A new principle of cell cultivation: No air-liquid interface but gas exchange across a synthetic membrane

Summary A completely liquid-filled growth chamber for axenic cultures ofTetrahymena pyriformis is described; gas exchange is ensured across a synthetic membrane. The chamber may be incorporated into a continuous flow system with inoculation and removal of cell samples under sterile conditions. Initially, the generation time of the cells was slightly prolonged, about 10%, but after some cell doublings decreased to 5%. The capacity of the cells to form food vacuoles (endocytosis) was unaltered during growth in the chamber. The synthetic membrane was highly permeable to O₂ and CO₂; however, cells grown in the chamber contained small refractive granules. The culture chamber permits the culture volume to be varied and it may be used for other protozoa, bacteria, and even tissue culture cells.     

Synergistic growth studies of Entamoeba gingivalis using an Ecologen.

A unique multiple diffusion growth chamber, an Ecologen, designed for the study of interactions among microorganisms, was introduced as a means of growing xenic cultures of Entamoeba gingivalis with Crithidia sp. or Yersinia enterocolitica. Entamoeba gingivalis was grown in the central diffusion reservoir of the Ecologen connected to separate growth chambers inoculated with the microorganisms to be evaluated. Growth of the accompanying bacteria in the E. gingivalis compartment was almost completely eliminated, except for sparse Pseudomonas sp. growth. The most vital E. gingivalis cultures were observed when either Crithidia sp. or Y. enterocolitica were added to the Ecologen 48 h prior to the E. gingivalis inoculum. The medium which provided the best growth of the oral protozoan in this system was the new improved E. gingivalis medium containing antibiotics.     

Stimulation of Lactic Acid Bacteria by a Micrococcus Isolate: Evidence for Multiple Effects

Growth of, and rate of acid production by, six cultures of lactic acid bacteria were increased in the presence of Micrococcus isolate F4 or a preparation of its capsular material. Concentrations of hydrogen peroxide found in pure cultures of the lactic acid bacteria were not detectable, or were greatly reduced, in mixed culture with Micrococcus isolate F4. The capsular material was not as effective as whole cells in preventing accumulation of H(2)O(2). Catalase stimulated growth of, and the rate of acid production by, the lactic acid bacteria, but not to the same extent as Micrococcus isolate F4 in some cultures. The existence of two mechanisms for micrococcal stimulation of the lactic acid bacteria is postulated. One mechanism involves removal of H(2)O(2); the other has not been characterized.     

An on-line technique for monitoring propionic acid fermentation

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Improved Culture Flask for Obligate Anaerobes

An improved flask system for the growth of extremely oxygen-sensitive bacteria in liquid culture is described. The improvement described utilizes an all-glass, neoprene-stoppered flask designed for growth of 50- to 1,000-ml cultures of bacteria with continuous gassing.     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

A Microperfusion Chamber for Studying the Growth of Bacterial Cells

A microperfusion chamber for studying the growth of bacterial cells is described. It can be used with unmodified phase contrast systems. Both temperature and the composition of the growth medium can be changed by the operator during observation of bacteria. The use of the chamber is illustrated with observations on the effect of penicillin on Arthrobacter globiformis .     

Consequences of protist-stimulated bacterial production for estimating protist growth efficiencies

The trophic link between bacteria and bacterivorous protists is a complex interaction that involves feedback of inorganic nutrients and growth substrates that are immeadiately available for prey growth. These interactions were examined in the laboratory and in incubations of concentrated natural assemblages of bacterioplankton. Growth dynamics of estuarine and marine bacterivorous protists were determined in laboratory culture using Vibrio natriegens as prey and were compared to growth of protists on bacterioplankton assemblages concentrated by tangential flow filtration from four northwest Florida Estuaries. Biomass transfers from bacteria to protists were monitored by tracing elemental carbon and nitrogen in particulate fractions of protist added and grazer free controls. Gross growth efficiencies of the protists on naturally occurring bacteria were within the range determined in lab estimates of growth efficiency on cultured bacteria (50%). However, bacterial response to protist excretion products was different in the lab and field incubations, and bacterial growth contributed to the biomass available to protists in the field incubations. As determined by radioisotope-labeled substrate incorporation, a time lag in bacterial reponse to protist excretion products was observed for laboratory batch cultures, allowing accurate estimation of growth efficiency. In incubations with concentrated natural bacterial assemblages, bacterial growth response coincided with protist growth and excretion. The additional bacterial production on protist excretion products reached a maximum of 2–3-fold higher than protist-free controls. In addition, ammonium concentrations increased with protist grazing and growth in lab cultures, but ammonium excreted by protists in concentrates did not accumulate. The C:N values for the bacterial concentrates suggests that these bacteria were nitrogen limited. It is speculated that dissolved organic carbon, concentrated by tangential flow filtration (> 100,000 MW membrane) with the bacterioplankton, was utilized by bacteria when nitrogen was supplied as ammonium and amino acids from protist excretion. Thus, estimates of protist growth efficiency on naturally occurring bacterioplankton, corrected for protist-stimulated bacterial production, were in the range of 13–21%.     

Growth of the extreme thermophile Sulfolobus acidocaldarius in a hyperbaric helium bioreactor

The relationship between pressure and temperature as it affects microbial growth and metabolism has been examined only for a limited number of bacterial species. Because many newly-discovered, extremely thermophilic bacteria have been isolated from pressurized environments, this relationship merits closer scrutiny. In this study, the extremely thermophilic bacterium, Sulfolobus acidocaldarius, was cultured successfully in a hyperbaric chamber containing helium and air enriched with 5% carbon dioxide. Over a pressure range of approximately 1-120 bar and a temperature range of 67-80 degrees C, growth was achieved in a heterotrophic medium with the air mixture at partial pressures up to 3.5 bar. Helium was used to obtain the final, desired incubation pressure. No significant growth was noted above 80 degrees C over the same range of hyperbaric pressures, or at 70 degrees C when pressure was applied hydrostatically. Growth experiments conducted under hyperbaric conditions may provide a means to study these bacteria under simulated in situ conditions and simultaneously avoid the complications associated with hydrostatic experiments. Results indicate that hyperbaric helium bioreactors will be important in the study of extremely thermophilic bacteria that are isolated from pressurized environments.     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.     

Development of a miniaturized selective counting strategy of lactic acid bacteria for evaluation of mixed starter in a model cassava fermentation

A miniaturized most probable number (MPN) method for the selective enumeration of three bacteria species ( Lactobacillus plantarum A6, Leuconostoc mesenteroides and Lactococcus lactis ) is described. This selective count method, based on specific consumption of carbon substrate and resistance to antibiotics, was used for the quantitative assessment of the three bacteria during mixed cultures in a model cassava fermentation. A typical microbial succession pattern was observed: (i) Lactococcus lactis and Leuc. mesenteroides dominated during the first hours of fermentation as their growth was very rapid ; (ii) from hour 12, Lactobacillus plantarum replaced the two latter strains and Lactococcus lactis disappeared gradually, followed by Leuc. mesenteroides . The growth rates of each strain appeared to be independent of the others, while acidification rates increased strongly in mixed cultures compared with pure cultures. No positive interactions resulting from the amylolytic character of Lactobacillus plantarum A6, and no negative interactions resulting from the Nis⁺ property of Lactococcus lactis , were revealed between the three strains under the model conditions used.     

A cell-culture system for long-term maintenance of elevated hydrostatic pressure with the option of additional tension

In cell stress research, there is still a need to apply long-term hydrostatic pressure without changing any other environmental condition. We present here a new, open, pressurized chamber system allowing long-term sustained and dynamic application of hydrostatic pressure with the option of additional tension. Based on the computer-controlled Flexcell Strain Unit, we designed a pressurized chamber with a dynamic airflow and a defined membrane extension, which can be regulated by spacers. During operation up to 26.6kPa, O(2) partial pressures and pH in the cell-culture medium do not change compared to control cultures kept at normal atmosphere.     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

Variability of the turgor pressure of individual cells of the gram-negative heterotroph Ancylobacter aquaticus.

Cells of Ancylobacter aquaticus were observed under phase microscopy in a chamber to which a measured pressure could be applied. The initial collapse pressure (Ca), i.e., the lowest pressure needed to collapse the most pressure-sensitive gas vesicles, was measured for 69 cells. The cells were taken from cultures in low-density balanced exponential growth, and the experiments were performed quickly so that the bacteria were in a uniform physiological state at the time of measurement. The turgor pressure, Pt, is the difference between the pressure, C, that would cause collapse of vesicles when removed from the cell and Ca. In this paper we focus on the variability of Pt from cell to cell. Part of the observed variability of Ca was due to the variability of the collapse pressure of individual vesicles (standard deviation [SD] = 90 kPa), but because there were about 100 vesicles per cell and because a change in refracted light after the fifth vesicle (approximately) collapsed probably could be detected by the human eye, the pressure would only have an SD of 18.6 kPa due to this type of sampling error. The observed SD of Pt was 42 kPa, indicating that turgor pressure did vary considerably from cell to cell. However, the turgor pressure was independent of cell size. Statistical analysis showed that Pt would decrease 6.9 kPa over a cell cycle, but with too large an SD (19.9 kPa) to be significant. This implies that the observed change in Pt over the cell cycle is not statistically significant.     

Norepinephrine as a growth stimulating factor in bacteria--mechanistic studies

Catecholamines (norepinephrine, epinephrine, dopamine) enhance the growth of several species of gram-negative bacteria. Since catechol rings are known siderophores in bacteria, the administration of catecholamines may enhance growth by improving iron uptake in growth-limiting media, serving as auxiliary siderophores. We have tested the iron content in bacterial growth media which are known to support rapid growth and "slow growth" media. Additionally, we have examined the uptake of 3H-norepinephrine, to determine whether the catecholamine is actually taken into the bacteria or is merely adsorbed to the outside of the bacteria. Finally, we have been examining the supernatants produced by culturing bacteria with norepinephrine. These supernatants have been shown to have the capacity to enhance growth of naive cultures of bacteria, and are suggested to contain an "autoinducer of growth". We have found that both fast-growth and slow-growth media contain similar concentrations of iron, and that these levels do not change in most supernatants from NE-supplemented bacterial cultures. Examination of culture supernatants from NE-supplemented bacteria under different temperature conditions reveals some interesting differences. First, culture supernatant from NE-treated Escherichia coli, cultured at 37 degrees C, when examined by HPLC, exhibits a change in the norepinephrine content over time which is not seen in supernatant from 21 degrees C cultures or other media treatments. Second, the 37 degrees C culture NE-supplemented E. coli supernatant was significantly more effective in enhancing growth of three bacterial species than any other culture method other than NE-supplementation itself (this includes supernatant from NE-supplemented cultures of the other two species as well as supernatants from unsupplemented cultures of all three species).     

Restricted pH ranges and reduced yields for bacterial growth under pressure

Hydrostatic pressure was found to cause a marked narrowing of pH ranges for growth and reductions in growth yields for a variety of bacteria. In many cases, reduced yields under pressure could be directly related to increased sensitivities to metabolic acids that accumulated in the enclosed culture vessels used. Magnesium and calcium ions partially reversed increases in sensitivities of representative gram-positive and gram-negative bacteria to low, but not high, pH. Growth inhibition of these organisms at both extremes of pH was associated with enhanced loss of K⁺ from pressurized cells. Inhibited cells in alkaline media also lysed under pressure, but microscopically observable lysis was clearly a secondary phenomenon because it occurred slowly. Apparent volumes for growth-inhibitory protonation-deprotonation reactions were calculated on the basis of measured shifts in inhibitory pH with pressure. The values ranged from 99 to 431 ml/mole, and their magnitudes indicated that growth inhibition by acids or bases involves cooperative changes in polymeric interactions such as those which accompany protein denaturation.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

New criteria for evaluation of soil bacterial complexes

The initial concentration of prokaryotic microorganisms, the type of their growth, doubling time, and the growth dynamics of bacteria and actinomycetes in three types of soil (meadow, chestnut, and soddy forest) were evaluated by the luminescence microscopic analysis of soil samples incubated in a humid chamber for 1 day. Soddy forest and chestnut soils differed in most of the parameters analyzed. Meadow soil was close to soddy forest soil in some parameters and to chestnut soil in other parameters. All soil suspensions exhibited high growth rates of bacteria and actinomycetes, indicating that the fraction of viable microorganisms in the soils was high.