scholarly journals Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

2003 ◽  
Vol 69 (10) ◽  
pp. 5787-5792 ◽  
Author(s):  
Raphael Ber ◽  
Emanuelle Mamroud ◽  
Moshe Aftalion ◽  
Avital Tidhar ◽  
David Gur ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Quantitative Evaluation ◽  
Fluorescent Protein ◽  
Agar Medium ◽  
Brain Heart Infusion ◽  
Selective Medium ◽  
Bacterial Populations ◽  
Medium Components ◽  
Selective Agar ◽  
Green Fluorescent

ABSTRACT Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.

scholarly journals Systemic Colonization of Potato Plants by a Soilborne, Green Fluorescent Protein-Tagged Strain of Dickeya sp. Biovar 3

2010 ◽  
Vol 100 (2) ◽  
pp. 134-142 ◽  
Author(s):  
Robert Czajkowski ◽  
Waldo J. de Boer ◽  
Henk Velvis ◽  
Jan M. van der Wolf
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Agar Medium ◽  
Lateral Roots ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Potato Plants ◽  
Soil Inoculation ◽  
Green Fluorescent

Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

A Simple Medium for Isolation of Coagulase-Positive Staphylococci in a Single Step

1982 ◽  
Vol 45 (3) ◽  
pp. 218-222 ◽  
Author(s):  
LEONIE MINTZER-MORGENSTERN ◽  
ELIY AHU KATZENELSON
Keyword(s):  
Agar Medium ◽  
Brain Heart Infusion ◽  
Egg Yolk ◽  
Food Samples ◽  
Selective Medium ◽  
Single Step ◽  
Isolation And Identification ◽  
Complete Reversal ◽  
Simple Medium ◽  
Coagulase Positive Staphylococci

An agar medium containing NaCl, egg yolk and tellurite for selective quantitative isolation of coagulase-positive staphylococci from food was developed. Isolation and identification of the staphylococci was achieved in a single step. A properly diluted food sample was spread over the medium and incubated for 24 h at 42 C. Coagulase-positive staphylococci appeared as small grey to dark-grey colonies surrounded by a dense white opacity. Coagulase-negative bacteria which, at times, grow on this medium, did not produce this reaction. The identification on this selective medium of isolates from 683 different food samples as coagulase-positive staphylococci was subsequently confirmed by the coagulase test. Comparative titrations of 29 various coagulase-positive staphylococcus strains on both the selective medium and nutrient agar yielded nearly identical titers. The growth of heat-stressed staphylococci was inhibited by the selective medium. Complete reversal of the inhibition was achieved by a 3-h pre-incubation in brain heart infusion at 37 C.

scholarly journals Pathogenesis of Yersinia pestis Infection in BALB/c Mice: Effects on Host Macrophages and Neutrophils

2005 ◽  
Vol 73 (11) ◽  
pp. 7142-7150 ◽  
Author(s):  
Roman A. Lukaszewski ◽  
Dermot J. Kenny ◽  
Rosa Taylor ◽  
D. G. Cerys Rees ◽  
M. Gill Hartley ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Fluorescent Protein ◽  
Virulent Strain ◽  
Late Phase ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Intracellular Bacteria ◽  
Necrosis Factor Alpha ◽  
Green Fluorescent

ABSTRACT The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 103 to 104 CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.

Heat Treatments To Enhance the Safety of Mung Bean Seeds

2004 ◽  
Vol 67 (6) ◽  
pp. 1257-1260 ◽  
Author(s):  
HAIJING HU ◽  
JOHN J. CHUREY ◽  
RANDY W. WOROBO
Keyword(s):  
Heat Treatment ◽  
Seed Germination ◽  
Fluorescent Protein ◽  
Germination Rate ◽  
Heat Treatments ◽  
Green Fluorescent Protein Gene ◽  
Bacterial Populations ◽  
Average Growth ◽  
E Coli ◽  
Green Fluorescent

Salmonella enterica serovars and Escherichia coli O157:H7 have been associated with contaminated seed sprout outbreaks. The majority of these outbreaks have been traced to sprout seeds contaminated with low levels of pathogens. E. coli O157: H7 strains can grow an average of 2.3 log CFU/g over 2 days during seed germination, and Salmonella can achieve an average growth of 3.7 log CFU/g. Therefore, it is important to find an effective method to reduce possible pathogenic bacterial populations on the seeds prior to sprouting. Our objective was to assess the effectiveness of various dry heat treatments on reducing E. coli O157:H7 and Salmonella populations on mung beans intended for sprout production and to determine the effect of these treatments on seed germination. Mung beans were inoculated with five-strain cocktails of E. coli O157:H7 and of Salmonella serovars harboring the green fluorescent protein gene and then air dried overnight. Heat treatments were performed by incubating the seeds at 55°C for various periods of time. Heat-treated seeds were then assessed for the efficacy of the heat treatment and the effects of heat treatment on germination rates. After inoculation and drying, 6 log CFU/g E. coli O157:H7 and 4 log CFU/g Salmonella were detected on the seeds. Following heat treatment, pathogenic bacterial populations on the seeds were below detectable levels (<1 log CFU/g), but the germination rate of the seed was not affected. Thus, the risk of contamination and the presence of pathogens in the finished sprouts were greatly reduced via the seed heat treatment process.

On the relation of colony variants to the time dependency of colony formation during adaptive mutation of Escherichia coli FC40

1997 ◽  
Vol 43 (9) ◽  
pp. 884-886 ◽  
Author(s):  
Patricia R. MacLeod ◽  
Robert A. MacLeod
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Pure Culture ◽  
Colony Formation ◽  
Agar Medium ◽  
Selective Medium ◽  
Adaptive Mutation ◽  
Time Dependency ◽  
Colony Type ◽  
Selective Agar

When Escherichia coli FC40 formed adaptive Lac+ revenants on a selective agar medium containing lactose as the carbon source, the colonies which accumulated over several days were of two readily distinguishable types. Colonies of both types appeared both early and late on the plates. Cells of colonies that appeared early and late on the plates, irrespective of the type, when grown in liquid medium and replated, all formed colonies on the selective medium within 48 h. Cells of each colony type gave rise to colonies of both types and attempts to isolate cells of each type in pure culture were unsuccessful. It was concluded that the presence of two colony types in the cultures plated did not contribute to the observed time dependency of colony formation during adaptive mutation. The proportions of the two colony types arising from cultures of the Lac+ revertants varied from culture to culture.Key words: Escherichia coli FC40, variants, adaptive mutation, colony accumulation.

scholarly journals A Semi-Selective agar medium to detect the presence of Xanthomonas axonopodis pv. malvacearum in naturally infected cotton seed

2005 ◽  
Vol 30 (5) ◽  
pp. 489-496 ◽  
Author(s):  
Yeshwant R Mehta ◽  
Cleide Bomfeti ◽  
Viviani Bolognini
Keyword(s):  
Cotton Seed ◽  
Agar Medium ◽  
Selective Medium ◽  
Detection Methods ◽  
Basic Medium ◽  
Infected Leaf ◽  
Xanthomonas Axonopodis ◽  
Selective Agar ◽  
Leaf Tissues ◽  
Final Composition

A semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam) in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selective medium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibiotics in different concentrations were tested alone or in combination with others. The final composition of the semi-selective agar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes and favour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid. The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on the semi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selective medium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was not always correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detect the presence of Xam in naturally infected cotton seed.

scholarly journals Yersinia pestis Can Reside in Autophagosomes and Avoid Xenophagy in Murine Macrophages by Preventing Vacuole Acidification

2009 ◽  
Vol 77 (6) ◽  
pp. 2251-2261 ◽  
Author(s):  
Céline Pujol ◽  
Kathryn A. Klein ◽  
Galina A. Romanov ◽  
Lance E. Palmer ◽  
Carol Cirota ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Fluorescent Protein ◽  
Viable Count ◽  
Activated Macrophages ◽  
Intracellular Replication ◽  
Microscopic Imaging ◽  
Murine Macrophages ◽  
Late Endosomes ◽  
Autophagic Process ◽  
Green Fluorescent

ABSTRACT Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstrated that Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to investigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages. Some YCVs contained double membranes characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that ∼40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (∼30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. We conclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification.

scholarly journals Engineering of vectors essential to derive chimeric proteins based on superfolder green fluorescent protein and harpins

2020 ◽  
Author(s):  
L. Tchebotarev ◽  
L. Valentovich
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Evaluation ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Chimeric Proteins ◽  
Qualitative And Quantitative ◽  
Enhanced Solubility ◽  
Green Fluorescent

Conjugation of harpins with green fluorescent protein is aimed at achieving enhanced solubility and stability of chimeric protein, facilitating qualitative and quantitative evaluation of its expression in the routine experiments.

scholarly journals Study on the isolation and identification of Pasteurella species from farm animal and human

2008 ◽  
Vol 2 (1) ◽  
pp. 81-90
Author(s):  
Sawsan salman Atia mubarak ◽  
Ismail khatham Shubar ◽  
Amar salim Rahiem
Keyword(s):  
Pasteurella Multocida ◽  
Agar Medium ◽  
Brain Heart Infusion ◽  
Farm Animal ◽  
Brain Heart Infusion Broth ◽  
Laboratory Animals ◽  
Isolation And Identification ◽  
Selective Agar

This study was interested in isolation and identification of Pasteuralla species in animals and human , also studing it's sensitivity to antibiotic with comparison to it's pathogenicity for laboratory animals . Tow groups of samples were collected and investigeted, 1st group consisting of one handered thirty six samples which were collected from animals ( cow .sheep , goats , and chikens ) . 2nd group was also consisting of 136 samples that were colleeted from human ( wound , urine , and sputum ) . These samples were cultured in selective enrichement brain heart infusion broth and then in the new Pasteurella multocida selective agar medium .

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