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Journal of Microbiological Methods

F A Silva Sergent, B Acosta-Hernández, J R Jaber, I Rosario Medina, F Real Valcárcel, D Padilla Castillo, S Déniz Suárez
This study evaluated the effect of skimmed milk as a cryoprotectant for the maintenance and long-term preservation of 70 Malassezia pachydermatis strains. An initial suspension of each strain was prepared in sterile distilled water with skimmed milk. The most effective method was cryopreservation that ensured 100% viability for 2years.
December 2, 2016: Journal of Microbiological Methods
Kjetill Østgaard, Viktoria Kowarz, Wang Shuai, Ingrid A Henry, Michal Sposob, Hildegunn Hegna Haugen, Rune Bakke
Mass produced plastic syringes may be applied as vessels for cheap, simple and large scale batch culture testing. As illustrated for the cases of denitrification and of biogas formation, metabolic activity was monitored by direct reading of the piston movement due to the gas volume formed. Pressure buildup due to friction was shown to be moderate. A piston pull and slide back routine can be applied before recording gas volume to minimize experimental errors due to friction. Inoculum handling and activity may be conveniently standardized as illustrated by applying biofilm carriers...
December 1, 2016: Journal of Microbiological Methods
Jonas D Van Belleghem, Maya Merabishvili, Bjorn Vergauwen, Rob Lavigne, Mario Vaneechoutte
Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography...
November 29, 2016: Journal of Microbiological Methods
Chen Li, Xiurong Ding, Zhizhong Liu, Juanjuan Zhu
The incidence of invasive fungal infections in immunocompromised patients has increased in recent decades. Rapid and accurate identification of these pathogenic fungi is crucial for initiating a timely, safe, and effective antifungal therapy. Here we developed a microarray based on flow-through hybridization gene chip technology. The microarray was tested for its specificity using a panel of reference and blinded clinical isolates. The results proved that this microarray was highly discriminative, leading to the unequivocal identification of each species, including Candida famata and the highly related species Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis...
November 29, 2016: Journal of Microbiological Methods
Danny Frauenstein, Katja Seidel, Lorenz Adrian
Microbial cells vary widely in size, specific density, shearing resistance, oxygen sensitivity and abundance so that differential harvesting and washing procedures are needed to efficiently recover cells from dilute suspensions. We here describe a mild, simple, variable and cost-efficient method to harvest cells on columns packed with silica beads. The method collects and concentrates 40-98% of the cells preserving enzymatic activity and cell viability. The method can be applied for strictly anaerobic microorganisms, is scalable to different culture volumes and can be multiplexed in standardized systems...
November 27, 2016: Journal of Microbiological Methods
Eric Jouy, Marisa Haenni, Laëtitia Le Devendec, Aurélie Le Roux, Pierre Châtre, Jean-Yves Madec, Isabelle Kempf
We have developed a phenotypic method suited to the systematic screening of resistance to colistin in E. coli, including those with the mcr-1 gene, by the absence of an inhibition zone after an application of a single drop of 8mg/L colistin solution on a previously inoculated Mueller-Hinton agar.
November 26, 2016: Journal of Microbiological Methods
Ken Graham, Rosemary Rea, Paul Simpson, Helena Stack
Enterococci show higher proteolytic activities than other lactic acid bacteria and thus have received considerable attention in scientific literature in recent years. Proteolytic enzymes of enterococci have warranted the use of some species as starter, adjuncts or protective cultures and as probiotics, while in some strains they have also been linked with virulence. Consequently, the isolation and identification of proteolytic enterococci is becoming of increasing interest and importance. However, current screening methods for proteolytic enterococci can be time consuming, requiring a two-step procedure which may take up to 96h...
November 25, 2016: Journal of Microbiological Methods
M Javed, N Baghaei-Yazdi, W Qin, S Amartey
Geobacillus species have potential applications in many biotechnological processes. They are fastidious in their vitamin and amino acid requirements. A new semi-defined agar medium (SDM) was developed which gave consistently high viable cell counts of various G. thermoglucosidasius strains (5×10(8)-6×10(8)cfu/ml) under aerobic conditions at 70°C.
November 22, 2016: Journal of Microbiological Methods
Jennifer L Hoeflinger, Daniel E Hoeflinger, Michael J Miller
Herein, an open-source method to generate quantitative bacterial growth data from high-throughput microplate assays is described. The bacterial lag time, maximum specific growth rate, doubling time and delta OD are reported. Our method was validated by carbohydrate utilization of lactobacilli, and visual inspection revealed 94% of regressions were deemed excellent.
November 20, 2016: Journal of Microbiological Methods
Lucía Calleros, Laura Betancor, Gregorio Iraola, Alejandra Méndez, Claudia Morsella, Fernando Paolicchi, Silvia Silveyra, Alejandra Velilla, Ruben Pérez
Campylobacter fetus is a Gram-negative, microaerophilic bacterium that infects animals and humans. The subspecies Campylobacter fetus subsp. fetus (Cff) affects a broad range of vertebrate hosts and induces abortion in cows and sheep. Campylobacter fetus subsp. venerealis (Cfv) is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus subsp. testudinum (Cft) has been isolated mostly from apparently healthy reptiles belonging to different species but also from ill snakes and humans...
November 17, 2016: Journal of Microbiological Methods
Arthur H Totten, Li Xiao, Donna M Crabb, Amy E Ratliff, Kevin Dybvig, Ken B Waites, T Prescott Atkinson
BACKGROUND: Mycoplasma pneumoniae (Mpn), one of the smallest self-replicating prokaryotes, is known to readily adhere to host cells and to form aggregates in suspension. Having only one cell membrane and no cell wall, mycoplasmas present questions as to optimal aggregate disruption method while minimizing cell death in vitro. We compared conventional vortex mixing with other methods for disruption of bacterial aggregates and for its effect on cell viability. METHODS: Strain UAB PO1, a clinical Mpn isolate, was dispersed using a conventional vortex mixer with or without nonionic detergent (0...
November 17, 2016: Journal of Microbiological Methods
Kageto Yamada, Machiko Kashiwa, Katsumi Arai, Noriyuki Nagano, Ryoichi Saito
We evaluated the effectiveness of carbapenem inactivation method (CIM) and modified CIM (mCIM). Our results indicated that mCIM with 4h incubation improved sensitivity and specificity for detecting carbapenemase-producing Enterobacteriaceae compared to CIM. Additionally, we developed a sodium mercaptoacetate-combination method (SMA-mCIM) to detect metallo-β-lactamase (MBL) with high sensitivity and specificity.
November 16, 2016: Journal of Microbiological Methods
Farhan Ahmad, Robert D Stedtfeld, Hassan Waseem, Maggie R Williams, Alison M Cupples, James M Tiedje, Syed A Hashsham
We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95°C for 5min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63°C...
November 14, 2016: Journal of Microbiological Methods
Michelle Ma, Tyler A Rice, Caroline M Percopo, Helene F Rosenberg
The silkworm larvae plasma (SLP) assay has been developed as a means to detect bacterial peptidoglycan as a surrogate for live bacteria. Here, we present results that indicate that generation of melanin by this assay is not fully reliable as a surrogate marker for bacterial count.
November 10, 2016: Journal of Microbiological Methods
Bruna Wurr Rodak, Douglas Siqueira Freitas, Soraya Marx Bamberg, Marco Aurélio Carbone Carneiro, Luiz Roberto Guimarães Guilherme
The symbiosis between legumes, arbuscular mycorrhizal (AM) fungi, and N2-fixing bacteria (NFB) provides mutual nutritional gains. However, assessing the nutritional status of the microorganisms is a difficult task. A methodology that could assess this status, in situ, could assist managing these organisms in agriculture. This study used X-ray microanalyses to quantify and locate mineral elements in structures formed in a tripartite symbiosis. Lima bean (Phaseolus lunatus L. Walp) was cultivated in pots under greenhouse conditions, to which we have added AM fungal isolates (Glomus macrocarpum and Acaulospora colombiana) and NFB (Bradyrhizobium japonicum) inocula...
November 9, 2016: Journal of Microbiological Methods
Eva Theres Gensberger, Tanja Kostić
Labeling of bacteria with marker genes, such as green fluorescent protein, is a useful and practicable tool for tracking and enumerating bacterial cells in a complex environment e.g. discrimination from the indigenous background population. In this study, novel TurboGFP prokaryotic expression vector was utilized for labeling of Yersinia species. Y. enterocolitica biovar 1A, biovar 2, biovar 4 and Y. pseudotuberculosis were successfully transformed with the vector and expressed bright green fluorescence that was even detectable visually by eye...
November 9, 2016: Journal of Microbiological Methods
Donna Hockman, Ming Dong, Hong Zheng, Sanjai Kumar, Matthew D Huff, Elena Grigorenko, Maureen Beanan, Robert Duncan
Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary for the validation of molecular diagnostic devices when a sufficient number of clinical specimens is unobtainable. Examples include rare pathogens, some of which are pathogens posing a biological weapon threat...
November 9, 2016: Journal of Microbiological Methods
Erich Heidenreich, Ferdinand Steinboeck
Mutations not only arise in proliferating cells but also in resting - thus non-replicating - cells. Such stationary-phase mutations may occasionally enable an escape from growth repression and e.g. contribute to cancerogenesis or development of drug resistance. The most widely used condition for the study of such adaptive mutations in the eukaryotic model organism Saccharomyces cerevisiae is the starvation for a single amino acid. To overcome some limitations of this experimental setup we developed a new adaptive mutation assay that allows a screening for mutagenic processes during a more regular cell cycle arrest induced by the lack of a fermentable carbon source...
November 9, 2016: Journal of Microbiological Methods
Jonathan Hk Chen, Vincent Cc Cheng, Kevin Kk She, Wing-Cheong Yam, Kwok-Yung Yuen
Species differentiation of nontuberculous mycobacteria (NTM) has long been a difficult task in clinical laboratories. This study demonstrated and evaluated a simple and cost-effective method using the real-time PCR with high-resolution melting (PCR-HRM) analysis technique, which could differentiate at least 14 different medically related NTM.
November 9, 2016: Journal of Microbiological Methods
Lydia-Marie Joubert, Jose Ag Ferreira, David A Stevens, Hasan Nazik, Lynette Cegelski
Aspergillus fumigatus biofilms consist of a three-dimensional network of cellular hyphae and extracellular matrix. They are involved in infections of immune-compromised individuals, particularly those with cystic fibrosis. These structures are associated with persistence of infection, resistance to host immunity, and antimicrobial resistance. Thorough understanding of structure and function is imperative in the design of therapeutic drugs. Optimization of processing parameters, including aldehyde fixation, heavy metal contrasting, drying techniques and Ionic Liquid treatment, was undertaken for an ultrastructural approach to understand cellular and extracellular biofilm components...
November 9, 2016: Journal of Microbiological Methods
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