Basal Plate Tissue in Narcissus Bulbs and in Shoot Clump Cultures: Its Structure and Role in Organogenic Potential of Single Leaf Cultures

Light microscopy demonstrated that the apparently amorphous,achlorophyllous tissue at the base of in vitro shoot clump cultureof Narcissus was comparable in structure to the basal plateof Narcissus bulbs. Both had very complex vascularisation andsmall, densely packed parenchymatous cells. In shoot clump cultures, primordia were produced by meristematiczones at the surface of this achlorophyllous tissue, very closeto the base of leaves. Single leaf units excised from the invitro shoot clump cultures with a wedge of basal achlorophylloustissue were highly organogenic when used as secondary explantsfor in vitro culture of Narcissus. No organogenesis occurredin the absence of the leaf base and achlorophyllous (basal plate)tissue and little organogenesis occurred unless the leaf baseand basal plate tissue were immersed in the culture medium (i.e.explants inoculated into liquid medium or upright in agar-solidifiedmedium). After two 5-week culture passages in liquid medium, more thanfive leaves were produced per leaf base inoculated. Thus rapidmicropropagation of Narcissus can be achieved using only thebase of single leaf units excised from shoot clump cultures.Copyright1993, 1999 Academic Press Anatomy, basal plate, bulb, in vitro, leaf culture, Narcissus, organogenesis     

Piggy-Back Microtransfer Technique

A new microtransfer technique is described which uses a modified, V-bottom, disposable, 96-cup plastic plate that permits serial dilutions and direct rapid transfer to a recipient micro cell culture plate.     

Microtissue Culture Plaque Assay for Herpesvirus saimiri

A microtissue culture method for the plaque assay of Herpesvirus saimiri has been developed. Virus titrations carried out in Microtest II tissue culture plates (Falcon) yielded reproducible results that agreed well with those obtained by employing macrocultures. The described method is quantitative, reproducible, economical, and suitable for routine assay of large numbers of virus samples.     

Microculture Plaque Assay for Human and Simian Cytomegaloviruses

The plaque assay for human and simian cytomegaloviruses routinely carried out in 60-mm petri dishes (macrocultures) has been adapted for use in microcultures in flat-bottom 16-mm circular wells of disposable plastic trays. Virus titrations and serum neutralization assays carried out in microcultures yielded reproducible results that were identical to those obtained in macrocultures.     

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 μL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96‐well microtiter plate, to preserve compatibility with the current high‐throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO₂ production rate was obtained from the online measurement of cumulative CO₂ production during the cultivation. The online data obtained by the sensor array and the CO₂ production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench‐scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro‐scale fermentations, e.g. using the 96‐well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Simple growth chambers for culturing microalgae with precision at different temperatures and irradiance

Profiles describing the relationships between growth, irradiance and temperature are important in the evaluation of microalgae and cyanobacteria for biomass production, as well as for their general characterization. To get correct results culturing chambers with plane parallel optical faces are needed. Only these give a defined light climate in the culture, but such devices are not found commercially. We here describe the testing of thermostated growth chambers with 200-mL culture volumes, easily constructed from disposable 144-mm diameter plastic Petri dishes. Their properties were examined by growing Spirulina platensis concurrently in 15 parallel cultures under identical conditions of illumination and temperature, showing efficient and reproducible growth between them. The chambers are naturally also very suitable for growing microalgae in general. This revised version was published online in August 2006 with corrections to the Cover Date.     

Titration and Neutralization of Poliovirus in Micro Tissue Culture Under Increased Carbon Dioxide

Modification of the micro tissue culture technique, including incubation under increased CO(2), resulted in prolongation of the viability of the cells. As a consequence, satisfactory titrations of poliovirus and of poliovirus antiserum have been achieved by the micro method. The technique offers a number of advantages over conventional methods.     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Microcalorimetry: A tool for the study of the biodegradability of straw by mixed bacterial cultures

Summary The biodegradability of straw by a mixed bacterial culture obtained from a pile of weeds was studied by microcalorimetry. All the cultures were grown at 30°C under anaerobic conditions in microcalorimetric vessels. The fermentation thermograms, obtained using well defined conditions, were very reproducible. The quantities of heat produced during straw degradation were found to be proportional to the quantity of straw introduced at the beginning of the fermentation.The recovered carbon was also found to be proportional to the initial quantity of straw. From both microcalorimetric and chemical analysis it was concluded that the limiting factor of the straw degradation was the cellulolytic activity of the mixed culture. This is supported by the fact that commercially available cellulase added to the growth medium increases the amount of straw degradation by about four times. The heat associated with fermentation of each cellulose monomer (C₆H₁₀O₅) was found to be 120 kJ, a value which is close to the heat associated with hexose fermentation by pure cultures. In conclusion, we propose that microcalorimetry can be used as a powerful tool for the analysis of the biodegradability of complex heterogeneous substrate by pure or mixed cultures.     

Analysis of volatile compounds released from embryogenic cultures and somatic embryos of sweet oranges by head space SPME

Volatile compounds released from callus and nucellar embryo tissues of ‘Valencia late’ and ‘Washington Navel’ sweet oranges (Citrus sinensis, L. Osbeck) were collected/concentrated by head space solid phase micro extraction and analysed by gas chromatography-mass spectrometry. Friable, white embryogenic cultures released a number of volatile compounds, including some essential oils. Different samples of the same embryogenic culture showed variability, possibly related to the presence of tissues undergoing differentiation. Analyses of the somatic embryos permitted the identification of several components, including limonene and methyl anthranilate. Considering the simplicity and the very small sample required (0.3 g of fresh tissue) head space solid phase micro extraction is suitable for studies and comparisons of volatile metabolites released from in vitro Citrus tissue cultures suggesting its potential in Citrus biochemical, genetic and breeding research. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chemical and sensory analysis of saffron produced through tissue cultures of Crocus sativus

Stigma-like structures were produced in tissue cultures (TC stigmas) from the ovary explants of C. sativus on MS medium supplemented with NAA and BA. The size of these structures was 2 to 3 cm in length. At higher concentrations of both NAA (54 µM) and BA (44 µM) white tubular abnormal structures were observed from ovary explants in addition to the TC stigmas. Crocin and picrocrocin, responsible for colour and bitter taste respectively, were found to be 6 and 11 times lower in TC stigmas than in the natural stigmas. The saffron obtained from tissue cultures was subjected to sensory analysis and compared with the data obtained from chemical analysis. The sensory data indicated that the saffron pigments produced in tissue cultures were one tenth that of natural stigmas. Sensory profile test showed that the tissue culture saffron was low in floral, spicy and fatty characteristics as compared to saffron obtained from flowers. This is the first report on the sensory analysis of a spice produced in tissue cultures.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

Fatty acid composition of lipids from differentiated tissues and cell cultures of Euonymus europaeus

The fatty acid patterns of Euonymus europaeus callus cultures and cell suspension cultures were analysed at the beginning of stationary growth phase and compared with those from the respective differentiated tissues. The lipid and fatty acid patterns in cell cultures differed remarkably from those in the tissues of the mother plant. No glycerol triacetate was detected in the callus cultures derived from differentiated tissues whereas in seeds this lipid compound amounts to 29%. In addition to fatty acids normally occurring in differentiated tissues, lipids in cultured cells also contained short-chain (C₁₂–C₁₄) as well as very long-chain fatty acids (C₂₀–C₂₄). In tissue culture cells the major fatty acids were found to be saturated, whereas in the mother cells unsaturated fatty acids were predominant. Palmitic acid is the most abundant fatty acid in most of the cultures. Lauric, myristic and palmitic acid amount to 50% in lipids of cell suspension cultures.     

Osteochondral tissue cultures: Between limits and sparks,the next step for advanced in vitro models

The increasing demand for reliable preclinical models and to reduce, refine and, if possible, replace animal studies have brought forth the development of complex tissue cultures in different research areas, including the musculoskeletal field. In this paper, we review the literature within last 10 years on the state of progress for in vitro models of osteochondral tissue cultures, taking into account the clinical relevance of the management and treatment of osteochondral lesions. According to the selected research criteria, 35 works, 27 of which with animal tissues and 8 with human tissues, resulted to be relevant for the purposes of this review. Data analyzed revealed a great heterogeneity among the proposed tissue culture models. The anatomical harvesting sites resulted to be mainly the knee stifle joint, both for animal (prevalently bovines) and human tissues derived from joint replacement surgery, and significant heterogeneity among culture conditions and media were found. To date, very few papers have focused on the set up of a reproducible in vitro model, applicable to a variety of studies, thus suggesting a relevant gap to fill in the development of advanced three-dimensional osteochondral culture models.     

Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.     

Fed-batch operation in special microtiter plates: a new method for screening under production conditions

Batch and fed-batch operation result in completely different physiological conditions for cultivated microorganisms or cells. To close the gap between screening, which is hitherto exclusively performed in batch mode, and fed-batch production processes, a special microtiter plate was developed that allows screening in fed-batch mode. The fed-batch microtiter plate (FB-MTP) enables 44 parallel fed-batch experiments at small scale. A small channel filled with a hydrogel connects a reservoir well with a culture well. The nutrient compound diffuses from the reservoir well through the hydrogel into the culture well. Hence, the feed rate can easily be adjusted to the needs of the cultured microorganisms by changing the geometry of the hydrogel channel and the driving concentration gradient. Any desired compound including liquid nutrients like glycerol can be fed to the culture. In combination with an optical measuring device (BioLector), online monitoring of these 44 fed-batch cultures is possible. Two Escherichia coli strains and a Hansenula polymorpha strain were successfully cultivated in the new FB-MTP. As a positive impact of the fed-batch mode on the used strains, a fourfold increase in product formation was observed for E. coli. For H. polymorpha, the use of fed-batch mode resulted in a strong increase in product formation, whereas no measurable product formation was observed in batch mode. In conclusion, the newly developed fed-batch microtiter plate is a versatile, easy-to-use, disposable system to perform fed-batch cultivations at small scale. Screening cultures in high-throughput under online monitoring are possible similar to cultivations under production conditions.     

Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures

We describe the use of the neutral protease Dispase for the dissociation of neonatal rat brain tissue for the preparation of primary monolayer astrocyte cultures. The method involves 5 to 6 successive extractions with careful separation of sedimenting, undissociated tissue. This method gives an initial cell suspension of high viability (93.7±1.7% cells exclude trypan blue). In comparison trypsin (0.25%) dissociated tissue gave a cell suspension that showed a lower viability of 58.2±7.6%. Identical saturation densities of 1.1 to 1.2×10⁴ cells/cm² after two weeks in culture were obtained for a range of seeding densities from 1 to 4×10⁴ cells/cm² of the Dispase dissociated tissue. Staining for glial fibrillary acidic protein showed that 90–100% cells were positive for this astroglial marker. Thus, the use of Dispase for the initial dissociation of rat brain tissue seems to give primary astrocyte cultures which are very reproducible and homogeneous.     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.     

A human placental polydeoxyribonucleotide (PDRN) may promote the growth of human corneal fibroblasts and iris pigment epithelial cells in primary culture

The optimal concentration of a human placental polydeoxyribonucleotide (PDRN) preparation (100 microg/ml) enhances the growth of human corneal fibroblasts in primary culture depending upon the donor age. In particular, this effect is very consistently reproducible with donors over 60 years of age (p = 0.0028), suggesting a selective benefit of PDRN in the tissue culture of senescent cells. Moreover, this drug may promote the development of human iris pigment epithelium (IPE) cells with much lower concentrations of fetal bovine serum than those suitable for the culture of IPE. Lastly, the use of a 'gauze disk' on the pieces of the corneal explants improves the efficiency of growth of the control fibroblast primary cultures.     

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l^?1 and 2.0 day^?1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions.     

Osmotic microenvironments within tissue culture well plates are an important cause of variability in plant protoplast cultures

Summary To determine if the medium osmolality of plant protoplast cultures in 24-well tissue culture plates changes sufficiently during the culture period to affect development of the protoplasts the osmolality and the division of the protoplasts was monitored within the plates over a three week period. Large increases in osmolality were measured in cell-free experiments indicating that overall evaporation from the plates was substantial. The amount of evaporation from a given culture well depended on the position of the well within the plate; three microenvironments corresponding to the corner, edge, and middle positions of the plate consistently developed. Water placed in the recessed area between each culture well moderated the desiccation of the medium but did not eliminate the formation of microenvironments. The osmolality of the medium in protoplast cultures was higher than in the cell-free experiments but similar trends in terms of plate position were recorded. After 3 weeks of incubation of plates with water added between the wells, the osmolality of the medium in the protoplast cultures had increased 209 mOsm in the corner wells, had increased 77 mOsm in the edge wells, and had decreased 39 mOsm in the middle wells. As a result, there was a three-fold higher incidence of division of the protoplasts in the middle wells than in the corner wells. The non-uniformity of medium osmolality in protoplast cultures within tissue cultures well plates is important in experimental design, in the reproducibility of procedures between different laboratories, and in the preparation of replenishment medium for protoplast cultures to minimize osmotic shock. This work was supported by the College of Agriculture and Life Sciences and the Graduate School, University of Wisconsin-Madison; by McIntire-Stennis project WIS 3082; and by the USDA-Forest Service, North Central Forest Experiment Station (B. Haissig, Project Leader).