Simple and convenient method for culturing anaerobic bacteria.

A simple and convenient method for culturing anaerobic bacteria is described. Cultures can be grown in commercially available flasks normally used for preparation of sterile external solutions. A special disposable rubber flask closure maintains anaerobic conditions in the flask after autoclaving. Growth of a variety of anaerobic oral bacteria was comparable to that obtained after anaerobic incubation of broth cultures in Brewer Anaerobic Jars.     

The growth of mycobacteria in liquid medium is influenced by the dimension of glass wall in contact with the medium

The growth of the type strain of Mycobacterium aurum was compared between flask and tube, both of which contained the same concentration of bacteria in synthetic media and had the same air-contact dimension but had different dimensions of the glass wall of vessels contacting the medium. Much better growth occurred in the flask, which had a smaller dimension of the glass wall contacting the medium, than in the tube, which had a larger dimension. The finding shows that the growth of bacteria is influenced by the dimension of the glass wall of vessels. It was considered that a high chance of the attack shock for bacteria given by the Brownian movement lowers the multiplication of bacteria.     

Effect of hydrostatic tensile stress on the growth of Escherichia coli and Bacillus cereus.

The specific growth rates of Escherichia coli and Bacillus cereus were measured for growth media in a flask, a lens-plate arrangement simulating an isolated capillary space, and a lens-plate arrangement under hydrostatic tensile stress. The specific growth rates of the bacteria were the same for the flask and lens-plate arrangement without hydrostatic tensile stress, but were enhanced when the growth media were subjected to hydrostatic tensile stress. The enhanced specific growth rates reached steady values at a tensile stress of 40 pascals. The effect was observed up to tensile stresses of around 100 pascals. The maximum increase in specific growth rate was 25% for E. coli and 22% for B. cereus.     

A modified plastic culture flask for microscopic observation of fungi

A plastic culture flask modified for the direct microscopic observation of fungi is described. The use of this flask allows for in situ observation of fungal morphology and monitoring of development of different stages of fungal growth. The flask provides a safe, rapid, and inexpensive culture technique for the clinical laboratory.     

Effect of hydrostatic tensile stress on the growth of Escherichia coli and Bacillus cereus.

The specific growth rates of Escherichia coli and Bacillus cereus were measured for growth media in a flask, a lens-plate arrangement simulating an isolated capillary space, and a lens-plate arrangement under hydrostatic tensile stress. The specific growth rates of the bacteria were the same for the flask and lens-plate arrangement without hydrostatic tensile stress, but were enhanced when the growth media were subjected to hydrostatic tensile stress. The enhanced specific growth rates reached steady values at a tensile stress of 40 pascals. The effect was observed up to tensile stresses of around 100 pascals. The maximum increase in specific growth rate was 25% for E. coli and 22% for B. cereus.     

A DEVICE TO ASSIST IN ESTIMATING SMALL NUMBERS OF BACTERIA IN LIQUIDS

SUMMARY: An apparatus is described which assists the determination of small numbers of micro-organisms in liquids by enabling volumetric samples to be taken without risk of aerial contamination. Three sets of 5 containers of 5, 50 and 500 ml are mounted in a common vessel, and are filled as part of the sampling process, and one 2,500 ml sample is contained in an inter-connected flask. The number of containers showing bacterial growth is used in conjunction with probability tables, to estimate the number of bacteria in the liquid.     

Enhanced protease production in a polymethylmethacrylate conico-cylindrical flask by two biofilm-forming bacteria

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment was employed for protease production by two biofilm-forming bacteria, an intertidal gamma-Proteobacterium (DGII) and a chicken meat isolate, Virgibacillus pantothenticus. The flask design allowed comparison of protease production during cultivation with a hydrophilic (glass) or hydrophobic (PMMA) surface. Compared to the Erlenmeyer flask, the CCF allowed protease production that was 30% and 35% higher and growth that was 20% and 345% higher for DGII and V. pantothenticus, respectively. Protease production increased by 202% and 22% and growth by 19,275% and 940% for DGII and V. pantothenticus, respectively, in the presence of a hydrophobic as compared to a hydrophilic surface. This investigation pioneers the application of a vessel beyond the traditional shake-flask for enhancing protease production by biofilm-formers.     

Sterile Culture of Excised Tomato Roots in Sands of Different Grain Size

A simple technique to grow excised tomato roots in sand in sterileculture is described. The apparatus consists of a culture tubefilled with sand linked to a small flask containing the nutrientsolution. By raising the lower-placed flask the sand is flushedwith solution, thereby replenishing nutrients at the root surface.Lowering the flask again allows air to enter the sand, thuseffecting sufficient aeration. It is further demonstrated that growth of the roots is relatedto acration and not to mechanical impedance in sands of differentgrain size.     

Gas-Tight Flask for the Concurrent Measurement of Gas Metabolism and Growth in Methane-Oxidizing Bacteria

A flask is described for use in conjunction with a gas chromatograph and a spectrophotometer to follow methane and oxygen uptake, carbon dioxide production, and cell growth concurrently in methane-oxidizing cultures.     

A rapid method of determining growth characteristics of plant cell populations in batch suspension culture

A non-destructive, simple and accurate method of determining the relative growth rate (RGR) of the packed cell volume (PCV) of plant suspension cells in one Erlenmeyer flask at any time during the incubation period is described. The Erlenmeyer flask was tilted and the length of the chord formed by the surface of the packed cells across the bottom of the flask was measured. The chord length and the log PCV were correlated in a calibration line. The method enables the RGR during the exponential growth phase to be calculated by multiplying the slope of the linear part of the curve of the chord length in time with the slope of the calibration line. In order to investigate other growth parameters and to analyse the accuracy of the method statistically, a four-parameter function for the chord length and a computer program were used. The RGR during the exponential growth phase of cell suspensions of Solanum tuberosum and Haplopappus gracilis appeared to be independent of the PCV of the inoculum. The method appeared to be sufficiently accurate.     

An automated home-built low-cost fermenter suitable for large-scale bacterial expression of proteins in Escherichia coli

We have developed an automated fermentation system for cost-efficient upscaling of protein expression in bacteria. The system, built for use by nonbiotechnologists, can be assembled mostly from standard laboratory equipment and allows a largely unattended growth of bacteria to OD 25 (at 600 nm) in a 12 L vessel. The typical yield of 250-350 g of wet weight cell pellet per run, which is equivalent to the biomass obtained from 250 shake flask cultures containing 400 mL Luria-Broth medium each, facilitates the production of large amounts of purified recombinant protein without the laborious need for optimization of expression and purification conditions.     

Measurement of limestone biodeterioration using the Ca2+ binding fluorochrome Rhod-5N

Limestone and marble have been used extensively in the construction of modern and historic buildings. Microbial colonization and growth on these stone structures is common. Microbial deterioration of stone has been assessed by measuring Ca2+ released from the stone, using ion selective electrodes and titration with EDTA. In this study, the calcium binding fluorochrome Rhod-5N was used to measure Ca2+ released from limestone by endolithic bacteria as an indicator of biodeterioration. In a 17 d flask experiment, Ca2+ released by endolithic bacteria was twice that of uninoculated controls. Rhod-5N is a rapid and accurate method for measuring microbial biodeterioration of stone.     

Kinetics and modelling of cell growth and substrate uptake inCentella asiatica cell culture

In this study, we have conducted kinetics and modelling studies ofCentella asiatica cell growth and substrate uptake, in an attempt to evaluate cell growth for a better understanding and control of the process. In our bioreactor cultivation experiment, we observed a growth rate of 0.18/day, a value only 20% higher than was seen in the shake flask cultivation trial. However, the observed maximum cell dry weight in the shake flask, 10.5 g/L, was 14% higher than was achieved in the bioreactor. Ninety seven percentage confidence was achieved via the fitting of three unstructured growth models; the Monod, Logistic, and Gompertz equations, to the cell growth data. The Monod equation adequately described cell growth in both cultures. The specific growth rate, however, was not effectively predicted with the Logistic and Gompertz equations, which resulted in deviations of up to 73 and 393%, respectively. These deviations in the Logistic and Gompertz models may be attributable to the fact that these models were developed for substrate-independent growth and fungi growth, respectively.     

Studies on phosphate uptake by Acinetobacter calcoaceticus under aerobic conditions

Acinetobacter calcoaceticus was cultivated in a well-aerated stirred tank reactor and its phosphate uptake capacity was investigated. Statistical media optimization was done to figure out favourable growth conditions of Acinetobacter calcoaceticus NRRLB-552. Plackett–Burman design was used to figure out the key nutrients (sodium acetate, ammonium chloride and calcium chloride) featuring high growth and/or uptake of phosphate. The optimal concentrations for these nutrients were (sodium acetate 5.0 g/l, ammonium chloride 0.67 g/l, calcium chloride 0.05 g/l) obtained by central composite design (CCD) protocols and verified in shake flask cultivations. Predicted and experimental dry cell weights obtained using the optimized media were 2.046 and 2.54 g/l indicating 97% agreement. The optimal values of pH and temperature for growth and phosphate uptake were found to be 7.69 and 31.86 °C, respectively, using CCD. Batch kinetics was also established in shake flask and fermenter using optimized medium and environmental conditions. Phosphate uptakes of 21 mg/g biomass and 36 mg/g biomass were obtained in shake flask and fermenter, respectively. The possible inhibition of nutrients (carbon, nitrogen and phosphate) was also established under shake flask cultivation conditions. Growth of the bacteria was inhibited at a concentration higher than 0.4% carbon and 0.6% nitrogen. However increasing concentration of phosphate did not show any inhibitory effect on growth. The above kinetics and inhibition data will serve as suitable database for the development of a mathematical model for growth and its use will be able to facilitate appropriate reactor design for the removal of phosphates from industrial effluents.     

Extended Monod equation for batch cultures with multiple exponential phases

The batch growth curves of Laclobacillus delbreuckii exhibit several exponential phases. From the results of a series of shaker flask experiments, the position of the slope changes in the growth curve and the overall bacterial yield is affected by the initial amount of yeast extract in the medium. It is postulated that this behavior is due to several growth enhancing substances that are initially in the yeast extract and are consumed by the bacteria during the course of the fermentation. Using a Monod-type expression to represent the effect of growth enhancing components in a proposed growth rate expression, a mathematical model of the system is set up and solved on the analog computer.     

Ferrous ion oxidation by Thiobacillus ferrooxidans immobilized in calcium alginate

Summary Thiobacillus ferrooxidans was immobilized by entrapment into calcium alginate matrix. The immobilized bacteria were used in packed-bed column reactors for the continuous oxidation of ferrous ion at pH 1.5. The presence of mineral salts resulted in a shorter lag period before a steady-state of about 95% iron oxidation was achieved. Parallel shake flask experiments were used to evaluate pH, mineral salts, and alginate toxicity as factors influencing biological iron oxidation. Manometric experiments indicated that the previous growth history of T. ferrooxidans was important in determining the rate of iron oxidation. Scanning electron microscopy and energy dispersive analysis of X-rays were used to characterize bacteria entrapped in calcium alginate and the enrichment of iron in the matrix.     

Alternative methodology for isolation of biosurfactant-producing bacteria.

Wide biosurfactant application on biorremediation is limited by its high production cost. The search for cheaper biossurfactant production alternatives has guided our study. The use of selective media containing sucrose (10 g x L(-1)) and Arabian Light oil (2 g x L(-1)) as carbon sources showed to be effective to screen and maintain biosurfactant-producing consortia isolated from mangrove hydrocarbon-contaminated sediment. The biosurfactant production was assayed by kerosene, gasoline and Arabian Light Emulsification activity and the bacterial growth curve was determined by bacterial quantification. The parameters analyzed for biosurfactant production were the growth curve, salinity concentration, flask shape and oxygenation. All bacteria consortia screened were able to emulsify the petroleum derivatives tested. Biosurfactant production increased according to the incubation time; however the type of emulsification (non-aqueous phase or aqueous phase) did not change with time but with the compound tested. The methodology was able to isolate biosurfactant-producing consortia from superficial mangrove sediment contaminated by petroleum hydrocarbons and was recommended for selection of biosurfactant producing bacteria in tropical countries with low financial resources.     

寡糖素对西洋参和人参愈伤组织培养的影响

红花、人参和黑节草寡糖素(简称CO、GO和DO)分别加入到培养基中,均能影响西洋参和人参愈伤组织生长和皂甙的合成。CO、GO和DO促进西洋参愈伤组织皂甙合成的最适浓度分别为5ppm、15ppm和10ppm,皂甙产率分别为14.89mg/flask、11.24mg/flask和14.53mg/flask,均明显高于对照(8.22mg/flash).CO、GO和DO促进人参愈伤组织皂甙合成的最适浓度分别为5ppm、20ppm和5ppm,皂甙产率分别为11.79mg/flask、11.20mg/flask和10.48mg/flask,均明显高于对照(6.65mg/flask),它们在适度浓度下对人参愈伤组织的生长均有促进作用。     

Axenic culture of Gyrodinium impudicum strain KG03, a marine red-tide microalga that produces exopolysaccharide

An exopolysaccharide-producing microalgal dinoflagellate was isolated from a red-tide bloom and designated strain KG03. A bacteria-free culture of strain KG03 was achieved using a modified wash with phototaxis and antibiotic treatment. Combined treatment with neomycin and cephalosporin was the most effective for eliminating the bacteria associated with the microalgae. Strain KG03 was identified as Gyrodinium impudicum by analyzing the ITS regions of the 5.8S rDNA, 18S rDNA, morphological phenotype and fatty acid composition. The exopolysaccharide production and cell growth in a 300-ml photobioreactor were increased 2.7- and 2.4-fold, respectively, compared with that in a flask culture at the first isolation step.     

Anaerobic methane oxidation and sulfate reduction in bacterial mats of coral-like carbonate structures in the Black Sea

A detailed study of the processes of anaerobic methane oxidation and sulfate reduction in the bacterial mats occurring on coral-like carbonate structures in the region of methane seeps in the Black Sea, as well as of the phenotypic diversity of sulfate-reducing bacteria developing in this zone, has been performed. The use of the radioisotopic method shows the microbial mat structure to be heterogeneous. The peak activity of the two processes was revealed when a mixture of the upper (dark) and underlying (intensely pink) layers was introduced into an incubation flask, which confirms the suggestion that methanotrophic archaea and sulfate-reducing bacteria closely interact in the process of anaerobic methane oxidation. Direct correlation between the rate of anaerobic methane oxidation and the methane and electron acceptor concentrations in the medium has been experimentally demonstrated. Several enrichment and two pure cultures of sulfate-reducing bacteria have been obtained from the near-bottom water and bacterial mats. Both strains were found to completely oxidize the substrates to CO2 and H2S. The bacteria grow at temperatures ranging from -1 to 18 (24) degrees C, with an optimum in the 10-18 degrees C range, and require the presence of 1.5-2.5% NaCl and 0.07-0.2% MgCl2 x 6H2O. Regarding the aggregate of their phenotypic characteristics (cell morphology, spectrum of growth substrates, the capacity for complete oxidation), the microorganisms isolated have no analogues among the psychrophilic sulfate-reducing bacteria already described. The results obtained demonstrate the wide distribution of psychrophilic sulfate-reducing bacteria in the near-bottom water and bacterial mats covering the coral-like carbonate structures occurring in the region of methane seeps in the Black Sea, as well as the considerable catabolic potential of this physiological group of psychrophilic anaerobes in deep-sea habitats.