Discover the most talked about and latest scientific content & concepts.

Concept: Neutral red


Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities.

Concepts: Bacteria, Microbiology, Yeast, Agar, Agar plate, Growth medium, Formazan, Neutral red


Non-predatory dead variability in zooplankton remains poorly quantified worldwide. Here, we make the first estimation of the percentage of dead organisms in coastal zooplankton communities in the Humboldt Current System (HCS) under in situ conditions. The study was conducted in four coastal sites of the southern HCS (between 36 and 37°S) over a period of one year. Percentages of dead organisms were based on the classification as live or dead of 158,220 holoplankton and 17,591 meroplankton individuals using neutral red staining technique. The percentage of dead organisms in total-zooplankton was between 4.3% in Coronel Bay (summer) and 76.9% in Llico (autumn). The percentage of dead total-holoplankton varied from 4.2% (Itata River Mouth; autumn) to 77.6% (Llico; autumn), while the percentage of dead total-meroplankton ranged from 1.5% to 56.8% in Coronel Bay and Coliumo Bay, respectively. The most abundant taxa analyzed were the copepods Acartia sp., Paracalanus sp., Calanoides sp., Cladocera, Polychaeta, and the eggs of anchoveta Engraulis ringens. Among these taxa, there was a high degree of interspecific variability in the estimation of the dead organisms. The Pearson correlation shows significant relationships between maximum temperature, and minimum salinity, with the percentage of dead individuals of Acartia sp. and Paracalanus sp. Environmental factors explaining those relationships were: the El Niño 2015-2016 event, and freshwater river runoff. The use of vital staining to estimate non-predatory death for total-zooplankton and selected sentinel species is a promising tool to establish baselines to evaluate natural perturbations (e.g. ENSO), and anthropogenic alterations in coastal pelagic ecosystems.

Concepts: Statistics, Biology, River, Staining, Freshwater, Gram staining, Plankton, Neutral red


Tendons are dense, fibrous connective tissues which carry out the essential physiological role of transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy is very common, both within the sporting arena and amongst the sedentary population. Studies have shown that light therapy may stimulate tendon healing, and more recently, intense pulsed light (IPL) has attracted attention as a potential treatment modality for tendinopathy; however, its mechanism of action and effect on the tendon cells (tenocytes) is poorly understood. The present study therefore investigates the influence of IPL on an in vitro bovine tendon model. Tenocytes were irradiated with IPL at different devise settings and under variable culture conditions (e.g. utilising cell culture media with or without the pH indicator dye phenol red), and changes in tenocyte viability and migration were subsequently investigated using Alamar blue and scratch assays, respectively. Our data demonstrated that IPL fluencies of up to 15.9 J/cm(2) proved harmless to the tenocyte cultures (this was the case using culture media with or without phenol red) and resulted in a significant increase in cell viability under certain culture conditions. Furthermore, IPL treatment of tenocytes did not affect the rate of cell migration. This study demonstrates that irradiation with IPL is not detrimental to the tenocytes and may increase their viability under certain conditions, thus validating our in vitro model. Further studies are required to elucidate the effects of IPL application in the clinical situation.

Concepts: Collagen, Muscle, Cell culture, Connective tissue, Tendon, PH indicator, Growth medium, Neutral red


This study sought to characterize the composition and morphology of acellular mineralization occurring in thermally and chemically gelable hydrogels comprising copolymers of hydrophobic N-isopropylacrylamide as a function of hydrogel hydrophobicity and culture medium formulation. The deposition of calcium phosphate (CaP) mineral was hypothesized to occur with increasing hydrogel hydrophobicity and presence of serum proteins in the culture medium. Two hydrogel compositions with a solid content of 15 and 20 wt% were examined in serum-containing and non-serum-containing media for 0, 14, 28, and 56 days. Using biochemical assays, calcium, but not phosphate content, was found to significantly increase over time in hydrophobic hydrogels soaked in cell culture medium with fetal bovine serum. Significant increases in the calcium to phosphate ratio were observed within these hydrogels from day 0 to 56, with mineralization indicated by von Kossa histological staining. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy/energy dispersive x-ray spectroscopy (SEM/EDX) were used to analyze CaP mineral characteristics. No crystalline apatitic reflection peaks were observed using XRD, which was supported by the lack of observable mineral deposits as observed using SEM/EDX. However, FTIR showed the presence of new absorption peaks in the serum-containing samples at 28 and 56 days which suggested the formation of an immature apatitic-like mineral. The ability to undergo hydrophobicity-dependent and protein-mediated mineralization demonstrates the potential of these dual-gelling hydrogels as acellular self-mineralizing materials for bone tissue engineering. This article is protected by copyright. All rights reserved.

Concepts: Spectroscopy, Bone, Dietary mineral, Cell culture, Mineral, Infrared spectroscopy, Fourier transform spectroscopy, Neutral red


Local administration of insulin from a titanium surface has been demonstrated to increase bone formation in non-diabetic rats. The authors hypothesized that insulin was released from the titanium surface and with preserved biological activity after the release. Thus, in the present in vitro study, human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro-chemiluminescence immunoassay. Neutral Red uptake assay and mineralization assay were used to evaluate the biological effects of the released insulin on human osteoblast-like MG-63 cells. The results confirmed that insulin was released from titanium surfaces during a six-week period. Etching the disc prior to insulin coating, thickening of the insulin coating and incubation of the discs in serum-enriched cell culture medium increased the release. However, longer storage time decreased the release of insulin. Furthermore, the released insulin had retained its biological activity, as demonstrated by the significant increase in cell number and a stimulated mineralization process, upon exposure to released insulin. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

Concepts: DNA, Bacteria, Glucose, Cell biology, Cell culture, In vitro, Growth medium, Neutral red


Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.

Concepts: Chondroitin sulfate, Extracellular matrix, Proteoglycan, Cartilage, Autologous chondrocyte implantation, Chondrocyte, Boer goat, Neutral red


Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco’s modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

Concepts: Bone, Vitamin D, Bacteria, Cell culture, Vitamin C, Ascorbic acid, Growth medium, Neutral red


Objective To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein(P-gp)and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology. Methods The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium(pH 6.6),while the control group was treated with a neutral cell culture medium(pH 7.4). The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile,the cytotoxicity of daunomycin was analyzed by cell live/dead fluorescence staining method. Results Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However,the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile,the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil. Conclusions Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multi-drug resistance reversal agents.

Concepts: Gene expression, Cancer, Bacteria, Cell culture, Growth medium, Neutral red


In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB- (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB-) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199)+10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5µgmL(-1) of FSH, 5mgmL(-1) of LH, and 2µgmL(-1) of E2; the T2 group was matured in the presence of 1µgmL(-1) of FSH, 6mgmL(-1) of LH, and 2.5µgmL(-1) of E2; and the T3 group was matured in the presence of 1.5µgmL(-1) of FSH, 7mgmL(-1) of LH, and 4.5µgmL(-1) of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB-, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB-, and not exposed to BCB respectively). However, BCB- oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1µgmL(-1) of FSH, 6mgmL(-1) of LH, and 2.5µgmL(-1) of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

Concepts: Africa, Polar body, Neutral red


The viability of conidia of Esteya vermicola, a potentially important biocontrol agent against the pinewood nematode Bursaphelenchus xylophilus, is usually determined by cultivation for 18-48 h in culture medium. As an alternative to this labor-intensive method, we have developed a rapid, simple, and low-cost staining method for assessing E vermicola conidia survival rates. A mixture of neutral red and methylene blue was found to be the most optimal among several stains that also included safranin O and Janus green B. This mixture stained nonviable conidia blue, in contrast to viable conidia, which were stained red in the cytoplasm and blue in the cell wall. This method may be particularly useful for traditional research laboratories, as it provides rapid results using common, relatively inexpensive laboratory equipment.

Concepts: Cell, Bacteria, Nematode, Nematodes, Stain, Bursaphelenchus xylophilus, Bursaphelenchus, Neutral red