg of DHA) daily from the capsules. Total fat intake from the capsules was 4 g/d. The fatty acid composition of PBMC phospholipids was significantly changed in the GLA, ARA, DHA and FO groups. Lymphocyte proliferation was not significantly affected by the placebo, ALNA, ARA or DHA treatments. GLA and FO caused a significant decrease (up to 65%) in lymphocyte proliferation. This decrease was partly reversed by 4 wk after stopping the supplementation. None of the treatments affected the production of interleukin-2 or interferon-gamma by PBMC and none of the treatments affected the number or proportion of T or B lymphocytes, helper or cytotoxic T lymphocytes or memory helper T lymphocytes in the circulation. We conclude that a moderate level GLA or EPA but not of other (n-6) or (n-3) PUFA can decrease lymphocyte proliferation but not production of interleukin-2 or interferon-gamma.

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Weak-acid preservatives, such as sorbic acid and acetic acid, are used in many low pH foods to prevent spoilage by fungi. The spoilage yeast Zygosaccharomyces bailii is notorious for its extreme resistance to preservatives and ability to grow in excess of legally-permitted concentrations of preservatives. Extreme resistance was confirmed in 38 strains of Z. bailii to several weak-acid preservatives. Using the brewing yeast Saccharomyces cerevisiae as a control, tests showed that Z. bailii was ~ 3-fold more resistant to a variety of weak-acids but was not more resistant to alcohols, aldehydes, esters, ethers, ketones, or hydrophilic chelating acids. The weak acids were chemically very diverse in structure, making it improbable that the universal resistance was caused by degradation or metabolism. Examination of Z. bailii cell populations showed that extreme resistance to sorbic acid, benzoic acid and acetic acid was limited to a few cells within the population, numbers decreasing with concentration of weak acid to < 1 in 1000. Re-inoculation of resistant sub-populations into weak-acid-containing media showed that all cells now possessed extreme resistance. Resistant sub-populations grown in any weak-acid preservative also showed ~ 100% cross-resistance to other weak-acid preservatives. Tests using 14C-acetic acid showed that weak-acid accumulation was much lower in the resistant sub-populations. Acid accumulation is caused by acid dissociation in the higher pH of the cytoplasm. Tests on intracellular pH (pHi) in the resistant sub-population showed that the pH was much lower, ~ pH 5.6, than in the sensitive bulk population. The hypothesis is proposed that extreme resistance to weak-acid preservatives in Z. bailii is due to population heterogeneity, with a small proportion of cells having a lower intracellular pH. This reduces the level of accumulation of any weak acid in the cytoplasm, thus conferring resistance to all weak acids, but not to other inhibitors.

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