Application of the novel fluorescent dye Beljian red to the differentiation of Giardia cysts

Beljian red (BR) is a novel long Stokes shift fluorescent dye that fluoresces orange when illuminated with UV or blue light. Due to its long Stokes shift, and the fact that it is excitable at 488 nm, BR has particular utility in multi-colour applications with short Stokes shift fluorophores such as fluorescein. Here we have demonstrated that BR can be used to discriminate Giardia cysts seeded into water samples from those naturally present in the sample. We show that the dye does not interfere with other staining methods such as DAPI, and is compatible with mAb-FITC staining in a multi-colour fluorescence technique. This should be useful in determining the specific recovery of protozoan parasites from environmental samples.     

Inducible gene expression by nonculturable bacteria in milk after pasteurization

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Plasmid R46-mediated protection against bleomycin is poLA+-dependent

Strains of Escherichia coli deficient in post-replication recombination repair were more sensitive to bleomycin than wild-type, repair-proficient strains. Mutants lacking excision repair functions were no more sensitive to bleomycin than the wild-type strains, indicating that this pathway is not involved in the repair of bleomycin-damaged DNA. Plasmid R46 not only protected repair-proficient strains but also those with recB, recC, uvrA or lig genotypes, suggesting that R46 protection against bleomycin is independent of these host repair functions. However, R46 protection was abolished in recA or polA strains, indicating that these gene functions are necessary for plasmid-mediated protection. It is suggested that protection may be due to a recA+-dependent interaction of a plasmid-encoded product with host DNA polymerase I, resulting in an increase in the DNA repair capacity of cells.     

A flow cytometry method for rapid detection and enumeration of total bacteria in milk

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (/= 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (P < 0.01) and a good agreement (r = 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was 相似文献   

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.     

Quantification of the magnification and distortion effects of a pediatric flexible video-bronchoscope

Background

Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.

Methods

Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.

Results

Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.

Conclusion

We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements.     

Comparison of fermentative capacities of industrial baking and wild-type yeasts of the species Saccharomyces cerevisiae in different sugar media

AIMS: To compare the fermentative capacity of wild and domesticated isolates of the genus Saccharomyces. METHODS AND RESULTS: The fermentative capacity of yeasts from a variety of wild and domesticated sources was tested in synthetic dough media that mimic major bread dough types. Domesticated yeast strains were found to have better maltose-utilizing capacity than wild yeast strains. The capacity to ferment sugars under high osmotic stress was randomly distributed amongst wild and baking strains of Saccharomyces. CONCLUSION: The domestication of bakers' yeast has enhanced the ability of yeasts to ferment maltose, without a similar impact on the fermentative capacity under high osmotic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, combined with molecular studies of both wild and domesticated yeast, showed that domestication of bakers' yeast has resulted in improved maltose utilization, apparently via the duplication and mutation of the MAL genes.     

Diversity of natural enemies in Central Otago,New Zealand apple orchards: a practical measure of sustainability in pest management?

The diversity of the arthropod fauna in apple orchards in Central Otago was recorded from 1994 to 1999 using beating trays, pitfall traps, and sticky traps and the data analysed by the Shannon–Wiener Index. Three different fruit production systems were compared, conventional (CFP), integrated (IFP), and biological (BFP), to determine whether total arthropod diversity and/or the diversity of natural enemies (predators and parasitoids) could be used to measure the sustainability of their pest management practices. The contribution of natural enemies to the diversity indices was also examined to determine if they reflected the key species essential for sustainable pest management. The diversity of natural enemies in beating trays was found to be the measure most sensitive to changes in pest management. The diversity of both the total arthropod fauna and natural enemies was much lower under CFP than under either IFP or BFP, due to the use of broad-spectrum pesticides. This practice made the CFP programme unsustainable and it is no longer used. During the transition from CFP to IFP, the diversity indices rose to become similar to that in the BFP programme by the third season. The introduction of frequent applications of fungicides in BFP, including lime sulphur, appeared to reduce total arthropod and natural enemy diversity. Falls in the diversity indices for natural enemies were shown to reflect reduced numbers, range, and evenness of natural enemies but not necessarily the key species known to be critical for sustainable pest management. It is proposed that a Shannon–Wiener Index for natural enemies in beating trays of 0.2 or less in summer is strongly indicative of unsustainable pest management in Otago apple orchards. On the other hand, indices of 0.3–0.8, as found under IFP and BFP, do not give unequivocal or quantitative indications of the sustainability of pest management. A suite of other measures of sustainability are more useful, notably combining lower pesticide use, reduced pest damage and presence at harvest, the greater roles of key natural enemies, more effective plant resistance to pests and diseases, and higher profitability.