Culture Methods for the Selection and Isolation of Stable Antibody-Producing Murine Hybridomas

1987 ◽  
pp. 181-194
Author(s):  
R. Thomas Taggart
Keyword(s):  
Culture Methods

Some Aspects of the Polyhexamethyleneguanidine Salts Effect on Cell Cultures

2014 ◽  
Vol 1 (1) ◽  
pp. 62-67 ◽  
Author(s):  
M. Mandygra ◽  
A. Lysytsia
Keyword(s):  
Proliferative Activity ◽  
Cell Monolayer ◽  
Eukaryotic Cell ◽  
Culture Methods ◽  
Cellular Processes ◽  
Negative Effect ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Anion Type ◽  
Cell Culture Methods

Aim. To investigate the effect of polyhexamethyleneguanidine (PHMG) to eukaryotic cell culture. Methods. The passaged bovine tracheal cells culture (TCC) and primary culture of chicken embryo fi broblasts (FCE) were used in the experiments. TCC and FCE monolayers were treated with aqueous solutions of PHMG chloride or succinate. The method of PHMG polycation adsorption to the cells’ plasma membrane together with microscopy were applied. Results. The dependence of PHMG effect on the eukaryotic cells on the agent concentration, duration of exposure and the anion type has been fi xed. The PHMG concentration of 10 –5 per cent (0.1 μg/ml) never causes degradation of the previously formed cell monolayer, while the higher concentrations damage it. The conditions of the PHMG chloride and succinate’s negative effect on cell proliferation and inhibition of monolayer formation were determined. The hypothesis that under certain conditions PHMG stimulates the proliferative activity of the cells has been confi rmed. Stimulation may be associated with non-specifi c stress adaptation of cells. In this case, it is due to modifi cations of the cell membrane after PHMG adsorption to it. Conclusions. PHMG polycation binds with the membrane’s phosphoglycerides fi rmly and irreversibly. A portion of the lipids are removed from participation in the normal cellular processes at that. At the same time, the synthesis of new lipids and membrane-bound enzymes is probably accelerated. The phospholip ids’ neogenesis acceleration can stimulate mitosis under certain conditions. The obtained results can be used in the biotechnologies.

Small Molecules Effective Against Liver and Blood Stage Malarial Infection

2019 ◽  
Vol 18 (23) ◽  
pp. 2008-2021 ◽  
Author(s):  
Snigdha Singh ◽  
Neha Sharma ◽  
Charu Upadhyay ◽  
Sumit Kumar ◽  
Brijesh Rathi ◽  
...  
Keyword(s):  
Drug Targets ◽  
Malaria Parasite ◽  
Blood Stage ◽  
Culture Methods ◽  
Liver Stage ◽  
Malarial Infection ◽  
Dual Stage ◽  
New Treatment ◽  
Global Threat

Malaria is a lethal disease causing devastating global impact by killing more than 8,00,000 individuals yearly. A noticeable decline in malaria related deaths can be attributed to the most reliable treatment, ACTs against P. falciparum. However, the cumulative resistance of the malaria parasite against ACTs is a global threat to control the disease and, therefore the new effective therapeutics are urgently needed, including new treatment approaches. Majority of the antimalarial drugs target BS malarial infection. Currently, scientists are eager to explore the drugs with potency against not only BS but other life stages such as sexual and asexual stages of the malaria parasite. Liver Stage is considered as one of the important drug targets as it always leads to BS and the infection can be cured at this stage before it enters into the Blood Stage. However, a limited number of compounds are reported effective against LS malaria infection probably due to scarcity of in vitro LS culture methods and clinical possibilities. This mini review covers a range of chemical compounds showing efficacy against BS and LS of the malaria parasite’s life cycle collectively (i.e. dual stage activity). These scaffolds targeting dual stages are essential for the eradication of malaria and to evade resistance.

scholarly journals Cytomegalovirus antigen detection in peripheral blood leukocytes after allogeneic marrow transplantation

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1358-1364 ◽  
Author(s):  
M Boeckh ◽  
RA Bowden ◽  
JM Goodrich ◽  
M Pettinger ◽  
JD Meyers
Keyword(s):  
Marrow Transplantation ◽  
Gastrointestinal Disease ◽  
Marrow Transplant ◽  
Cmv Infection ◽  
Culture Methods ◽  
Blood Leukocytes ◽  
Shell Vial ◽  
Allogeneic Marrow Transplantation ◽  
Peripheral Leukocytes ◽  
Allogeneic Marrow

Abstract Detection of cytomegalovirus (CMV) antigenemia was compared with shell vial centrifugation cultures for rapid detection of CMV infection. In a prospective study, 59 CMV seropositive patients were monitored weekly during the first 100 days after allogeneic marrow transplantation for virus excretion from urine, throat, and blood and for antigenemia by direct staining of peripheral leukocytes using an antibody pool directed against pp 65. Antigenemia was present in 21 of 22 patients with culture-proven CMV infection and in 3 of 37 without culture-proven CMV infection (sensitivity 95%, specificity 91%). The median time of onset of antigenemia and shell vial cultures was day 47 and 55 after transplant, respectively (P = .0006). Among patients who developed CMV disease without preceding cultures, antigenemia was detected in all patients with CMV pneumonia (N = 6) and in two of three patients with gastrointestinal disease by a median of 10 and 7 days, respectively, before the onset of disease (P = .0002). Levels of antigenemia were significantly higher in patients with disease or viremia than in patients with excretion from urine or throat (P less than .05). Whether the antigenemia assay is more sensitive than rapid culture methods to focus antiviral prophylaxis in marrow transplant patients must be determined in controlled studies.

scholarly journals Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 20
Author(s):  
Luigi De Grossi ◽  
Davide Santori ◽  
Antonino Barone ◽  
Silvia Abbruzzese ◽  
Matteo Ricchi ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Liquid Culture ◽  
Culture Media ◽  
Small Ruminants ◽  
Culture Method ◽  
Mycobacterium Avium Subsp ◽  
Type I ◽  
Culture Methods ◽  
Specific Pcr ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

scholarly journals Evaluating Metagenomic Analysis for Pathogen Transmission in Healthcare Settings

2020 ◽  
Vol 41 (S1) ◽  
pp. s224-s224
Author(s):  
Curt Hewitt ◽  
Katharina Weber ◽  
Danielle LeSassier ◽  
Anthony Kappell ◽  
Kathleen Schulte ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Dna Analysis ◽  
Pathogen Transmission ◽  
Species Level ◽  
Metagenomic Analysis ◽  
Skin Microbiome ◽  
Limited Information ◽  
Culture Methods ◽  
Data Set ◽  
Skin Contact

Background: The prevalence of healthcare-acquired infections (HAIs) and rising levels of antimicrobial resistance place a significant burden on modern healthcare systems. Cultures are typically used to track HAIs; however, culture methods provide limited information and are not applicable to all pathogens. Next-generation sequencing (NGS) can detect and characterize pathogens present within a sample, but few research studies have explored how NGS could be used to detect pathogen transmission events under HAI-relevant scenarios. The objective of this CDC-funded project was to evaluate and correlate sequencing approaches for pathogen transmission with standard culture-based analysis. Methods: We modeled pathogen transfer via hand contact using synthetic skin. These skin coupons were seeded with a community of commensal organisms to mimic the human skin microbiome. Pathogens were added at physiologically relevant high or low levels prior to skin-to-skin contact. The ESKAPE pathogens: E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp plus C. difficile were employed because they are the most common antibiotic resistant HAIs. Pathogen transfer between skin coupons was measured following direct skin contact and fomite surface transmission. The effects of handwashing or fomite decontamination were also evaluated. Transferred pathogens were enumerated via culture to establish a robust data set against which DNA and RNA sequence analyses of the same samples could be compared. These data also provide a quantitative assessment of individual ESKAPE+C pathogen transfer rates in skin contact scenarios. Results: Metagenomic and metatranscriptomic analysis using custom analysis pipelines and reference databases successfully identified the commensal and pathogenic organisms present in each sample at the species level. This analysis also identified antibiotic resistance genes and plasmids. Metatranscriptomic analysis permitted not only gene identification but also confirmation of gene expression, a critical factor in the evaluation of antibiotic resistance. DNA analysis does not require cell viability, a key differentiator between sequencing and culturing reflected in simulated handwashing data. Sensitivity remains a key limitation of metagenomic analysis, as shown by the poor species identification and gene content characterization of pathogens present at low abundance within the simulated microbial community. Species level identification typically failed as ratios fell below 1:1,000 pathogen CFU:total community CFU. Conclusions: These findings demonstrate the strengths and weaknesses of NGS for molecular epidemiology. The data sets produced for this study are publicly available so they can be employed for future metagenomic benchmarking studies.Funding: NoneDisclosures: None

Urinary microbiota of women with recurrent urinary tract infection: collection and culture methods

2021 ◽  
Author(s):  
Baylie R. Hochstedler ◽  
Lindsey Burnett ◽  
Travis K. Price ◽  
Carrie Jung ◽  
Alan J. Wolfe ◽  
...  
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Recurrent Urinary Tract Infection ◽  
Culture Methods ◽  
Urinary Microbiota

scholarly journals Phenotypic and phylogenetic characterization of Lactobacillus species isolated from traditional Lighvan cheese

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Haleh Forouhandeh ◽  
Sepideh Zununi Vahed ◽  
Hossein Ahangari ◽  
Vahideh Tarhriz ◽  
Mohammad Saeid Hejazi
Keyword(s):  
16S Rrna ◽  
Random Amplified Polymorphic Dna ◽  
Bacterial Species ◽  
16S Rrna Sequencing ◽  
Lactobacillus Species ◽  
Culture Methods ◽  
Phylogenetic Characterization ◽  
Rrna Sequencing ◽  
Specific Culture ◽  
Lighvan Cheese

Abstract Lighvan cheese (Lighvan panir) is among the most famous traditional cheese in Iran for its desired aroma and flavor. Undoubtedly, the lactic acid bacteria especially the genus Lactobacillus are the critical factors in developing the aroma, flavor, and texture in Lighvan cheese. In this study, the Lactobacillus population of the main Lighvan cheese was investigated. The Lactobacillus of the main Lighvan cheese was isolated using specific culture methods according to previously published Guidelines. Then, the phylogenetic features were investigated and the phenotypic characteristics were examined using specific culture methods. Twenty-eight Gram-positive bacterial species were identified belonged to the genus Lactobacillus. According to the same sequences as each other, three groups (A, B, and C) of isolates were categorized with a high degree of similarity to L. fermentum (100%) and L. casei group (L. casei, L. paracasei, and L. rhamnosus) (99.0 to 100%). Random amplified polymorphic DNA (RAPD) fingerprint analysis manifested the presence of three clusters that were dominant in traditional Lighvan cheese. Cluster І was divided into 4 sub-clusters. By the result of carbohydrate fermentation pattern and 16S rRNA sequencing, isolates were identified as L. rhamnosus. The isolates in clusters II and III represented L. paracasei and L. fermentum, respectively as they were identified by 16S rRNA sequencing and fermented carbohydrate patterns. Our result indicated that the specific aroma and flavor of traditional Lighvan cheese can be related to its Lactobacillus population including L. fermentum, L. casei, L. paracasei, and L. rhamnosus. Graphical abstract

scholarly journals Next Generation Sequencing for the Prediction of the Antibiotic Resistance in Helicobacter pylori: A Literature Review

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 437
Author(s):  
Ilaria Maria Saracino ◽  
Matteo Pavoni ◽  
Angelo Zullo ◽  
Giulia Fiorini ◽  
Tiziana Lazzarotto ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Next Generation Sequencing ◽  
Literature Review ◽  
Molecular Mechanisms ◽  
World Health ◽  
Next Generation ◽  
Culture Methods ◽  
Phenotypic Resistance ◽  
H Pylori ◽  
Generation Sequencing

Background and aims: Only a few antimicrobials are effective against H. pylori, and antibiotic resistance is an increasing problem for eradication therapies. In 2017, the World Health Organization categorized clarithromycin resistant H. pylori as a “high-priority” bacterium. Standard antimicrobial susceptibility testing can be used to prescribe appropriate therapies but is currently recommended only after the second therapeutic failure. H. pylori is, in fact, a “fastidious” microorganism; culture methods are time-consuming and technically challenging. The advent of molecular biology techniques has enabled the identification of molecular mechanisms underlying the observed phenotypic resistance to antibiotics in H. pylori. The aim of this literature review is to summarize the results of original articles published in the last ten years, regarding the use of Next Generation Sequencing, in particular of the whole genome, to predict the antibiotic resistance in H. pylori.Methods: a literature research was made on PubMed. The research was focused on II and III generation sequencing of the whole H. pylori genome. Results: Next Generation Sequencing enabled the detection of novel, rare and complex resistance mechanisms. The prediction of resistance to clarithromycin, levofloxacin and amoxicillin is accurate; for other antimicrobials, such as metronidazole, rifabutin and tetracycline, potential genetic determinants of the resistant status need further investigation.

scholarly journals Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1102
Author(s):  
Galina N. Raldugina ◽  
Sergey V. Evsukov ◽  
Liliya R. Bogoutdinova ◽  
Alexander A. Gulevich ◽  
Ekaterina N. Baranova
Keyword(s):  
Choline Oxidase ◽  
Culture Methods ◽  
Gene Encoding ◽  
Oxidase Gene ◽  
Bacterial Enzyme ◽  
Transgenic Lines ◽  
High Regeneration ◽  
Whole Plants ◽  
Implicit Response

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

scholarly journals 3D Clumps/Extracellular Matrix Complexes of Periodontal Ligament Stem Cells Ameliorate the Attenuating Effects of LPS on Proliferation and Osteogenic Potential

2021 ◽  
Vol 11 (6) ◽  
pp. 528
Author(s):  
Spoorthi Ravi Banavar ◽  
Swati Yeshwant Rawal ◽  
Shaju Jacob Pulikkotil ◽  
Umer Daood ◽  
Ian C. Paterson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Periodontal Ligament ◽  
3D Culture ◽  
Osteogenic Potential ◽  
Culture Methods ◽  
Periodontal Ligament Stem Cells ◽  
Raman Analysis ◽  
Atp Production ◽  
Tnf Α ◽  
Inflammatory Milieu

Background: The effects of lipopolysaccharide (LPS) on cell proliferation and osteogenic potential (OP) of MSCs have been frequently studied. Objective: to compare the effects of LPS on periodontal-ligament-derived mesenchymal stem cells (PDLSCs) in monolayer and 3D culture. Methods: The PDLSCs were colorimetrically assessed for proliferation and osteogenic potential (OP) after LPS treatment. The 3D cells were manually prepared by scratching and allowing them to clump up. The clumps (C-MSCs) were treated with LPS and assessed for Adenosine triphosphate (ATP) and OP. Raman spectroscopy was used to analyze calcium salts, DNA, and proline/hydroxyproline. Multiplexed ELISA was performed to assess LPS induced local inflammation. Results: The proliferation of PDLSCs decreased with LPS. On Day 28, LPS-treated cells showed a reduction in their OP. C-MSCs with LPS did not show a decrease in ATP production. Principal bands identified in Raman analysis were the P–O bond at 960 cm−1 of the mineral component, 785 cm−1, and 855 cm−1 showing qualitative changes in OP, proliferation, and proline/hydroxyproline content, respectively. ELISA confirmed increased levels of IL-6 and IL-8 but with the absence of TNF-α and IL-1β secretion. Conclusions: These observations demonstrate that C-MSCs are more resistant to the effects of LPS than cells in monolayer cell culture. Though LPS stimulation of C-MSCs creates an early pro-inflammatory milieu by secreting IL-6 and IL-8, PDLSCs possess inactivated TNF promoter and an ineffective caspase-1 activating process.

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