scholarly journals Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores

2022 ◽  
Author(s):  
Wen Jie Wu ◽  
Jinhui Chang
Keyword(s):  
Phase Contrast ◽  
Dipicolinic Acid ◽  
Half Time ◽  
Bacillus Atrophaeus ◽  
Germination Process ◽  
Germination Speed ◽  
Contrast Microscopy ◽  
Oxygen Sensitive ◽  
Effect Of Oxygen ◽  
Fundamental Mechanism

AbstractThe effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

scholarly journals A Polysaccharide Deacetylase Gene (pdaA) Is Required for Germination and for Production of Muramic δ-Lactam Residues in the Spore Cortex of Bacillus subtilis

2002 ◽  
Vol 184 (21) ◽  
pp. 6007-6015 ◽  
Author(s):  
Tatsuya Fukushima ◽  
Hiroki Yamamoto ◽  
Abdelmadjid Atrih ◽  
Simon J. Foster ◽  
Junichi Sekiguchi
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Northern Hybridization ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Contrast Microscopy ◽  
Acid Release

ABSTRACT The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. β-Galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by EσG RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of l-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic δ-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic δ-lactam is discussed.

DEVELOPMENT OF FINE STRUCTURE, THERMOSTABILITY, AND DIPICOLINATE DURING SPOROGENESIS IN A BACILLUS

1960 ◽  
Vol 6 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Tadayo Hashimoto ◽  
Samuel H. Black ◽  
Philipp Gerhardt
Keyword(s):  
Heat Resistance ◽  
Phase Contrast ◽  
Dipicolinic Acid ◽  
Maximum Level ◽  
Threshold Level ◽  
Acid Synthesis ◽  
Phase Contrast Microscopy ◽  
Outer Coat ◽  
Contrast Microscopy ◽  
Functional Features

A correlative study was made of some structural and functional features of a reasonably synchronized culture of Bacillus cereus strain terminalis during spore formation and maturation. The successive stages of development could be recognized by phase contrast microscopy and in electron micrographs of ultra-thin-sectioned cells. Attempts were made to correlate these changes with the acquisition of heat resistance and the synthesis of dipicolinic acid. The outer coat of the spores was observed to be formed first around the forespore; the exosporium, cortex, and inner coat then appeared sequentially and independently of existing sporangial membranes. Dipicolinic acid synthesis began in the early transitional stage, just after forespore formation, and reached one third of the maximum level before an increase of heat resistance in the population was detectable, indicating the possibility of a correlation only above a threshold level of the compound.

scholarly journals Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation

2003 ◽  
Vol 185 (17) ◽  
pp. 5306-5309 ◽  
Author(s):  
Samuel Amiteye ◽  
Kazuo Kobayashi ◽  
Daisuke Imamura ◽  
Shigeo Hosoya ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Dipicolinic Acid ◽  
Diacylglycerol Kinase ◽  
Phase Contrast Microscopy ◽  
Electron Microscopic ◽  
Contrast Microscopy ◽  
Microscopic Studies ◽  
Essential Function ◽  
Kinase Homologue

ABSTRACT The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

scholarly journals Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores

2006 ◽  
Vol 72 (10) ◽  
pp. 6808-6814 ◽  
Author(s):  
Hannah S. Shafaat ◽  
Adrian Ponce
Keyword(s):  
Phase Contrast ◽  
Dipicolinic Acid ◽  
Ice Core ◽  
Greenland Ice Sheet ◽  
Ice Cores ◽  
Phase Contrast Microscopy ◽  
Standard Curve ◽  
Viability Assay ◽  
Contrast Microscopy ◽  
Arctic Ice

ABSTRACT We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by l-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% ± 3.8% and 48.9% ± 4.5%, respectively), while only 27.8% ± 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 ± 19 germinable spores/ml and 369 ± 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% ± 9.3%), and the second core contained 131 ± 4 germinable spores/ml and 162 ± 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% ± 8.8%), whereas only 2 CFU/ml were detected by culturing.

Detachment of biofilm bacteria due to variations in nutrient supply

1998 ◽  
Vol 37 (4-5) ◽  
pp. 211-214 ◽  
Author(s):  
Linda K. Sawyer ◽  
Slawomir W. Hermanowicz
Keyword(s):  
Phase Contrast ◽  
Digital Image Analysis ◽  
Specific Growth ◽  
Nutrient Supply ◽  
Surface Shear Stress ◽  
Surface Shear ◽  
Nutrient Limitations ◽  
Contrast Microscopy ◽  
Biofilm Bacteria ◽  
Observable Parameter

Growth and detachment rates of an environmental isolate of Aeromonas hydrophila attached to a surface were determined under varying nutrient supply conditions in a complex medium. Growth and detachment of cells were observed in real time using phase contrast microscopy in glass parallel plate flow chambers. Surface shear stress was controlled in all experiments at 3 N m−2. Images were taken every 15 min. Digital image analysis was used to determine specific growth and detachment rates. An observable parameter proportional to the nutrient depletion at the surface due to transfer limitations was used to indicate nutrient limitations. Specific detachment rates increased as the depletion parameter increased, indicating that nutrient limitations cause this bacterium to detach at greater rates.

scholarly journals THU0406 IDENTIFICATION OF INTRACELLULAR VACUOLES IN SYNOVIAL FLUID WITH CALCIUM PYROPHOSPHATE AND MONOSODIUM URATE CRYSTALS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 440.1-441
Author(s):  
M. L. Peral ◽  
I. Calabuig ◽  
A. Martín-Carratalá ◽  
M. Andrés ◽  
E. Pascual
Keyword(s):  
Clinical Practice ◽  
Synovial Fluid ◽  
Phase Contrast ◽  
Calcium Pyrophosphate ◽  
Monosodium Urate ◽  
Fluid Analysis ◽  
Cytoplasmic Vacuoles ◽  
Contrast Microscopy ◽  
Ordinary Light ◽  
Msu Crystals

Background:Synovial fluid analysis using polarized microscopy is the gold standard for the diagnosis of crystal-related arthritis. In our experience, we have noted that, when calcium pyrophosphate (CPP) crystals are observed, they sometimes appear within intracellular vacuoles. However, this phenomenon is not seen in those samples containing monosodium urate (MSU) crystals. This finding has been scantly reported in the literature, but may be useful in clinical practice to ensure accurate crystal identification.Objectives:Our study aims to assess whether the presence of vacuoles contributes to identifying the type of crystal, and also to gauge the frequency of their presentation.Methods:We conducted an observational study in a rheumatology unit between February and June of 2019. Synovial fluids containing CPP or MSU crystals, obtained in daily clinical practice, were consecutively included for analysis. Two observers simultaneously analyzed the presence of vacuoles by ordinary light and phase contrast microscopy in less than 24 hours after their extraction, using a microscope equipped with two viewing stations. The primary study variable was to determine whether CPP and MSU crystals are seen inside intracellular vacuoles, and to calculate the frequency of this finding for each type of crystal, estimating their 95% confidence interval (95% CI) and comparing rates using Fisher’s exact test.Results:Twenty-one samples were obtained. Data is given in the Table. MSU crystals were present in 7 (33.3%) and CPP crystals in 14 (66.6%). Interestingly, none of the MSU samples showed crystal-containing vacuoles (95% CI 0-35.4%). On the contrary, cytoplasmic vacuoles containing crystals were present in all of the CPP samples (95% CI 78.5-100%). The findings were confirmed by phase-contrast microscopy. Differences were statistically significant (p<0.001).Table.SAMPLES ACCORDING TO TYPE OF MICROCRYSTAL(n=21)SAMPLES WITH VACUOLS(UNDER ORDINARY LIGHT)SAMPLES WITH VACUOLS(UNDER PHASE CONTRAST)CPP (14; 66.6%)14 (100%)(95%CI 78.5-100%)14 (100%)(95%CI 78.5-100%)MSU (7; 33.3%)0 (0%)(95%CI 0-35.4%)0 (0%)(95%CI 0-35.4%)Conclusion:The presence of vacuoles may be a useful and easy way to differentiate MSU and CPP crystals when performing synovial fluid microscopy in clinical practice, since it appears to be a distinctive feature in CPP crystal fluids.References:[1]Kohn NN, Hughes RE, McCarty DJ Jr, Faires JS. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: the «pseudogout syndrome». II. Identification of crystals. Ann InternMed. 1962 May;56:738-45.[2]Pascual E, Sivera F, Andrés M. Synovial Fluid Analysis for Crystals. CurrOpRheumatol 2011;23:161-169.[3]McCarty DJ, Koopman WJ. Arthritis and allied conditions: A textbook of rheumatology, volumen 1. Lea &amp;Febiger. 1993.[4]Pascual E, Sivera F. Synovial fluid crystal Analysis. En Gout and other crystal arthropathies. Terkeltaub R ed. Elsevier; 2012: p.20-34.[5]Hwang HS, Yang CM, Park SJ, Kim HA. Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process. Int J Mol Sci. 2015 Dec 8;16(12):29265-77.Image 1. Microscopy with ordinary light. Cells with cytoplasmic vacuoles are observed, as well as abundant intra and extracellular CPP crystals.Image 2. Microscopy with phase contrast technique. Cells with intracellular vacuoles are observed inside which have microcrystals with parallelepiped morphology, compatible with CPP.Disclosure of Interests: :None declared

scholarly journals Rough Dental Implant Surfaces and Peri-Implantitis: Role of Phase-Contrast Microscopy, Laser Protocols, and Modified Home Oral Hygiene in Maintenance. A 10-Year Retrospective Study

2021 ◽  
Vol 11 (11) ◽  
pp. 4985
Author(s):  
Gianluigi Caccianiga ◽  
Gérard Rey ◽  
Paolo Caccianiga ◽  
Alessandro Leonida ◽  
Marco Baldoni ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Phase Contrast ◽  
Vertical Movement ◽  
Subgingival Plaque ◽  
Phase Contrast Microscopy ◽  
Implant Surface ◽  
Total Percentage ◽  
Contrast Microscopy

The aim of this study was to evaluate two different kinds of rough implant surface and to assess their tendency to peri-implantitis disease, with a follow-up of more than 10 years. Data were obtained from a cluster of 500 implants with Ti-Unite surface and 1000 implants with Ossean surface, with a minimum follow-up of 10 years. Implants had been inserted both in pristine bone and regenerated bone. We registered incidence of peri-implantitis and other causes of implant loss. All patients agreed with the following maintenance protocol: sonic brush with vertical movement (Broxo), interdental brushes, and oral irrigators (Broxo) at least two times every day. For all patients with implants, we evaluated subgingival plaque samples by phase-contrast microscopy every 4 months for a period of more than 10-years. Ti-Unite surface implants underwent peri-implantitis in 1.6% of the total number of implants inserted and Ossean surface implants showed peri-implantitis in 1.5% of the total number of implants. The total percentage of implant lost was 4% for Ti-Unite surfaces and 3.6% for Ossean surfaces. Strict control of implants leads to low percentage of peri-implantitis even for rough surfaces dental implants.

Organic Crystals in the Tracheids of Torreya Yunnanensis

IAWA Journal ◽  
1996 ◽  
Vol 17 (4) ◽  
pp. 393-403 ◽  
Author(s):  
Yuki Kondo ◽  
Tomoyuki Fujii ◽  
Yoshioki Hayashi ◽  
Atsushi Kato
Keyword(s):  
Phase Contrast ◽  
Growth Ring ◽  
Uv Spectra ◽  
Resonance Spectroscopy ◽  
Organic Crystals ◽  
X Ray Diffraction ◽  
Cinnamic Aldehyde ◽  
X Ray ◽  
Polarised Light ◽  
Contrast Microscopy

Organic crystals were found in tracheid lumina of some samples of Torreya yunnanensis Chen ' L. K. Fu imported from Yunnan, China. Tracheids with crystals were found in short to long tangential bands along the growth ring boundaries. Because the crystals were rapidly dissolved with ethanol and xylene, cross and tangential sections were mounted in de-ionized water without staining and observed by biological, polarised light, and phase-contrast microscopy. The crystals were sublimated under vacuum during routine sample preparation for conventional SEM and only the peripheral parts remained. With the aid of low vacuum-SEM and modified cryo-SEM procedure, the shape of the crystals was revealed. Some were styloid and large enough to fill tracheid lumina, while others were stacked appearing as slates filling tracheid lumina. X-ray diffraction applied to sections and isolated crystals showed that they were single crystals and orientated along the cell wall. UV spectra on isolated crystals and methanol dissolution of crystals suggested that they were composed of phenolic compounds. Crystals that were recrystallized from methanol were analysed by 1H and l3C nuclear magnetic resonance spectroscopy. These two techniques revealed that the major and minor components were o-methoxy cinnamic acid and o-methoxy cinnamic aldehyde.

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