Sterile Liquid Medium Research Articles

Automatic inoculation of bacterial cultures using low-melting point, wax-like coconut oil

A simple method for the automatic inoculation of broth culture tubes is described. An incubator is plugged into an electrical outlet timer. Heating of the incubator causes wax-like coconut oil to melt and allow an inoculum-coated bead to drop into sterile liquid medium.

Aggregate Formation in a Kaolinite Suspension during Microbiological Modification of Clay Surface

An aggregate-forming effect of the microorganisms that modify a hydrophilic surface of minerals to an amphiphilic one was demonstrated in a model experiment: a sterile liquid medium was supplemented with the microorganism Bacillus velezensis washed off from river sand and with kaolinite, which contains potassium ions (up to 1%) in a sorbed state and in admixtures. An increase in the protein, carbon, and nitrogen contents was observable over two months of incubation; the C : N ratio reached a value close to 5. It was assumed that the microorganisms consuming potassium ion necessary for their vital activity produce protein compounds in the form of specific secretions of glycoprotein and polysaccharide natures. These compounds hydrophobized the surface of minerals (the contact angle increased from 20° to 40°), changed the specific surface area, and increased the share of microaggregate fractions (50–250 and 250–500 µm) by 5.4 and 1.5%, respectively. The incubation with B. velezensis hydrophobized the kaolinite surface as compared with the control specimens, most likely because of an amphiphilic nature of the microbial products. In turn, the hydrophobization of mineral surface induced a hydrophobic interaction between mineral particles thereby forming organomineral microaggregates.

Effects of plant growth regulators on in vitro seed germination and seedling development of Enhalus acoroides, a tropical seagrass

The effects of plant growth regulators (PGRs) on seed germination and seedling development of Enhalus acoroides were investigated under axenic conditions. Seeds were cultured in sterile liquid medium supplemented with different concentrations of 6-benzyladenine (BA) × α-naphthylacetic acid (NAA) and N-phenyl-N’-(1,2,3-thidiazol-5-yl) urea (TDZ) × 2,4-dichlorophenoxyacetic acid (2,4-D). All seeds fully germinated (100% germination rates) within 1 d on liquid media without PGRs, while germination of seeds in liquid media supplemented with PGRs was delayed with 94–100% germination within 2–7 d. Embryogenic callus was obtained after 2 wk when cultured in liquid medium supplemented with 18 μM BA and 160 μM NAA. The addition of TDZ and 2,4-D inhibited root emergence. The results showed a significant effect of the culture medium on seedling development, but not on seed germination.

Rapid detection and toxicity assessment of ochratoxin A by Photobacterium leiognathi in drinking water

SummaryThis study was aimed to develop a rapid technique for detection of ochratoxin A (OTA) by Photobacterium leiognathi (P. leiognathi) based on its inhibition of luminescence on P. leiognathi. The freeze‐dried powder of P. leiognathi was incubated and grown aerobically in sterile liquid medium at 28 °C for 20 h. Optical cell density (OD) at wavelength of 610 nm was measured using UV spectrometer every 2 h. Different concentrations of standard OTA solution were used to measure its luminescence inhibition and calculate its half maximal inhibitory concentration (IC50). Scanning electron microscopy (SEM), flow cytometry (FCM), sodium dodecyl sulphonate polyacrylamide gel electrophoresis (SDS‐PAGE), DNA extraction and gel electrophoresis were used to observe the performance of P. leiognathi under the treatment of OTA. A correlation (R2 > 0.98) was obtained between the relative luminosity unit of P. leiognathi and OTA concentration in the range of 0.01–20 mg L−1 with recoveries of 80.8–87.4%. The effects of OTA on P. leiognathi are time‐dependent, and the IC50 value of 12.71 mg L−1 at 30 min demonstrated its good sensitivity to OTA. The cells of P. leiognathi under 40 mg L−1 OTA exposure for 30 mins showed morphological alterations, protein damage, apoptosis and necrosis. The aforementioned results indicate that biological assay is a promising and alternative method used for rapidly monitoring the sudden pollution of OTA in the early emergency warning of drinking water system.

Optimasi Produksi Inokulan Cair Trichoderma harzianum Berbasis Molase

Trichoderma harzianum as a potential biological agent and are widely used to control diseases in various agricultural crops and plantations. This research goal of getting the best growth of Trichoderma harzianum on molasses-based liquid medium. The treatments tested was added 0.05%, 0.1% and 1% liquid pure culture of Trichoderma harzianum each in 500 ml of sterile liquid medium containing 5% molasses (v/v) and 5% commercial yeast (b / v), and incubated for five days. The results showed that Trichoderma harzianum growth with the highest number of spores occur if the molasses-based liquid medium supplemented with 1% Trichoderma harzianum.

Effect of bone mesenchymal stem cells transplantation on neurological function and learning and memory of traumatic brain injury rats by the external carotid artery

Objective To study the effect of bone mesenchymal stem cells(BMSC) transplantation into traumatic brain injury(TBI) rats by the external carotid artery on neurological function and learning and memory. Methods Ten adult SD rats were randomly divided into TBI group (n=5) and BMSC transplantation group (n=5). Feeney free falling method was used to establish TBI models. The experimental rats were administrated with BMSC via external carotid artery (ECA), while TBI rats were injected with sterile liquid medium of equal volume via right ECA.Neurological function were evaluated according to the modified neurological severity score (NSS) at 1, 3, 7, 15 days.Morris water maze test was used to observe the animal capabilities of place navigation and space exploration at 15 days, then animals were sacrificed. Survival and migration of implanted BMSC in brains under fluorescence microscope. Result After traumatic brain, varying degrees convulsions, paralysis, loss of balance function in rats were found.Compared with TBI group, BMSC transplantation decreased significantly NSS (P<0.01). BMSC transplantation significantly decreased on escape latency((20.48±2.29)s) than the TBI group((85.93±47.48)s) (P<0.01). Moreover, BMSC group in the target quadrant dwell time ((28.62±1.72)%) and distance ((29.05±3.08)%) as well as the number of passing the platform (8.00±2.45) were significantly higher than the TBI group ((19.37±2.81)%, (21.78±3.06)%, (2.00±1.87) respectively, P<0.01). Transplanted BMSC could survive and migrate around injury brain through Hochest mark immunofluorescence. Conclusion BMSC can survive and migrate around injury brain by transplantation of external carotid artery, which results in a significant neurological function improvement and learning and memory increase in rats with traumatic brain injury. Key words: Brain trauma; BMSC graft; External carotid artery; Neurological function; Learning and memory

Field experimental evidence that stochastic processes predominate in the initial assembly of bacterial communities.

To assess the relative importance of environmental selection, dispersal and stochastic processes in structuring ecological communities, we conducted a bacterial community assembly experiment using microcosms filled with sterile liquid medium under field conditions in the Inner Mongolian grasslands. Multiple replicate microcosms containing different carbon substrates were placed at nine locations across three spatial scales (10, 300 and 10 000 m distance between locations) in such a way that the environment of microcosms varies independently of the geographical distance. The operational taxonomic units within the experimental communities were assessed via the terminal restriction fragment length polymorphism techniques on the 10th and 17th days after the onset of the experiment. We found no evidence of distance decay in community similarity, and communities within a given location were more similar to each other regardless of environment than communities at other locations within the same spatial scale. Variance partitioning indicated that location explained more compositional variation in microbial communities than environment, particularly on the 17th day, despite that environment and location in combination could only explain less than half of the total variation. These results suggest that bacterial dispersal is not limited by distance in this experiment, and community assembly in microcosms is not environmentally determined but governed by stochastic processes.

The Volatiles from Fermentation Product of &amp;lt;i&amp;gt;Tuber formosanum&amp;lt;/i&amp;gt;

The mycelium of T. formosanum (characterized by DNA analysis) grown in a sterile liquid medium produced some VOCs. The VOCs were analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 23 compounds were identified and quantified. Among them, the main compounds were Dimethyl sulfide (19.82%), Isopropyl alcohol (9.84 ng/l), 2-Butanone (9.24%), Ethanol (7.84%), and 1, 3-Pentadiene (5.46%).

Isolation and characterization of Aeromonas schubertii from diseased snakehead, Channa maculata (Lacepède)

Pure bacterial cultures were isolated from diseased snakeheads, Channa maculata (Lacepède), suffering high mortality in a farm in Zhongshan, southern China. Three isolates, namely ZS20100725, ZS20100725-1 and ZS20100725-2, were identified as Aeromonas schubertii. All the isolates showed high 16S rRNA sequence similarities with A. schubertii. The isolates exhibited strong virulence to snakeheads in experimental challenges with LD(50) ranging between 1.4 × 10(4) and 6.4 × 10(6) CFU g(-1). Two of the isolates were positive for haemolysin, elastase, lipase and lecithinase by phenotypic determination, which was further confirmed by PCR amplification of the haemolysin and elastase genes. In sterile liquid medium, the best growth conditions of strain ZS20100725 were 30 °C, pH 7 and 0.5% salinity (w/v). Antibiotic susceptibility tests showed that strain ZS20100725 was susceptible to cefoxitin, cefoperazone and chloramphenicol. Furthermore, histopathology of diseased snakeheads infected with A. schubertii showed necrosis and congestion in liver, kidney and spleen and also damage to the cardiac muscle, intestine and gills.

Cytoskeleton and mitochondrial morphology of saprotrophs and the pathogen Heterobasidion annosum in the presence of Suillus bovinus metabolites

Ectomycorrhizal fungi are known to synthesize antifungal compounds both in vitro and in symbiosis with the host-plants. Culture filtrates of the ectomycorrhizal fungus Suillus bovinus (at pHs of 2.5–6) showed antifungal activity towards saprotrophs Trichoderma harzianum, and Trichoderma virens and the pathogen Heterobasidion annosum, by significantly suppressing their growth relative to sterile liquid medium at the same pHs. In the presence of the culture filtrates, hyphae of the saprotrophs and the pathogen were characterized by distensions, irregular and frequent branching, tip damage and cytoplasm coagulation. Since hyphal abnormalities may be evoked by disruptions in the cytoskeleton and mitochondria, their structural changes were also examined. Depolymerization of microtubules was confirmed for all of the fungi. Serious damage to mitochondria morphology may cause significant functional impairment. Growth of mycelia was inhibited in the lower pH S. bovinus culture filtrate, and the mitochondrial morphology was altered. This suggests that the activity of antifungal compounds synthesized by ectomycorrhizal fungus is significantly affected by pH.

Preparation and characterization of a hydroxyapatite–collagen composite as component for injectable bone substitute

The paper presents an improved sol–gel technique for the preparation of hydroxyapatite–atelocollagen composites suitable as precursor for osseous regeneration. This technique includes a premineralization step of atelocollagen providing a suspension of microfibrils in which the synthesis of the inorganic component was conducted at low temperature. The characterization of the composite was performed by FTIR spectroscopy, X-ray diffraction and scanning electron microscopy. The particle size distribution and the zeta potential were determined by Zetasizer measurements. The time stability of the composite was evaluated by turbidimetric measurements and macroscopic observation. The composites can be stored for a long time in sterile liquid media without a major risk of aggregation or sedimentation. From the final composite a slurry was obtained by sedimentation in centrifugal field and oscillatory rheological experiments were made to get information about its flow behavior. The slurry could be used as an injectable osteoconductive component, able to act as stock form in orthopaedic kits.

Plasticity and Constraint in Growth and Protein Mineralization of Ectomycorrhizal Fungi under Simulated Nitrogen Deposition

Ectomycorrhizal fungi allow their host plants access to organic forms of N through enzymatic mineralization of the substrate and enhanced absorption of amino acids and mineral N. The cost to the plant is carbohydrates that support fungal growth and metabolism. Enrichment of soils with mineral N, as through atmospheric deposition, may affect the growth and function of these fungi by direct effects of increased N availability on fungi and indirect effects through reduced plant C allocation to roots. We tested the potential of N enrichment and altered carbohydrate supply to affect the growth and protein mineralization activity of 10 ectomycorrhizal fungi in sterile liquid media. Nitrogen treatments consisted of organic N only vs organic plus mineral N. Carbon treatments consisted of 5 g per liter glucose vs. no glucose added. Fungi differed widely in their growth and mineralization responses to these variables. Seven of 10 fungi had at least 20% reduced growth with reduced carbohydrates. Only 2 of 10 increased growth by 20% or more with increased mineral N. Carbohydrates affected growth more in a purely organic N environment suggesting an energy limitation to mineralization. Protein mineralization activity tended to be depressed by reductions in carbohydrates and increased by increased mineral N. The high sensitivity of fungal growth to carbohydrates suggests important indirect effects of N enrichment via altered C allocation in host trees. Principal Components analysis separated most fungal species along an axis representing a gradient from high protein mineralization efficiency to high intrinsic growth rate. Those fungi with slow growth and efficient mineralization activity corresponded closely to fungi often cited as late successional species, while fungi with high growth rates and low mineralization efficiency are often categorized as early successional. One fungus, Cenococcum geophillum, separated from others on an axis representing strong N dependence in growth. Nitrogen enrichment has the potential to alter the composition and function of the ectomycorrhizal fungus community. Physiological differences among species provide a starting point for predicting community responses and anticipating ecosystem consequences.

Plasticity and constraint in growth and protein mineralization of ectomycorrhizal fungi under simulated nitrogen deposition

Ectomycorrhizal fungi allow their host plants access to organic forms of N through enzymatic mineralization of the substrate and enhanced absorption of amino acids and mineral N. The cost to the plant is carbohydrates that support fungal growth and metabolism. Enrichment of soils with mineral N, as through atmospheric deposition, may affect the growth and function of these fungi by direct effects of increased N availability on fungi and indirect effects through reduced plant C allocation to roots. We tested the potential of N enrichment and altered carbohydrate supply to affect the growth and protein mineralization activity of 10 ectomycorrhizal fungi in sterile liquid media. Nitrogen treatments consisted of organic N only vs organic plus mineral N. Carbon treatments consisted of 5 g per liter glucose vs. no glucose added. Fungi differed widely in their growth and mineralization responses to these variables. Seven of 10 fungi had at least 20% reduced growth with reduced carbohydrates. Only 2 of 10 increased growth by 20% or more with increased mineral N. Carbohydrates affected growth more in a purely organic N environment suggesting an energy limitation to mineralization. Protein mineralization activity tended to be depressed by reductions in carbohydrates and increased by increased mineral N. The high sensitivity of fungal growth to carbohydrates suggests important indirect effects of N enrichment via altered C allocation in host trees. Principal Components analysis separated most fungal species along an axis representing a gradient from high protein mineralization efficiency to high intrinsic growth rate. Those fungi with slow growth and efficient mineralization activity corresponded closely to fungi often cited as late successional species, while fungi with high growth rates and low mineralization efficiency are often categorized as early successional. One fungus, Cenococcum geophillum, separated from others on an axis representing strong N dependence in growth. Nitrogen enrichment has the potential to alter the composition and function of the ectomycorrhizal fungus community. Physiological differences among species provide a starting point for predicting community responses and anticipating ecosystem consequences.

The volatile organic compounds from the mycelium of Tuber borchii Vitt.

The mycelium of T. borchii (characterized by DNA analysis) grown in sterile liquid medium produced some VOCs. The VOCs were retained on carbographs by passing a flow of helium, isolated and characterized in a GC–MS equipment after a thermal desorption. The compounds present in the VOCs from the mycelium cultures, but not in the VOCs from the control cultures, contained 29 compounds. The main compounds were 1,3-ditertbutylbenzene (16.1 ng/l), 3-methylheptane (9.2 ng/l), butan-2-one (8.8 ng/l), ethynylbenzene (5.6 ng/l), and octan-3-one (4.9 ng/l).

Ascorbic Acid: A Precursor of Oxalate in Crystal Idioblasts ofYucca torreyiin Liquid Root Culture

Liquid‐cultured primary roots of Yucca torreyi L. (Agavaceae), which are similar to its intact roots, develop uninterrupted files of calcium oxalate crystal idioblasts with raphide bundles in their cortex, beginning just proximal to the terminal meristem. Each single file of idioblasts displays a basipetal ontogenetic sequence. [1‐14C]glycolic acid, [1‐14C]glyoxylic acid, and l‐[1‐14C]ascorbic acid, all of which are potential precursors of oxalate, were each added to different flasks that contained a sterile liquid medium and isolated roots and were allowed to interact with the roots for 45 min. After thorough washing, the roots grew for periods that extended from 1.6 h to 24 h postincorporation before being fixed for light microscope autoradiography. Autoradiography of root sections with the l‐[1‐14C]ascorbic acid at the 1.6–6.0‐h incorporation times showed concentrations of silver grains over the idioblasts, primarily over the vacuole crystal bundles and cytoplasmic plastids. The [1‐14C]glyoxalic acid– and [1‐14C]glycolic acid–labeled root sections showed a smaller amount of silver grains distributed over the entire sections, but these grains were not concentrated over the crystal idioblasts. These results strongly indicate that the l‐[1‐14C]ascorbic acid is the immediate precursor of oxalate in the crystal idioblasts of Y. torreyi primary roots and support more recent biochemical data regarding oxalate synthesis in higher plants. The use of roots in liquid culture containing uninterrupted files of developing crystal idioblasts could serve as a model system for additional biochemical, physiological, and molecular studies that seek to understand the formation and functional significance of crystal idioblasts in higher plant organs.

Pitting corrosion by bacteria on carbon steel, determined by the scanning vibrating electrode technique

The scanning vibrating electrode technique (SVET) has provided a non-destructive, on-line method for locating the presence and position of anodic electrochemical activity on the surface of carbon steel coupons. Using the SVET, changes in current densities over the steel with time were mapped in the presence and in the absence of bacteria. In a sterile liquid medium, the maps showed highly localized anodic current densities, which subsequently became inactive. Analysis of current maps and the open circuit potential (OCP) showed the potential transients were due to pit initiation and repassivation processes. When in the same bulk fluid, an aerobic bacterium, isolated from a corrosion tubercle, was grown, similar trends of pit initiation and repassivation were observed for several hours. However, after extended exposure to bacteria, local anodic activity did not repassivate. The corrosion then propagated and spread, until a large area of the sample was anodic. Recovery of the spent growth medium after growth of the bacteria, when used as the bulk solution, did not induce this irreversible pitting. These results indicated that the growth of bacteria altered surface conditions and prevented the initiated anodic sites from repassivating. The on-line non-destructive localized measurements possible by SVET can provide insight into the mechanisms of microbial influenced corrosion (MIC).

Effects of Bacteroides gingivalis culture products on human polymorphonuclear leucocyte morphology

This study aimed to determine whether leucocytes exposed to Bacteroides gingivalis culture supernatant exhibited consistent morphological changes and whether measurement of such changes could provide a simple screening assay for bacterial products with potential biological activity. Glass-adherent polymorphonuclear leucocytes were obtained from clotted blood preparations and from blood after purification by centrifugation through Ficoll-Hypaque. These were then incubated in Hanks' balanced salt solution, a sterile liquid medium (BM) and B. gingivalis (W83) culture supernatant. Polymorphonuclear leucocytes were classified by their shape into small non-polar (< 18 μm diameter), large non-polar (⩾ 18 μm diameter), bipolar and hyperpolar types. Treatment with B. gingivalis culture supernantant consistently increased large non-polar cells by 150% to over 300% ( p < 0.01), when compared with polymorphonuclear leucocytes incubated with the sterile liquid medium (control). This change was accompanied by smaller decreases in small non-polar, bipolar and hyperpolar cells, these being significant for bipolar cells in clot preparations ( p < 0.01) and small non-polar cells after Ficoll-Hypaque isolation ( p < 0.01). Neither the liquid medium nor the B. gingivalis culture supernatant was toxic to the cells as indicated by trypan blue exclusion tests. Lipopolysaccharide and short-chain fatty acids were not responsible for the changes in polymorphonuclear leucocyte shape. However, the activity of the culture supernatant was destroyed by heating at 80 °C for 30 min, indicating that proteolytic enzymes may have been involved.

Decomposition of14C-Labeled Maple and Spruce Lignin by Marine Fungi

Fungi occur on driftwood, intertidal wood, manila rope, and other lignocellulosic substrates in marine and estuarine environments (Johnson and Sparrow, 1961; Hughes, 1975; Kohlmeyer and Kohlmeyer, 1979). Several Ascomycetes and Deuteromycetes cause soft rots of wood submerged in seawater (Barghoorn and Linder, 1944; Henningsson, 1976). The marine basidiomycete Nia vibrissa Moore & Meyers causes a white rot (Leightley and Eaton, 1979). Relatively few fungi can decompose lignin, an extremely complex aromatic polymer (Crawford and Crawford, 1980; Crawford, 1981). Fukuzumi et al. (1975) demonstrated that the marine hyphomycete Dendryphiella salina (Sutherland) Pugh & Nicot could degrade sodium lignosulfonate, an industrially modified lignin. Henningsson (1976), using sulfuric acid hydrolysis to measure lignin content, found a strain of Zalerion maritimum (Linder) Anastasiou that degraded 12% of the lignin in birch wood in six months. Methods involving the use of 14C-labeled lignocellulosic substrates have made it possible to determine the amounts of lignin degraded by microorganisms to CO2 and water-soluble products (Crawford and Crawford, 1976). Using [lignin'4C]-lignocelluloses prepared from Norway maple and blue spruce as substrates, we measured the degradation of hardwood and softwood lignin by pure cultures of 12 marine and estuarine fungi. The fungi tested were Asteromyces cruciatus Moreau & Moreau ex Hennebert (F-156), Ceriosporopsis halima Linder (R-592), Corollospora maritima Werdermann (R-19), Dendryphiella salina (DS-1), Diplodia oraemaris Linder (F-162), Halosphaeria mediosetigera Cribb & Cribb (R-575), Heleococcum japonense Tubaki (R-692), Humicola alopallonella Meyers & Moore (F-164), Monodictys pelagica (Johnson) Jones (F-80), Nia vibrissa (RDB-8), Papulaspora halima Anastasiou (F-167), and Zalerion maritimum (R-6). Twigs of Norway maple (Acer platanoides L.) and blue spruce (Picea pungens Engelm.) were incubated with a solution of L-[U-14C]-phenylalanine to label the lignin (Crawford and Crawford, 1976). [Lignin-l4C]-lignocelluloses were prepared from the twigs by grinding and extracting the phloem layer (Crawford and Crawford, 1976). Specific activities of the maple and spruce lignocelluloses were 539 and 902 dpm mg-l, respectively. Triplicate culture tubes containing 50 mg of labeled lignocellulose each were autoclaved and 15 ml of a sterile liquid medium [oven-dried Rila Marine Salts (Rila Products, Teaneck, New Jersey) 20 g, yeast

A Technique for obtaining Clone Cultures of Soil Amœbæ in Sterile Liquid Medium

ON previous occasions free-living and parasitic amœbae have been obtained free from their associated bacteria by sterilization of the cysts either by washing in bactericidal chemicals1,2 or in antibiotics3,4. The technique I have used for isolating clones of soil amœbae and growing them in sterile peptone solution involves the treatment of the trophozoite stage with antibiotics, making use of the fact that the amœbae are more tolerant to many antibiotics than are certain bacteria.