Crystalline Reddishi Orange Pigment of Manufactured Black Tea

The author has carried out studies concerning the effects of different conditions on the gamma-ray resistivity of E. coli. This report deals with the influence of the heating procedure applied to irradiated samples after irradiation, the gamma-ray resistivities of strains stocked for long periods, and the influences of the addition of heat sterilized cells of the same strain and chemicals on mediums.     

Effect of Gamma-ray upon Food Microorganisms

The author has carried out a series of studies on the effects of various conditions on the survival of E. coli irradiated with cobalt-60 gamma-ray. This report deals with the survivals of the strain irradiated in the medium containing each of the components of various types of fish-meats and meats.     

Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author     

Identification of mutable slender glume gene in rice (Oryza sativa L.).

The segregation pattern and chromosomal location of a slender glume mutation, induced by gamma-ray irradiation, was investigated. The mutation is genetically unstable: in the selfed progenies of slender glumed plants, not only plants with normal glumes but also plants that are chimeric for glume shape almost always appear at low frequency. The results showed that the mutation is controlled by a single recessive, mutable mutant gene slg. The frequency of reversion of slg to its wild-type state was little affected by crossing, backcrossing, genetic background or cytoplasmic factors. Conventional trisomic and linkage analyses revealed that the slg locus was located close to the rfs (rolled fine stripe leaf) locus on chromosome 7. In a subsequent RFLP analysis, slg was found to be located between the two RFLP loci XNpb20 and XNpb33, with recombination values of 3.0 and 3.2%, respectively. Southern analysis indicated that the mutability of slg is caused by none of the known transposable elements in rice. From these results, we infer that slg has a novel transposable DNA insert in its vicinity, which was possibly activated by gamma-ray irradiation. Received: 28 September 1998 / Accepted: 18 December 1998     

A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation

Summary A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the -galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.     

Towards a one-step Enterobacter sakazakii enrichment

Aims: The current international standard method for detection of Enterobacter sakazakii from milk products is by the International Organization for Standardization and the International Dairy Federation documented method, a procedure involving two-step enrichment. This study aimed to assess enrichment of E. sakazakii using a one-step enrichment. Methods and Results: Enrichment of four strains of E. sakazakii was compared using five different media, with stressed or unstressed cells, and at three levels of competing microflora, which were included to assess their effects on the positive isolation of E. sakazakii. Enrichment of milk powders, prepared by spray-drying milk seeded with E. sakazakii, was assessed using one-step enrichment for detection of E. sakazakii, followed by confirmation of positive isolates by real-time PCR. Conclusions: Current media are unsuitable for enrichment and detection of all E. sakazakii isolates, in particular, when high levels of background microflora are present in the sample matrix, and new defined media are needed for successful one-step enrichment. Significance and Impact of the Study: These findings provide further analysis of one-step enrichment processes for E. sakazakii in the presence of competing microflora, and show that further formulation is needed for a universal E. sakazakii enrichment medium, with careful selection of both nutrients and selective agents.     

浒苔遥感监测研究进展

浒苔大规模集聚形成的绿潮灾害是海洋生态系统主要生态环境问题之一,基于卫星遥感影像监测浒苔及其扩展动态已成为一种及时有效的手段。对国内外浒苔遥感监测方面文献进行归纳整理,认为光学遥感数据、多波段比值法是最常用的遥感数据和监测方法。对遥感监测浒苔机理进行了阐述,并对分类方法进行评价认为监督分类法解译精度不高。目前单波段阈值法和多波段比值法应用广泛,但在监测漂浮浒苔和混合象元解译存在不足。辐射传输模型法能有效提高信息解译的精度,但还处于起步阶段。遥感监测浒苔灾害的未来发展需要提高影像空间分辨率,深入研究监测方法,进行多种平台和多源遥感数据相结合,并由定性走向定量,从而建立健全遥感监测预警系统。     

Levofloxacin susceptibility testing for Helicobacter pylori in China: comparison of E-test and disk diffusion method

Background: The aims of this study were to compare disk diffusion with E‐test method for levofloxacin susceptibility testing of Helicobacter pylori and standardized breakpoints for disk diffusion as a stable and reliable method for determining qualitative levofloxacin susceptibility. Materials and Methods: We determined the levofloxacin susceptibility of 45 H. pylori strains isolated from Chinese patients by the E‐test method. Disk diffusion was evaluated as an alternative method to determine susceptibility and compared with the E‐test results by linear regression analysis. Results: The minimum inhibitory concentration (MIC) values tested by E‐test method ranged from 0.047 to 32 μg/mL. Resistance to levofloxacin was detected in 16 (35.6%) isolates. The levofloxacin disk zone sizes obtained by disk diffusion method correlated well (r² = .877) with the MICs obtained by E‐test method. As a consequence of regression analysis, isolates with inhibition diameters <12 mm were considered resistant to levofloxacin. There was 100% agreement between the two methods for levofloxacin, applying the regression‐based breakpoints. Conclusions: The disk diffusion method is equivalent to the E‐test method for testing levofloxacin susceptibility of H. pylori strains; it is more practical and inexpensive, and it is suitable for the analysis of a small number of isolates compared with the E‐test method.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Preparation of Four Geometric Isomers of (11E)-4,6,11-Hexadecatrienal and Their Effect toward Male Eri-Silk Moths

Four geometric isomers of (11E)-4,6,11-hexadecatrienal were prepared, and their pheromone activity towards male eri-silk moths was evaluated. The EAG activity of each isomer was determined by the EAG-GLC method in order of increasing activity to be (4Z,6E,11E)- and (4E,6E,11E)?? (4E,6Z,11E) ?? (4Z,6Z,11E)-hexadecatrienal.     

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.     

Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization,direct viable counting and solid phase cytometry

Aims: We developed an improved Fluorescent In Situ Hybridization FISH‐based method to detect viable Escherichia coli cells by solid phase cytometry (SPC), and results were compared to those obtained by the standard culture method. Methods and Results: The method includes a direct viable count (DVC) assay, multi‐probes labelled and unlabelled (helpers) to detect specifically viable E. coli cells and to enhance SPC cell counts. We demonstrate that helpers increase the fluorescence intensity of hybridized E. coli cells as detected by SPC and assess the high specificity of the DVC–FISH procedure on a large panel of cultured strains. Application to seawater, freshwater and wastewater samples showed a good correlation between SPC cells counts and standard plate counts. Conclusion: The high specificity of the procedure was demonstrated as well as its accuracy for detecting and counting viable E. coli cells in environmental samples. Significance and Impact of the Study: The developed approach may be used to monitor faecal contamination sources and to investigate the occurrence of viable E. coli in natural environments.     

Evaluation of Petrifilm™ EC method for enumeration of E. coli from soil

Aims: To evaluate the suitability of commercially available Petrifilm? EC plates for enumeration of Escherichia coli from soil. Methods and Results: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10², 10³, 10⁵ CFU g^?1 of soil. The efficiency of recovery on Petrifilm? EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m‐FC basal medium supplemented with 3‐bromo‐4‐chloro‐5‐indoyl‐β‐d ‐glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4‐methylumbelliferyl‐β‐d ‐glucuronide (EC‐MUG) broth. Petrifilm? EC and m‐FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between Petrifilm? EC, m‐FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure‐applied field soil samples showed a significant difference (P < 0·05) between the Petrifilm? EC method and the m‐FC method in enumerating E. coli possibly as a result of false positives on m‐FC. Conclusion: The Petrifilm? EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g^?1 soil. Significance and Impact of the Study: The commercially available Petrifilm? EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.     

Genetic diversity of chromosomal metallo-β-lactamase genes in clinical isolates of <Emphasis Type="Italic">Elizabethkingia meningoseptica</Emphasis> from Korea

This study was performed to characterize the chromosomal metallo-β-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5α. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla _GOB genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla _GOB gene, including 10 novel variants (bla _GOB-8 to bla _GOB-17). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5α. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

Methods available for the detection of Escherichia coli O157 in clinical,food and environmental samples

Because of the potential severity of infections caused by Escherichia coli O157 it is important that the most sensitive laboratory methods are used both for outbreak investigation and surveillance. Selective culture of E. coli O157 remains the detection method of choice, particularly in investigation of outbreaks where strains isolated from various sources may need to be compared by various typing methods. Strains of E. coli O157 do not normally ferment sorbitol, whereas many other serogroups of E. coli do, and sorbitol MacConkey agar, or modified forms of this medium, have become widely used for their isolation. Detection of small numbers of E. coli O157 may be facilitated by enrichment culture which may include a recovery period during which selective agents are not added to the medium. Immunomagnetic separation of E. coli O157 after enrichment culture enhances sensitivity still further and has the potential to be fully automated. Alternatives to culture include immunoassays and PCR, both of which are available as commercial detection kits. The last 15 years has seen many advances in detection of E. coli O157 and has been accompanied by a plethora of reports in the scientific literature. However, it is an area which is continually developing and we are still far away from a universally accepted method for this purpose. This revised version was published online in November 2006 with corrections to the Cover Date.     

The chromosomes of some North American chipmunks (sciuridae) belonging to the genera tamias and eutamias

Summary The somatic chromosomes of T. striatus lysteri, E. minimus operarius, E. speciosus frater, E. quadrivittaius hopiensis and E. umbrinus montanus have been analyzed utilizing a method employing pretreatment with colchicine and hypotonic citrate followed by staining and squashing.Each animal had a modal diploid chromosome number of 38.A preliminary chromosome classification in chipmunks, based on chromosome size and centromere position, has been proposed. Utilizing this classification, karyograms of T. striatus lysteri, E. minimus operarius, E. quadrivittatus hopiensis and E. umbrinus montanus were analyzed. Karyograms of E. umbrinus montanus and E. quadrivittatus hopiensis were similar and differed from those of E. minimus operarius and from those of T. striatus lysteri. Differences in karyograms were distinct; however, a very close relationship was noted between species as well as between genera.X and Y chromosomes were identified in all animals.Utilization of karyograms for the purpose of evaluating possible evolutionary pathways and as an aid to systematic zoology is discussed.This work was supported by grants from the Leukemia Society, Inc., and the American Cancer Society.Trainee in Hematology, U.S. Public Health Service.     

Isolation and PCR detection of <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis> in South African food products,specifically infant formula milks

Enterobacter sakazakii has recently been identified as an opportunistic pathogen. The current culture-dependent detection methods for these bacteria are time-consuming and in this study a PCR method for the detection of E. sakazakii in South African food products, including an internal amplification control (IAC) was developed. DNA was isolated and amplified from the products and they were plated on selective growth media after pre-enrichment and enrichment in Enterobacteriaceae enrichment broth. Four of the 22 products tested positive for the presence of E. sakazakii, confirmed by PCR detection and growth on selective media. The PCR method proved effective in detecting E. sakazakii in South African products after three days and could serve as an alternative for traditional microbiological techniques.