Enriched Growth Medium Research Articles

Evaluation of blood culture samples for contamination and assessment of possible causes

Blood is an enriched growth medium for the growth of microorganisms. Blood culture is the key test for diagnosis of bacteremia and fungemia in patients. A false positive blood culture (blood culture contaminants) is a challenge for both consultants and microbiologists as well. This study based on highlighting source of blood culture contamination and ways of prevention in laboratory and hospitals.

Modified SHI medium supports growth of a disease-state subgingival polymicrobial community in vitro.

Developing a laboratory model of oral polymicrobial communities is essential for in vitro studies of the transition from healthy to diseased oral plaque. SHI medium is an enriched growth medium capable of supporting in vitro biofilms with similar diversity to healthy supragingival inocula; however, this medium does not maintain the diversity of gram‐negative bacteria more associated with subgingival plaque. Here, we systematically modified SHI medium components to investigate the impacts of varying nutrients and develop a medium capable of supporting a specific disease‐state subgingival community. A diseased subgingival plaque sample was inoculated in SHI medium with increasing concentrations of sucrose (0%, 0.1%, 0.5%), fetal bovine serum (FBS) (0%, 10%, 20%, 30%, 50%), and mucin (0.1, 2.5, 8.0 g/L) and grown for 48 hrs, then the 16S rRNA profiles of the resulting biofilms were examined. In total, these conditions were able to capture 89 of the 119 species and 43 of the 51 genera found in the subgingival inoculum. Interestingly, biofilms grown in high sucrose media, although dominated by acidogenic Firmicutes with a low final pH, contained several uncultured taxa from the genus Treponema, information that may aid culturing these periodontitis‐associated fastidious organisms. Biofilms grown in a modified medium (here named subSHI‐v1 medium) with 0.1% sucrose and 10% FBS had a high diversity closest to the inoculum and maintained greater proportions of many gram‐negative species of interest from the subgingival periodontal pocket (including members of the genera Prevotella and Treponema, and the Candidate Phyla Radiation phylum Saccharibacteria), and therefore best represented the disease community.

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-regeneration capacity. Nevertheless, some limitation related to donor age, muscle condition, expansion hitch, and myogenic potentiality maintenance have limited their use as therapeutic tool. To overcome this hindrance, different stem cells population with myogenic capabilities have been investigated to evaluate their real potentiality for therapeutic approaches, but, as of today, the perfect cell candidate has not been identified yet. In this work, we analyze the characteristics of skeletal muscle-derived human Mesenchymal Stem Cells (hMSCs), showing the maintenance/increment of myogenic activity upon differential culture conditions. In particular, we investigate the influence of a commercial enriched growth medium (Cyto-Grow), and of a medium enriched with either human-derived serum (H.S.) or human Platelet-rich Plasma (PrP), in order to set up a culture protocol useful for employing this cell population in clinical therapeutic strategies. The presented results reveal that both the enriched medium (Cyto-Grow) and the human-derived supplements (H.S. and PrP) have remarkable effects on hMSCs proliferation and myogenic differentiation compared to standard condition, uncovering the real possibility to exploit these human derivatives to ameliorate stem cells yield and efficacy.

Quantification of tumor virulence based upon competition between dissimilar subcultured cancer cell lines

Purpose: A direct quantitative metric that represents the intrinsic virulent properties of a particular tumor is absent, and the development of such a numeric modifier would balance the disproportionate weight of qualitative elements that presently monopolize the nomenclature of cancer. To address this shortcoming, an in vitro model was developed to study the competitive nature of dissimilar cancer cell lines. Methods: A prospective series of surgically excised and pathologically confirmed malignant tumors were separated from their adherent state and subcultured according to international standards. After a total of 7 different tumor cell lines were established, each cell line was paired with a dissimilar cancer cell line, and by using all possible combinations, a total of 21 unique tumor pairs were assessed over 7 days after plating on diametrically opposed sections of gridded Petri dishes containing enriched growth medium. Each dish was then examined for changes in tumor cell position on the grid, and a scoring system was established that awarded points for aggressive behaviors. Results: Ovarian cancer, lymphoma, and lung cancers demonstrated overall dominance, with wins in at least 2/3 of their matches. Breast and kidney cancers were intermediate in performance, and gastric and colon cancers were subordinate in the majority of their matches. Discussion: This pilot experiment revealed that intercellular competition exists between previously untreated, subcultured, and dissimilar tumor cells in this in vitro assay. Moreover, such intercellular competition and the unilateral dominance of specific cancer tissue types have not been previously reported in the literature.

PH modulates arsenic toxicity in Bacillus licheniformis DAS-2

The toxic characteristics of arsenic species, As(V) and As(III) result in ecological risks. Arsenic tolerant bacterium was isolated and identified as the Bacillus licheniformis DAS-2 through 16SrDNA sequencing. B. licheniformis DAS-2 was efficient to tolerate and remove both the As(V)[MIC 8mM] and As(III)[MIC 6mM] from the growth medium. The potential for the removal/uptake of arsenic from the 3, 5 and 7mM As(V) enriched growth media was 100%, 60% and 35% respectively and from the 1, 3 and 5mM As(III) enrichment it was 100%, 99% and 58% respectively at neutral pH. 80% of uptake As(V) was reduced to As(III) in 3mM As(V) enrichment which was gradually decreased to only 17% at 7mM As(V) enrichment at neutral pH. The arsenic toxicity in B. licheniformis DAS-2 was found modulated by pH and was examined through alteration in growth, uptake/removal, reduction and measurement of chemical toxicity.

An assessment of growth media enrichment on lipid metabolome and the concurrent phenotypic properties of Candida albicans.

A critical question among the researchers working on fungal lipid biology is whether the use of an enriched growth medium can affect the lipid composition of a cell and, therefore, contribute to the observed phenotypes. One presumption is that enriched medias, such as YPD (yeast extract, peptone and dextrose), are likely to contain lipids, which may homogenize with the yeast lipids and play a role in masking the actual differences in the observed phenotypes or lead to an altered phenotype altogether. To address this issue, we compared the lipids of Candida albicans, our fungus of interest, grown in YPD or in a defined media such as YNB (yeast nitrogen base). Mass spectrometry-based lipid analyses showed differences in the levels of phospholipids, including phosphatidylinositol, phosphatidylglycerol, lyso-phospholipids; sphingolipids, such as mannosyldiinositolphosphorylceramide; and sterols, such as ergostatetraenol. Significant differences were observed in 70 lipid species between the cells grown in the two media, but the two growth conditions did not affect the morphological characteristics of C. albicans. The lipid profiles of the YNB- and YPD-grown C. albicans cells did vary, but these differences did not influence their response to the majority of the tested agents. Rather, the observed differences could be attributed to the slow growth rate of the Candida cells in YNB compared to YPD. Notably, the altered lipid changes between the two media did impact the susceptibility to some drugs. This data provided evidence that changes in media can lead to certain lipid alterations, which may affect specific pathways but, in general, do not affect the majority of the phenotypic properties of C. albicans. It was determined that either YNB or YPD may be suitable for the growth and lipid analysis of C. albicans, depending upon the experimental requirements, but additional precautions are necessary when correlating the phenotypes with the lipids.

Bacterial Treatment and Metal Characterization of Biomedical Waste Ash

Biomedical waste ash generated due to the incineration of biomedical waste contains large amounts of heavy metals and polycyclic aromatic hydrocarbons (PAHs), which is disposed of in regular landfills, and results in unfavorable amounts of hazardous materials seeping into the ground and may pollute surface water and groundwater. Therefore, it is essential to remove the toxicity of ash before disposal into landfills or reutilization. Environmental characteristic analysis of BMW ash showed increased hardness (1320 mg/L) and chloride (8500 mg/L) content in leachate compared to World Health Organization (WHO) and Environment Protection Agency (EPA) guidelines for drinking water (hardness, 300 mg/L; chloride, 250 mg/L). The alkalinity and pH of the ash leachate were 400 mg/L and 8.35, respectively. In this paper, study was carried out to investigate the metal tolerance level of bacterial isolates isolated from soil. The isolate Bacillus sp. KGMDI can tolerate up to 75 mg/L of metal concentration (Mn, Mo, Cr, Fe, Cu, and Zn) in enriched growth medium. This shows that the isolated culture is capable of growing in presence of high concentration of heavy metals and acts as potential biological tool to reduce the negative impact of BMW ash on the environment during landfilling.

Antagonistic Bacillus cereus TC-1 Isolated from Solar Salt Work in Southern India

Bacillus cereus TC-1 was isolated from condenser pond of manmade solar salt pan in Thamaraikulam, Tamilnadu, India effectively suppressed the shrimp bacterial pathogens of Vibrio harveyi, V. parahaemolyticus, V. anguillarum, V. alginolyticus and V. vulnificus by In vitro antagonistic assay at 9 to 15 mm of zone of inhibition. Phylogenetic analysis and evolutionary distance revealed that, B. cereus TC-1 was highly similar to that the Bacillus sp. and other B. cereus strains. Their optimum growth rate was ranged between of 4 to 6% of NaCl in the growth media. The alkaline protease production was significantly (P< 0.001) differed among the different NaCl enriched growth media. Based on the antagonistic activity of the B. cereus TC-1 against the shrimp pathogens and its antimicrobial factors, it may be used as probiotics and developing novel antimicrobial bioactive substances against aquatic pathogens.

Overproduction or Absence of the Periplasmic Protease DegP Severely Compromises Bacterial Growth in the Absence of the Dithiol: Disulfide Oxidoreductase DsbA

Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in photosynthesis but are defective in respiration especially in enriched growth medium at 35 degrees C. They also exhibit severe pleiotropic phenotypes extending from motility defects to osmofragility and oxidative stresses. In this work, using a combined proteomics and molecular genetics approach, we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originates from the overproduction of the periplasmic protease DegP, which renders them temperature-sensitive for growth. The DsbA-null mutants reverted frequently to overcome this growth defect by decreasing, but not completely eliminating, their DegP activity. In agreement with these findings, we showed that overproduction of DegP abolishes the newly restored respiratory growth ability of the revertants in all growth media. Structural localizations of the reversion mutations in DegP revealed the regions and amino acids that are important for its protease-chaperone activity. Remarkably although R. capsulatus DsbA-null or DegP-null mutants were viable, DegP-null DsbA-null double mutants were lethal at all growth temperatures. This is unlike Escherichia coli, and it indicates that in the absence of DsbA some DegP activity is required for survival of R. capsulatus. Absence of a DegQ protease homologue in some bacteria together with major structural variations among the DegP homologues, including a critical disulfide bond-bearing region, correlates well with the differences seen between various species like R. capsulatus and E. coli. Our findings illustrate the occurrence of two related but distinct periplasmic protease families in bacterial species.

ECONOMIZATION INTENSIVE OUTDOOR MASS PRODUCTION OF THE GREEN ALGA SCENEDESMUS SP.

The green alga Scenedesmus sp. was out door scaled-up within three cement open ponds with a final capacity of 15m3 for each one. The used nutrient solution was composed of tap water enriched by macronutrients (NPK) from commercial sources. Dilution of cultures was done according to growth rate and nutrients supplementation as growth reached their maximum dry weight. Continuous centrifugation was carried out at the end of each batch and the obtained biomass was calculated. A comparison was performed; during long term successive season; as growth was employed under the recommended amount of NPK. It was found that electric power represented the maximum cost and the net cost was found in contrast with the volume of growth unit. In addition, enriched growth medium by extra amount of urea and micronutrients led to yield maximization. Maximum cost was observed during inoculums preparation and the lowest cost was found with cultures grown in large scale.

The Zn- and Cd-Clusters of Recombinant Mammalian MT1 and MT4 Metallothionein Domains Include Sulfide Ligands

Recombinant (E. coli ) synthesis of mammalian MT1 and MT4 domains as separate peptides in Zn(II) and Cd(II) enriched growth media has rendered metal complexes containing sulfide anions as additional ligands. The Cd preparations show higher sulfide content than the Zn preparations. Also, the betaMT1 and betaMT4 fragments exhibit higher sulfide/peptide ratios than the respective alpha fragments. Titration of Zn3-betaMT1 with Cd(II) followed by addition of several sodium sulfide equivalents shows that the Cd(II)-betaMT1 species can incorporate sulfide ligands in vitro, with a concomitant evolution of their UV-vis and CD fingerprints to those characteristic of the Cd-S2- chromophores. Current results have also provided full understanding of previous data collected by this group in the characterization of the Cd-betaMT1 preparations obtained from large-scale fermentor synthesis by allowing identification of at least 2S2- ligands per Cd-betaMT1 species. Furthermore, the results here presented have revealed that synthesis of betaMT4 in Cd-supplemented cultures yielded Cd,S(2-)-containing clusters instead of the proposed heterometallic Zn,Cd-betaMT4 complexes. Finally, a global evaluation of our results suggests that the higher the Cu-thionein character of a MT peptide, the higher is its tendency to harbor nonproteic ligands (i.e., sulfide anions) when building divalent metal clusters, especially Cd-MT complexes.

Efficient purification of unique antibodies using peptide affinity-matrix columns

Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85–99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells. Investigations of the fine specificity of the ER6.1 peptide demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER6.1 peptide.

Rapid automated antimicrobial susceptibility testing of Streptococcus pneumoniae by use of the bioMerieux VITEK 2.

The VITEK 2 is a new automated instrument for rapid organism identification and susceptibility testing. It has the capability of performing rapid susceptibility testing of Streptococcus pneumoniae with specially configured cards that contain enriched growth medium and antimicrobial agents relevant for this organism. The present study compared the results of testing of a group of 53 challenge strains of pneumococci with known resistance properties and a collection of clinical isolates examined in two study phases with a total of 402 and 416 isolates, respectively, with a prototype of the VITEK 2. Testing was conducted in three geographically separate laboratories; the challenge collection was tested by all three laboratories, and the unique clinical isolates were tested separately by the individual laboratories. The VITEK 2 results of tests with 10 antimicrobial agents were compared to the results generated by the National Committee for Clinical Laboratory Standards reference broth microdilution MIC test method. Excellent interlaboratory agreement was observed with the challenge strains. The overall agreement within a single twofold dilution of MICs defined by the VITEK 2 and reference method with the clinical isolates was 96.3%, although there were a number of off-scale MICs that could not be compared. The best agreement with the clinical isolates was achieved with ofloxacin and chloramphenicol (100%), and the lowest level of agreement among those drugs with sufficient on-scale MICs occurred with trimethoprim-sulfamethoxazole (89.7%). Overall there were 1.3% very major, 6.6% minor, and no major interpretive category errors encountered with the clinical isolates, although >80% of the minor interpretive errors involved only a single log(2) dilution difference. The mean time for generation of susceptibility results with the clinical isolates was 8.1 h. The VITEK 2 provided rapid, reliable susceptibility category determinations with both the challenge and clinical isolates examined in this study.

Effect of turbulence and inorganic carbon supply on growth ofDunaliella viridis Teodoresco

Turbulence is known to enhance growth in microalgae. We studied the possible effects of the extra inorganic carbon supplied to the cultures when air bubbling is the source of turbulence. A set of experiments was carried out to separate the effects of the subsidiary action of agitation on growth from those due to possible carbon enrichment from air CO2 when air bubbling was the source of mixing. It was found that air bubbling does not induce an increase of the concentration of inorganic carbon in HCO 3 − -enriched growth media, and that no differences appear between air-bubbled and CO2-free air-bubbled cultures. On the other hand, photosynthetic O2 was accumulated in the medium in non-mixed cultures, which showed higher respiration and lower photosynthetic rates, probably due to photoinhibition induced by oversaturation of O2. Growth rate, maximal cell density and photosynthetic efficiency were higher in aerated cultures than in non-aerated ones.

Overcoming Inclusion Body Formation in a High-Level Expression System

Attempts at overexpressing T4-phage deoxycytidylate deaminase using the pET3c/BL21(DE3)/pLysS system resulted in this enzyme being part of an inactive inclusion-body complex. However, by employing an enriched growth medium it was found that the deaminase could be induced in a soluble active form to at least 20% of this organism′s cellular protein. Insoluble inclusion bodies were obtained with less rich media. This procedure was employed successfully with other highly expressed proteins that formed inclusion bodies. The use of a rich growth medium during the course of protein induction may be a valuable adjunct to limiting inclusion body formation with this as well as other expression systems.

Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans.

Growth inhibition and cell viability assays demonstrate that the histidine-rich polypeptides isolated from human parotid saliva are bacteriostatic and bactericidal for strains of Streptococcus mutans belonging to the serotype b and c classifications. Both inhibition of growth and cell division are enhanced by preincubation of bacteria with these polypeptides in low-ionic-strength buffers of acidic and neutral pH before dilution into enriched growth media. With prior exposure at pH 6.8, inhibition by these polypeptides of the serotype c strains, S. mutans GS5 and SB, as well as the serotype b strain, S. mutans BHT, is reversible over time under the experimental conditions selected. With similar exposure at pH 5.2, however, irreversible damage is manifested by complete inhibition of both growth and cell viability. At concentrations of 250 micrograms of the mixture of histidine-rich polypeptides per 5 X 10(5) bacterial cells per ml in the acidic preincubation buffer, bacterial lethality is maintained for a period of 48 h in the enriched growth media. At a 50-micrograms/ml concentration of these salivary agents, approximately 80% killing of S. mutans SB is noted after a 24-h incubation; however, surviving bacteria multiply and reach turbidities of untreated control cells when examined at the 48-h growth point. Similarly, hen egg white lysozyme is also found to be bactericidal for these microorganisms when preincubation is carried out under acidic conditions. However, in contrast to the histidine-rich polypeptides, lysozyme under these experimental conditions does not inhibit growth of S. mutans SB at neutral pH, although it does inhibit growth of both S. mutans BHT and S. mutans GS5 at this pH. Preexposure of S. mutans SB to the peptides in buffer at ionic strengths of 0.025 to 0.125, followed by either viability assays under nongrowing conditions or growth inhibition studies, suggests that there is very little effect of ionic strength on the antibacterial function of these peptides. In contrast to the inhibition of viability noted under growing conditions, lower concentrations of the histidine-rich polypeptides were required to elicit immediate cell death under nongrowing conditions.

Alteration of Acrylonitrile-Methylacrylate-Butadiene Terpolymer by Nocardia rhodochrous and Penicillium notatum

[C]Barex-210, a terpolymer of acrylonitrile, methylacrylate, and butadiene, was tested for bioconversion. Powdered samples of polymer, each specifically C labeled at different carbon atoms of the polymer, were incubated with either Nocardia rhodochrous or Penicillium notatum in an enriched growth medium for various periods of time. After 6 months of incubation, the C-labeled polymer was transformed from a high-molecular-weight material completely soluble in dimethyl formamide (DMF) into both a lower-molecular-weight form still soluble in DMF and a second form that was no longer soluble in DMF. The amount of C-labeled carbon atoms converted into DMF-insoluble material was 8% of the backbone carbon-carbon atoms and 12% of the side-chain nitrile and acrylate atoms from the acrylonitrile-methylacrylate copolymer and 60% of the elastomer (acrylonitrile-butadiene copolymer) atoms. Metabolism of the polymer was not established from measurements of metabolic CO(2). Evolution of CO(2) amounted to only 0.3, 0.6, 1.8, and 3.3% of these four fractions, respectively. Although the transformation of high-molecular-weight polymer into DMF-insoluble material was rapid in the early stages of microbial growth, the accompanying CO(2) evolution was much slower. Further evidence of polymer alteration was indicated by the infrared spectrum of the insoluble material, which showed a disappearance of the nitrile and methylacrylate peaks.

Participation of the intracellular enzymes in the control of mutational processes

The influence of UV-specific endonuclease and medium composition on the frequency and spectrum of genic mutations in Escherichia coli KI2 uvr (+) (with normal repair enzymes) and urv A6 (defective in UV-specific endonuclease) was studied. Mutations at the locus glu (gene controlling assimilation of glucose) were induced by ultra-violet irradiation and hydroxylamine treatment. To identify mutant colonies, triphenyl tetrazolium chloride (TTC) was added to the medium since it coloured the mutant colonies bright crimson and readily permitted distinction between pure mutant clones (complete mutations) and mixed clones (mosaic or sector mutations).A maximum mutation frequency after UV-irradiation was observed in E. coli uvr (+) cells but not in the E. coli uvr A6 strain. The curve of mutagenesis with a maximum was found in both studied strains after treatment by hydroxylamine which did not cause DNA damage recognized by UV-specific endonuclease.The highest frequency of mutations (at the point of maximum) in the series of experiments with enriched growth medium was almost 10 times higher than in the series of the experiments with poor medium.It was established that in bacteria with normal repair enzymes the frequency of complete mutations was higher than the frequency of mosaic mutations. It was also observed that the rate of UV-mutagenesis was higher in the case of E. coli uvr (+).The study of the distribution of mosaic mutant sectors in experiments with bacteria suspended in either a nutrient broth or a buffer during UV-irradiation revealed that the size of mutant sectors was rather variable and that the differences in the number of nucleoids per cell did not always determine the distribution of mutant sector sizes.

Complete differentiation of adipocyte precursors. A culture system for studying the cellular nature of adipose tissue.

Evidence for the complete morphological maturation of precursor cells into adipocytes in vitro is presented. Cells were isolated from the stromal fraction of adipose tissue from adult humans and from rats and were grown in culture. Abdominal skin fibroblasts were used as controls. All cell strains were initially fusiform and replicated. On reaching monolayer confluency, they were transferred to an enriched growth medium in which the human and rat adipocyte precursors differentiated into a homogeneous population of cells, morphologically indistinguishable from mature adipocytes. In contrast, skin fibroblasts from the same person or animal, and grown under identical culture conditions, did not accumulate lipid and retained their fusiform contour. The same results were obtained in the first six subcultures that were studied. Thus, there is firm evidence that fat tissue of adult humans and rats contains adipocyte precursors that differentiate into mature fat cells. The culture system that has been described will facilitate the elucidation of the factors involved in replication and differentiation of adipocyte precursors.

Age-dependent Excystment of the Protozoan Acanthamoeba castellanii

Summary: Axenic cyst cultures of Acanthamoeba castellanii (Neff strain) maintained in salt medium undergo excystment when transferred into enriched growth medium containing yeast extract and proteose peptone. Both the synchrony and extent of excystment induced in this manner are critically dependent upon the length of time cysts are aged in salt medium prior to the initiation of excystment. Cysts sustained in salt medium for 50 h or less characteristically show only about 10 % excystment even after 70 h in the enriched growth medium. With progressively longer periods of ageing the synchrony and degree of excystment improve such that for cysts of age ≥ 800 h, more than 85 % of the organisms germinate within a period of 12 h. Thus more complete and relatively synchronous excystment can be achieved by this simple experimental manipulation.