Method for Assessment of Viability and Morphological Changes of Bacteria in the Early Stage of Colony Formation on a Simulated Natural Environment

ABSTRACT A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.

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Morphological aspects of self-repair of lesions caused by internal growth stresses in stems of Aristolochia macrophylla and Aristolochia ringens

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2010.0075 ◽

2010 ◽

Vol 277 (1691) ◽

pp. 2113-2120 ◽

Cited By ~ 26

Author(s):

Sebastian Busch ◽

Robin Seidel ◽

Olga Speck ◽

Thomas Speck

Keyword(s):

Cell Wall ◽

Laser Scanning ◽

Early Stage ◽

Morphological Changes ◽

Secondary Growth ◽

Laser Scanning Microscopy ◽

Morphological Data ◽

Confocal Laser ◽

Growth Stresses ◽

Self Repair

This study reveals in detail the mechanism of self-repair during secondary growth in the vines Aristolochia macrophylla and Aristolochia ringens based on morphological data. For a comprehensive understanding of the underlying mechanisms during the self-repair of lesions in the sclerenchymatous cylinder of the stem, which are caused by internal growth stresses, a classification of morphological changes in the cells involved in the repair process is required. In an early stage of self-repair, we observed morphological changes as a mere extension of the turgescent cortex cells surrounding the lesion, whereby the cell wall extends locally through visco-elastic/plastic deformation without observable cell wall synthesis. Later stages involve typical cell growth and cell division. Several successive phases of self-repair were investigated by light microscopy of stained samples and confocal laser-scanning microscopy in fluorescence mode. The results indicate that A. macrophylla and A. ringens respond to lesions caused by internal growth stresses with a sophisticated self-repair mechanism comprising several phases of different repair modes.

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Rapid Assessment of the Physiological Status of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102 by Flow Cytometry

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.2031-2035.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 2031-2035 ◽

Cited By ~ 36

Author(s):

Yoshinori Hiraoka ◽

Kazuhide Kimbara

Keyword(s):

Flow Cytometry ◽

Polychlorinated Biphenyl ◽

Rapid Assessment ◽

Live Cells ◽

Physiological Status ◽

Comamonas Testosteroni ◽

Double Staining ◽

Permeabilized Cells ◽

Degrading Bacterium ◽

Double Staining Method

ABSTRACT The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R 2 = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = −0.9776 x + 98.36; R 2 = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (≤1 h) and distinguishes correctly among live, dead, and permeabilized cells.

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Rapid Detection of Single Cell Bacteria as a Novel Approach in Food Microbiology

Journal of AOAC International ◽

10.1093/jaoac/85.4.979 ◽

2002 ◽

Vol 85 (4) ◽

pp. 979-983 ◽

Cited By ~ 13

Author(s):

Eva D′Haese ◽

Hans J Nelis

Keyword(s):

Single Cell ◽

Rapid Detection ◽

Laser Scanning ◽

Solid Phase ◽

Membrane Filter ◽

Food Samples ◽

Food Microbiology ◽

Physiological Status ◽

Microbial Count ◽

Slow Growing

Abstract Solid-phase cytometry (SPC) is a novel technique that allows rapid detection of bacteria at the single cell level, without the need for a growth phase. After filtration of the sample, the retained microorganisms are fluorescently labeled on the membrane filter and automatically counted by a laser scanning device. Each fluorescent spot can be visually inspected with an epifluorescence microscope connected to the ChemScan by a computer-driven moving stage. Depending on the fluorogenic labels used, information on the identity and the physiological status of the microorganisms can be obtained within a few hours. Although SPC was originally recommended for the determination of the total viable microbial count in water and other liquid samples, it may also be a promising technique for the detection and enumeration of bacteria in food samples, provided they can be isolated from the unfilterable matrix. The short detection time inherent in this approach is a considerable advantage over conventional plate counting, especially for slow-growing microorganisms. The basic principles of SPC are discussed as well as its potential for the detection of Mycobacterium paratuberculosis, a model example of a slow-growing bacterium in milk.

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Connection of discontinuous segments in early functional recovery from thoracic ossification of the posterior longitudinal ligament treated with posterior instrumented surgery

Journal of Neurosurgery Spine ◽

10.3171/2019.8.spine19604 ◽

2020 ◽

Vol 32 (2) ◽

pp. 200-206

Author(s):

Kei Ando ◽

Kazuyoshi Kobayashi ◽

Masaaki Machino ◽

Kyotaro Ota ◽

Satoshi Tanaka ◽

...

Keyword(s):

Early Stage ◽

Morphological Changes ◽

Japanese Orthopaedic Association ◽

Multislice Ct ◽

Posterior Longitudinal Ligament ◽

Ct Images ◽

Rigid Fixation ◽

Disc Space ◽

Fusion Surgery ◽

Estimated Blood Loss

OBJECTIVEThe objective of this study was to investigate the relationship between morphological changes in thoracic ossification of the posterior longitudinal ligament (T-OPLL) and postoperative neurological recovery after thoracic posterior fusion surgery. Changes of OPLL morphology and postoperative recovery in cases with T-OPLL have not been examined.METHODSIn this prospective study, the authors evaluated data from 44 patients (23 male and 21 female) who underwent posterior decompression and fusion surgery with instrumentation for the treatment of T-OPLL at our hospital. The patients’ mean age at surgery was 50.7 years (range 38–68 years). The minimum duration of follow-up was 2 years. The location of thoracic ossification of the ligamentum flavum (T-OLF), T-OLF at the OPLL level, OPLL morphology, fusion range, estimated blood loss, operative time, pre- and postoperative Japanese Orthopaedic Association (JOA) scores, and JOA recovery rate were investigated. Reconstructed sagittal multislice CT images were obtained before and at 3 and 6 months and 1 and 2 years after surgery. The basic fusion area was 3 vertebrae above and below the OPLL lesion. All parameters were compared between patients with and without continuity across the disc space at the OPLL at 3 and 6 months after surgery.RESULTSThe preoperative morphology of OPLL was discontinuous across the disc space between the rostral and caudal ossification regions on sagittal CT images in all but one of the patients. Postoperatively, these segments became continuous in 42 patients (97.7%; occurring by 6.6 months on average) without progression of OPLL thickness. Patients with continuity at 3 months had significantly lower rates of diabetes mellitus (p < 0.05) and motor palsy in the lower extremities (p < 0.01). The group with continuity also had significantly higher mean postoperative JOA scores at 3 (p < 0.01) and 6 (p < 0.05) months and mean JOA recovery rates at 3 and 6 months (both p < 0.01) after surgery.CONCLUSIONSPreoperatively, discontinuity of rostral and caudal ossified lesions was found on CT in all patients but one of this group of 44 patients who needed surgery for T-OPLL. Rigid fixation with instrumentation may have allowed these segments to connect at the OPLL. Such OPLL continuity at an early stage after surgery may accelerate spinal cord recovery.

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Morphometric characteristics of the metacestode Echinococcus vogeli Rausch & Bernstein, 1972 in human infections from the northern region of Brazil

Journal of Helminthology ◽

10.1017/s0022149x14000376 ◽

2014 ◽

Vol 89 (4) ◽

pp. 480-486 ◽

Cited By ~ 3

Author(s):

F. Almeida ◽

F. Oliveira ◽

R. Neves ◽

N. Siqueira ◽

R. Rodrigues-Silva ◽

...

Keyword(s):

Laser Scanning ◽

Morphological Changes ◽

Northern Region ◽

Laser Scanning Microscopy ◽

Health Concern ◽

Confocal Laser ◽

Total Width ◽

Morphometric Characteristics ◽

Blade Area ◽

Echinococcus Vogeli

AbstractPolycystic echinococcosis, caused by the larval stage (metacestode) of the small-sized tapeworm, Echinococcus vogeli, is an emerging parasitic zoonosis of great public health concern in the humid tropical rainforests of South and Central America. Because morphological and morphometric characteristics of the metacestode are not well known, hydatid cysts from the liver and the mesentery were examined from patients following surgical procedures. Whole mounts of protoscoleces with rostellar hooks were examined under light and confocal laser scanning microscopy. Measurements were made of both large and small hooks, including the total area, total length, total width, blade area, blade length, blade width, handle area, handle length and handle width. The results confirmed the 1:1 arrangement of hooks in the rostellar pad and indicated, for the first time, that the morphometry of large and small rostellar hooks varies depending upon the site of infection. Light and confocal microscopy images displayed clusters of calcareous corpuscles in the protoscoleces. In conclusion, morphological features of large and small rostellar hooks of E. vogeli are adapted to a varied environment within the vertebrate host and such morphological changes in calcareous corpuscles occur at different stages in the maturation of metacestodes.

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Staphylococcal infection and pathomurfological characteristic of the pituitary-adrenal-thyroid system

NATURE AND SCIENCE ◽

10.36719/2707-1146/04/69-73 ◽

2020 ◽

Vol 03 (04) ◽

pp. 69-73

Author(s):

Samira Mammadhasan Yagubova ◽

◽

Elchin Chingiz Akbarov ◽

Tarana Nadir Mirzayeva ◽

◽

...

Keyword(s):

Structural Changes ◽

Adrenal Glands ◽

Early Stage ◽

Morphological Changes ◽

Endocrine System ◽

Staphylococcal Infection ◽

The Body ◽

Glandular Cells ◽

Exogenous Factors ◽

Specific Symptoms

During the staphylococcal infection, changes in the interaction of glandular cells, dystrophic and disorganizing pathologies in tissues, especially acute structural and hemodynamic changes in the stroma of the glands in the pituitary-adrenal-thyroid system, develop from the first day of the experiment. At the end of the experiment, on the background of a decrease in exudative processes, fibroplastic reactions are significantly activated, resulting in signs of incomplete regeneration – mainly sclerotic processes and cystic-atrophic changes in the parenchyma. Structural changes in tissues in the early stages of staphylococcal infection and the dynamics of development are characterized by specific symptoms in each of the glands. Since the pituitary gland is exposed to endogenous and exogenous factors earlier and more often than the adrenal glands, and the adrenal glands are earlier than the thyroid gland, dystrophic and destructive changes in the pituitary and adrenal glands are more pronounced at the early stage of the experiment. These morphological changes can change the hormonal status of the body and lead to dysfunction of the endocrine system as a whole – a decrease in the functional activity of the glands to some extent, and even inhibition of adenohypophyseal cells. Key words: staphylococcal infection, peritonitis, pituitary, adrenal and thyroid glands

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Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3407-3414.1992 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3407-3414

Author(s):

Y Yoshida ◽

M Kawata ◽

Y Miura ◽

T Musha ◽

T Sasaki ◽

...

Keyword(s):

Dna Synthesis ◽

Laser Scanning ◽

Morphological Changes ◽

Stress Fibers ◽

Confocal Laser ◽

3T3 Cells ◽

Membrane Ruffling ◽

Ras P21 ◽

Swiss 3T3 ◽

Gamma S

Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.

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Experimental Investigations of the Process of Carbonate Fracture Dissolution Enlargement under Reservoir Temperature and Pressure Conditions

Geofluids ◽

10.1155/2018/5971421 ◽

2018 ◽

Vol 2018 ◽

pp. 1-19

Author(s):

Jingxia Wang ◽

Qingchun Yu

Keyword(s):

Laser Scanning ◽

Early Stage ◽

Confining Pressure ◽

Carbonate Reservoir ◽

Dissolution Process ◽

Experimental Investigations ◽

Polynomial Type ◽

Calcite Veins ◽

Before And After ◽

Logarithmic Type

Karst is a central focus in the field of carbonate reservoir geology. Fracture dissolution enlargement is an important mechanism for the formation of high-quality reservoirs. This study performed four carbonate fracture dissolution enlargement (CFDE) experiments under a confining pressure of 20 MPa, and temperatures ranged from 40 to 60°C. CO2-saturated deionized water was injected into artificial carbonate fractures at approximately 11.5 ml/h for 96, 208, 216, and 216 hours. The water flowing out of the fractures was sampled every 8 h to monitor the concentration of Ca2+. SEM photomicrographs and 3D laser scanning images were taken before and after the CFDE experiments to observe the dissolution process of the fracture surfaces. After the CFDE experiment, the hydraulic apertures (Bh) of sample 1 (S1), sample 3 (S3), and sample 4 (S4) were enlarged by 3.4, 1.4, and 1.2 times, respectively. The aperture of sample 2 (S2) was slightly reduced in the early stage of the experiment. The experimental results of this study demonstrate that Bh can be divided into three categories as a function of time: S type, logarithmic type, and polynomial type. The laboratory dissolution rate of S1, S2, S3, and S4 were 2.50 × 10−6, 3.11 × 10−6, 2.70 × 10−6, and 3.04 × 10−6 mol/m2/s. The pattern of fracture dissolution is closely related to the Peclet and Damkohler numbers. The dissolution processes of high Peclet and Damkohler numbers lead to a pattern of obvious channelization. The Peclet and Damkohler numbers of the S3 CFDE experiment were the highest, and the channelizing dissolution is the most notable in S3 of the four fractures. A dissolution process at low temperature has a higher Peclet number and thus leads to obvious channelizing dissolution. Mineral heterogeneities in the rock also play a significant role in channelizing dissolution. A preferential channel typically develops in places where bioclasts are accumulated or the calcite veins are distributed.

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