Differential Staining of Bacteria: Endospore Stain

Reynolds, Jackie; Moyes, Rita B.; Breakwell, Donald P.

doi:10.1002/9780471729259.mca03js15

Cited by 17 publications

(10 citation statements)

References 1 publication

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“…To visualize the spores, colonies of the parent and sigK mutant strains were grown on TPGY plates for 7 days and stained on a glass slide with malachite green and safranin (28).…”

Section: Methodsmentioning

confidence: 99%

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

Kirk

Dahlsten

Zhang

et al. 2012

Appl Environ Microbiol

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ABSTRACTA key survival mechanism ofClostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organismBacillus subtilis, nothing is known about these mechanisms inC. botulinum.Using the ClosTron gene-knockout tool,sigK, encoding late-stage (stage IV) sporulation sigma factor K inB. subtilis, was disrupted inC. botulinumATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast,sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis ofsigKin the parent strain revealed expression at the late log growth phase in the parent strain. Analysis ofspo0A, encoding the sporulation master switch, in thesigKmutant and the parent showed significantly reduced relative levels ofspo0Aexpression in thesigKmutant compared to the parent strain. Similarly,sigFshowed significantly lower relative transcription levels in thesigKmutant than the parent strain, suggesting that the sporulation pathway was blocked in thesigKmutant at an early stage. We conclude that σKis essential for early-stage sporulation inC. botulinumATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organismB. subtilis. Understanding the sporulation mechanism ofC. botulinumprovides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

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“…To visualize the spores, colonies of the parent and sigK mutant strains were grown on TPGY plates for 7 days and stained on a glass slide with malachite green and safranin (28).…”

Section: Methodsmentioning

confidence: 99%

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

Kirk

Dahlsten

Zhang

et al. 2012

Appl Environ Microbiol

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“…To image spores in both spore-positive control and nanoparticle-treated cultures, optical microscopy with the differential staining method was used according to an established method. 27 Once cells were harvested by centrifugation at 750 × g for 10 minutes and resuspended in ultrapure water, a 10 μL aliquot was placed on a glass slide and allowed to air dry. B. subtilis on the microscope slide was then heat fixed and covered with a piece of filter paper flooded with 5% malachite green, and placed above boiling water for 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

Biological impact of nanoscale lithium intercalating complex metal oxides to model bacteriumB. subtilis

Feng

Miller

Linn

et al. 2019

Environ. Sci.: Nano

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The wide applications of lithium intercalating complex metal oxides in energy storage devices call for a better understanding of their environmental impact at the end of their life cycle. In this study, we examine the biological impact of a panel of nanoscale lithium nickel manganese cobalt oxides (LixNiyMnzCo1−y−zO2, 0 < x, y, z < 1, abbreviated to NMCs) to a model Gram-positive bacterium, Bacillus subtilis, in terms of cellular respiration and growth. A highly sensitive single-cell gel electrophoresis method is also applied for the first time to understand the genotoxicity of these nanomaterials to bacterial cells. Results from these assays indicate that the free Ni and Co ions released from the incongruent dissolution of the NMC material in B. subtilis growth medium induced both hindered growth and cellular respiration. More remarkably, the DNA damage induced by the combination of the two ions in solution is comparable to that induced by the NMC material, which suggests that the free Ni and Co ions are responsible for the toxicity observed. A material redesign by enriching Mn is also presented. The combined approaches of evaluating their impact on bacterial growth, respiration, and DNA damage at a single-cell level, as well as other phenotypical changes allows us to probe the nanomaterials and bacterial cells from a mechanistic prospective, and provides a useful means to an understanding of bacterial response to new potential environmental stressors.

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“…For confirmation of bacterial presence and identity, we used standard staining including Gram and acid-fast staining [ 33 , 34 ]. To prepare the bacteria culture slides, a volume of 1 mL of bacterial culture in 1.5 mL microcentrifuge tube was centrifuged at 13,000 RPMs for 2.5 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

et al. 2018

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BackgroundSeveral pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn’s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82.MethodsThe multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM).ResultsFollowing several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel.ConclusionWe developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease.

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Differential Staining of Bacteria: Endospore Stain

Cited by 17 publications

References 1 publication

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

Biological impact of nanoscale lithium intercalating complex metal oxides to model bacteriumB. subtilis

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

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