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PubMed1994

A novel two-compartment culture dish allows microscopic evaluation of two different treatments in one cell culture simultaneously. Influence of external pH on Na+/Ca2+ exchanger activity in cultured rat cardiomyocytes.

A new type of culture dish containing two separate compartments is described, that can be used in high-magnification microscopy. Using the dish, two halves of a single-cell culture, grown on a standard coverslip, can be exposed to different treatments simultaneously, allowing the effect of one treatment to be compared with that of the other treatment in the same culture. This way, the natural variability that might exist between different individual cultures is circumvented. In addition, by simultaneously conducting two experiments per dish, the number of experiments needed can be decreased. This both reduces the time to complete a series of experiments and allows the optimal use of specimens that are difficult to obtain, such as human material. We found there is an excellent barrier between the two compartments for lipophilic and hydrophilic compounds, and for low-molecular-mass cations. To illustrate the use of the dish we describe the influence of external pH on the activity of the Na+/Ca2+ exchanger in intact cultured neonatal rat ventricular cardiomyocytes. The intracellular free calcium concentration ([Ca2+]i) in the cardiomyocytes, measured using fura-2 and imaging fluorescence microscopy, was studied during sodium-free incubation. The resulting rise in [Ca2+]i at pH 7.4 in one compartment was compared with that in the other compartment in which the pH was either 6.0, 7.0, 7.4 or 8.0. It was found that below pH 7.4, Na+/Ca2+ exchanger activity was diminished, whereas at pH higher than 7.4 the Na+/Ca2+ exchanger activity was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Removal of endotoxin from culture media by a polymyxin B sepharose column. The activity of contaminating endotoxin in culture media measured by the interleukin 1 inducing effect on human monocyte cultures and by the Limulus test.

The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media and utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines. Many commercially liquid culture media were found to contain ET in concentrations above 25 X 10(-12) g/ml. A simple system for the removal of ET from media and solutions was established by use of a commercially available Polymyxin B Sepharose gel. To measure the lipopolysaccharide (LPS) binding capacity of the gel, known concentrations of LPS were added to culture media, which were passed through a column consisting of the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured by the Limulus amoebocyte lysate (LAL) test before and after passage of the column. The LPS-binding capacity of the gel was approximately 2.4 X 10(-6) g/10 ml. The biological activity of contaminating ET and added LPS in media, before and after passage of the column, was also characterized by the capacity of the media to induce interleukin 1 (IL-1) secretion in human Mo cultures. The content of IL-1 in Mo culture supernatants was determined by the mouse thymocyte costimulatory (LAF) assay. By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 X 10(-12) g/ml of ET stimulate human Mo cultures to IL-1 secretion.

Adult↗
PubMed1975

Lipids in plant tissue cultures. IV. The characteristic patterns of lipid classes in callus cultures and suspension cultures.

Lipids from callus cultures and suspension cultures of higher plants constitute 5 to 8% of the dry tissue's weight. The predominant lipid classes are the sterols, steryl esters, steryl glycosides and esterified steryl glycosides. Considerable amounts of a variety of sterylglycolipids, whose structures are not completely elucidated, are also present. Triglycerides and phospholipids occur in small proportions, whereas monogalactosyl diglycerides, digalactosyl diglycerides and sulfoquinovosyl diglycerides are present only in traces, if at all. Beta-Sitosterol is the predominant constituent sterol, stigmasterol and campesterol as well as a variety of as yet unidentified sterols occur in smaller proportions. The major constituent fatty acids are palmitic, oleic, linoleic and linolenic acids. Saturated very long-chain fatty acids are found in smaller proportions. Unusual fatty acids, such as epoxy acids, which occur in the seed lipids of certain plants, are not found in tissue cultures derived from these plants. Clucose and traces of galactose are the only sugars obtained by acid hydrolysis of the glycolipids occurring in plant tissue cultures.

Cell Division↗
PubMed1986

Bone marrow aspirate culture superior to streptokinase clot culture and 8 ml 1:10 blood-to-broth ratio blood culture for diagnosis of typhoid fever.

We compared the sensitivities of bone marrow aspirate culture (BMAC), 3 ml 1:4 and 8 ml 1:10 blood-to-broth ratio blood cultures (BC), 8 ml streptokinase clot culture (STKCC) and rectal swab culture (RSC) for isolating Salmonella typhi and S. paratyphi A from 61 patients with typhoid or paratyphoid fever in Jakarta, Indonesia. BMAC (92%) was significantly more sensitive than 8 ml BC (62%), 8 ml STKCC (51%), 3 ml BC (44%), RSC (56%) and the 19 ml combination of all three BC methods (71%). The combination of the three BC methods and RSC had an isolation rate of 87%. In Jakarta the diagnosis of typhoid fever cannot be confidently excluded unless a BMAC is done.

Adolescent↗
PubMedDate not supplied

[Comparison, using RAST-inhibition, of the allergenic potency of Dermatophagoides pteronyssinus extracts obtained from: whole mite culture, fraction of enriched mite culture, fraction of feces enriched culture, purified mite bodies, commercial extracts].

The RAST-inhibition potency has been obtained from five extracts prepared by: whole mite culture, mites-enriched culture, faeces-enriched culture, purified mite bodies, commercial extract. The present study demonstrates that extracts of: a) purified bodies, and b) faeces-enriched culture are both potent preparations in RAST-inhibition.

Allergens↗
PubMed2001

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Animals↗
PubMed2004

Cultural and ethical issues in working with culturally diverse patients and their families:the use of the culturagram to promote cultural competent practice in health care settings.

In all aspects of health and mental health care-the emergency room, the outpatient clinic, inpatient facilities, rehab centers, nursing homes, and hospices-social workers interact with patients from many different cultures. This paper will introduce an assessment tool for health care professionals to advance understanding of culturally diverse patients and their families.

Clinical Competence↗
PubMed2006

When personality and culture clash: the psychological distress of allocentrics in an individualist culture and idiocentrics in a collectivist culture.

Because humans need both autonomy and interdependence, persons with either an extreme collectivist orientation (allocentrics) or extreme individualist values (idiocentrics) may be at risk for possession of some features of psychopathology. Is an extreme personality style a risk factor primarily when it conflicts with the values of the surrounding society? Individualism-collectivism scenarios and a battery of clinical and personality scales were administered to nonclinical samples of college students in Boston and Istanbul. For students residing in a highly individualistic society (Boston), collectivism scores were positively correlated with depression, social anxiety, obsessive-compulsive disorder and dependent personality. Individualism scores, particularly horizontal individualism, were negatively correlated with these same scales. A different pattern was obtained for students residing in a collectivist culture, Istanbul. Here individualism (and especially horizontal individualism) was positively correlated with scales for paranoid, schizoid, narcissistic, borderline and antisocial personality disorder. Collectivism (particularly vertical collectivism) was associated with low report of symptoms on these scales. These results indicate that having a personality style which conflicts with the values of society is associated with psychiatric symptoms. Having an orientation inconsistent with societal values may thus be a risk factor for poor mental health.

Adolescent↗
PubMed1994

High extracellular potassium concentrations stimulate oxidative metabolism in a glutamatergic neuronal culture and glycolysis in cultured astrocytes but have no stimulatory effect in a GABAergic neuronal culture.

Rates of deoxyglucose accumulation and of CO2 production from [U-14C]glucose, or from [U-14C]lactate or [2-14C]pyruvate (as a determination of tricarboxylic acid (TCA) cycle activity) were determined in primary cultures of either astrocytes, cerebellar granule cell neurons (utilizing glutamate as their transmitter) or cerebral cortical interneurons (utilizing GABA as their transmitter) during control ('resting') conditions and during exposure to an elevated extracellular potassium concentration, mimicking functional activity. The elevation of the extracellular potassium concentration increased the rate of deoxyglucose accumulation, but not of TCA cycle activity in astrocytes and both deoxyglucose accumulation and TCA cycle activity in cerebellar granule cells, but had no stimulatory effect in cerebral cortical neurons. Based on these observations it is suggested that the increase in energy metabolism in the CNS in vivo during functional activity mainly reflects increased active accumulation of potassium ions and extrusion of sodium ions in neurons receiving excitatory input and in adjacent astrocytes in order to re-establish pre-stimulus ion distribution across cell membranes.

Animals↗
PubMed2000

Rapid detection of microorganisms in blood cultures of newborn infants utilizing an automated blood culture system.

BACKGROUND: Neonatal sepsis is a low incidence, high-risk disease with many sepsis work-ups performed to detect a single case. Seventy-two hours of antibiotic therapy have been traditionally recommended pending negative culture results. Improved culture media and new technology integrated into blood culture systems could shorten incubation time required to detect positive culture results. This would then change the length of antibiotic therapy in the management of the newborn infant with suspected sepsis. In addition, previous data supporting the 72-hour recommendation were retrospectively acquired, utilized nonautomated systems, and reported in an era with a different population of microorganisms cultured in special care nurseries. OBJECTIVE: Evaluate the time of incubation to detect positive blood cultures from newborn infants with suspected sepsis using a computer-assisted, automated blood culture system, ESP (Trek Diagnostic Systems, Inc, Westlake, OH). DESIGN: Prospective, observational study. PATIENTS AND SETTING: All positive blood culture results that were obtained from term and preterm newborn infants born from November 1993 through June 1997 at a publicly funded hospital with over 6000 live births per year. METHODS: As positive blood culture results were identified, data were prospectively obtained from the patient's medical record. The computer algorithm in the automated blood culture system determined the time to positivity. Time to positivity was determined for blood cultures obtained before the initiation antimicrobial therapy and compared with those cultures obtained after beginning therapy. Time to positivity was also evaluated for clinically important Gram-positive and Gram-negative bacteria and yeast. RESULTS: Four hundred fifty-five positive blood culture results were obtained from 222 patients. Gram-positive organisms accounted for 80% (366/455) of the positive culture results, Gram-negative organisms accounted for 11% (48/455), and yeast for 9% (41/455). Virtually all cultures growing clinically significant Gram-positive and Gram-negative organisms were positive by 24 to 36 hours of incubation. Cultures growing Staphylococcus epidermidis were virtually all positive after 36 to 48 hours of incubation. Of cultures growing yeast, 88% (36/41) were positive by 48 hours of incubation. There was no difference in time to positivity in pretherapy or posttherapy obtained positive blood cultures. Prenatally administered antibiotics did not affect time to positivity in positive cultures drawn on the first day of life. In a selected group of microorganisms that are the frequent cause of bacteremia in term infants, 97% and 99% of cultures were positive by 24 to 36 hours of incubation when only pretherapy cultures are evaluated. CONCLUSIONS: The ESP blood culture system identified 77%, 89% and 94% of all microorganisms at 24, 36, and 48 hours of incubation in aerobic cultures obtained from both term and preterm infants. Introduction of antimicrobial therapy did not affect time to positivity. Reducing duration of antibiotic therapy to 24 to 36 hours should be considered in term, asymptomatic newborn infants undergoing evaluation for suspected sepsis for maternal indications. Confirmation of similar rapidity of detection using other blood culture systems should be undertaken.

Anti-Bacterial Agents↗
PubMed2006

Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media.

This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.

Animals↗
PubMed1993

Differences in glycogen metabolism in astroglia-rich primary cultures and sorbitol-selected astroglial cultures derived from mouse brain.

Recently it has become possible by chemical selection using sorbitol instead of glucose in the culture medium to produce pure astroglial cultures from astroglia-rich primary cultures from mouse brain. The glycogen-degrading enzyme glycogen phosphorylase in brain is localized in astrocytes and ependymal cells. In view of this fact it appeared necessary to study the influence of glucose and other hexoses on the glycogen metabolism in these cultures lacking the influence of other cell types in comparison to the astroglia-rich primary cultures containing several types of cells. The sorbitol-fed selected cultures and the glucose-deprived astroglia-rich primary cultures contain less than 10% of the glycogen encountered in glucose-fed primary cultures. During incubation with glucose the glycogen content of the selected cultures and the glucose-deprived primary cultures increases by more than one order of magnitude. Nevertheless, not all cells are found to have accumulated glycogen. The time course of the replenishment of glycogen is similar in both types of culture, although maximal levels reached in the selected cultures are 3 times those in the astroglia-rich primary cultures. This difference might be explained by the fact that the ratio of the maximal activities of glycogen synthase and glycogen phosphorylase in selected cultures was found to be twice that in the unselected cultures. During glucose deprivation the glycogen content is reduced in both culture systems with half-maximal contents being reached at 15 min (primary culture) and 45 min (selected culture). Both types of culture can also utilize mannose for the synthesis of glycogen and the production of lactate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Optimizing culture conditions for the production of animal cells in microcarrier culture.

Cytodex 1 microcarriers have been used for the successful culture of more than 80 different types of animal cells--including primary cells, normal diploid cell strains and established or transformed cell lines. culture volumes have ranged from a few milliliters for diagnostic studies to over several hundred liters for vaccine production. Experience with this wide variety of cell types and culture volumes has enabled the identification of several parameters critical for obtaining maximum cell yields from microcarrier cultures. The most vital stage for the successful microcarrier culture of many cell types was the initial stage of the culture cycle. To achieve high cell yields, it was necessary to use culture procedures which maximized plating efficiency and final cell yield could be further increased by ensuring that the inoculation cell density exceeded a critical viable cell/microcarrier ratio. Modifications of the standard microcarrier culture procedures included reducing initial culture volume, reducing the initial stirring speed and/or supplementing the medium during the early stages of the culture cycle. Control of pH, nutrient supply, and gas tension were all critical throughout the culture cycle. Results with low-serum and serum-free media indicate that the requirement for fetal calf serum in the microcarrier culture of Vero and MRC-5 cells can be reduced or even eliminated. Large scale microcarrier culture equipment should take into account the modified culture procedures which are often required to achieve the full potential of this culture method. The design of a new flexible culture system suitable for pilot and production scale cultures is presented. This system accomodates a wide variety of culture and production procedures and through a recirculation system permits: (a) "in-line" monitoring and control of culture parameters; (b) provides an efficient gas exchange capacity which obviates the need for fermenter headspace and sparging; and (c) allows for maximal utilization of medium components and rapid harvesting of medium or cell products.

Animals↗
PubMed1997

Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium.

Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis.

Animals↗
PubMed1998

The yield of bone marrow biopsy and culture compared with blood culture in the evaluation of HIV-infected patients for mycobacterial and fungal infections.

PURPOSE: To compare the clinical utility of bone marrow biopsy and culture specimens with blood cultures for mycobacterial and fungal infections among human immunodeficiency virus (HIV)-infected patients. PATIENTS AND METHODS: All bone marrow biopsies obtained from HIV-infected patients at the University of Alabama at Birmingham (UAB) Medical Center during 1993 to 1995 were blindly reviewed in a standardized format. Bone marrow culture results and blood culture results obtained within 6 weeks of each bone marrow study were compiled. Medical records were reviewed to determine indications for performing bone marrow biopsies, empiric or prophylactic antimicrobial therapies preceding the biopsy, and CD4 counts. RESULTS: Eighty-two bone marrow studies were obtained from 76 patients. Most were performed during the evaluation of fever, cytopenia, or weight loss. Of 55 bone marrow mycobacterial cultures, 13 yielded Mycobacterium avium complex (MAC) and 2 yielded M tuberculosis (MTB). Of 51 bone marrow fungal cultures performed, 2 yielded Cryptococcus neoformans and 1 Histoplasma capsulatum. All patients with a bone marrow culture positive for MAC had a CD4 count of 20 cells/mm3 or less. The mean CD4 count in this group (+/-95% confidence interval) (8+/-3 cells/mm3) was lower than that of culture-negative cases (41+/-25 cells/mm3); P <0.015). When bone marrow cultures and mycobacterial blood cultures were concurrently obtained, results were usually in agreement between the two sites. The mean time until the report of positive mycobacterial bone marrow cultures (22+/-5 days) was similar to that for blood cultures (24+/-3 days). Most (84%) patients with multiple mycobacterial cultures had completely concordant results (all positive or all negative). When blood or bone marrow culture yielded mycobacteria, only 29% of the corresponding bone marrow examinations revealed stainable acid-fast bacilli (AFB). In contrast, all 3 cases with positive fungal bone marrow cultures also had stainable organisms on histologic examination. CONCLUSIONS: The combined use of bone marrow biopsy and culture as well as blood cultures provide the maximum diagnostic yield when evaluating patients with AIDS for mycobacterial or fungal infections. However, when mycobacterial infections were diagnosed, bone marrow results seldom provided more immediate or specific information than lysis centrifugation blood cultures. A single lysis centrifugation blood culture should be the first step in the routine evaluation of HIV-infected patients when disseminated MAC infection is suspected.

AIDS-Related Opportunistic Infections↗