Licea eremophila, a new myxomycete from arid areas of South America

A new stipitate species of myxomycete of the genus Licea is described based on material from arid areas in Argentina and Chile. It was isolated from moist chamber cultures and found fruiting on field collections, usually on the same substrate, Puya sp. (Bromeliaceae). It differs from all described species in the genus in that it has stipitate sporocarps with dehiscence by defined preformed platelets and a smooth inner peridial surface. The new species has polyhedral, yellow spores with a uniform thick spore wall and dense warts except on irregularly dispersed raised bands with fewer warts, visible by SEM, an ornamentation not previously observed in the genus. Life-cycle events are described and illustrated, from germination to sporulation, based on moist chamber and agar cultures. The morphology of the myxomycete specimens was examined with scanning electron microscopy and light microscopy, and both light and SEM micrographs of relevant details are included.     

New Methods for Cultivating,Harvesting, and Purifying Mass Cultures of the Hypotrich Ciliate Euplotes aediculatus1

A new method is described for mass cultivation of Euplotes aediculatus, a hypotrich ciliate containing omikron-symbionts. The ciliate cultures, continuously aerated in Erlenmeyer flasks (5000 ml) with 4500 ml medium, yield densities of 2300 cells/ml which are four to five times higher than cell densities of cultures grown in unaerated Fernbach flasks. Harvesting such cultures involves the application of 25-μm mesh sieves. Cells so concentrated can be purified by using columns or special chambers in which Euplotes migrates towards the cathodes in an electric field (field strength 7 V/cm).     

An Instrument for Continuous Viewing of Roll Tube Cultures under a Stereoscopic Microscope

An instrument which fits on the stage of a CTS M6100 stereoscopic microscope and provides a continuous field of view for roll tube cultures is described. It has been used to facilitate the rapid counting of colonies on soil extract agar.     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 ^1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure ^3,4.A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.     

A method of estimating the numbers of soil protozoa,especially amoebae,based on their differential feeding on bacteria

Cultural methods of estimating protozoal numbers in soil based on a dilution technique have suffered from two main sources of error: first, the use of too few replicate cultures and, secondly, the uncertain quality of the bacterial food supply. A method is described of using glass cells to set up 8 replicate cultures in one Petri dish, employing pure bacterial cultures spread over a non-nutrient agar base to provide a standard and suitable food for the Protozoa. The selection and testing of suitable bacterial strains as food supply is described. Tests for the 'recovery' of counted suspensions of Protozoa added to sterilized soil were made; the results of these are given and the limits of experimental error in the method are discussed.     

A method for obtaining mitotic figures from blood leucocyte cultures of rainbow trout, Salmo gairdneri

A method for obtaining dividing cells for chromosome analysis from blood cultures of the rainbow trout, Salmo gairdneri , is described. The effects of different phytohaemagglutinin and foetal calf serum concentrations, oxygenation of the cultures, and varying the initial volume of cell inoculum on the number of mitoses obtained are described and discussed.     

Life cycle and morphology of Physarum pusillum (Myxomycetes) on agar culture

Myxomycetes (slime molds) are unique eukaryotic microorganisms with both characteristics of fungi and amoebae. Artificial cultures grown under controlled conditions were used to study the life cycle and morphogenesis. Physarum pusillum was collected from the field. Spores were inoculated and cultured with the hanging drop method. The complete life cycle was observed from spore to spore on agar without adding any solid nutrients or bacteria as food. Life cycle morphological characteristics were described for spore germination, myxamoebae, zygote, plasmodium and sporangia formation.     

Culture techniques for studies on the growth, development and reproduction of copepods and cladocerans under laboratory and in situ conditions: a review

SUMMARY. This review deals with culture techniques in use to estimate individual growth, development and reproduction of copepods and cladocerans in cultures. The conceptual aspects of growth, reproduction and feeding behaviour are briefly summarized. Marine copepods are included, because the techniques employed for the cultivation of these animals are often more advanced than those used for freshwater crustaceans. Most of the studies reviewed here were carried out under defined laboratory conditions, but a few were under field conditions, generally in small in situ enclosures. The culture methods described in this review are based on 272 studies published between 1910 and 1987. A large number of different culture systems are critically discussed in relation to culture conditions and culture techniques and the taxa used. Particular attention is given to the effect of food quality, and the problem of how to apply laboratory measurements to field populations so as to limit errors to the minimum. A more detailed summary is given in the section on 'Recommendations' at the end of the review.     

Diversity and structure of AMF communities as affected by tillage in a temperate soil

Arbuscular mycorrhizal fungi (AMF) were studied in differently tilled soils from a long-term field experiment in Switzerland. Diversity and structure of AMF communities were surveyed either directly on spores isolated from the field soil or on spores isolated from trap cultures, planted with different host plants. Single-spore cultures were established from the AMF spores obtained from trap cultures. Identification of the AMF was made by observation of spore morphology and confirmed by sequencing of ITS rDNA. At least 17 recognised AMF species were identified in samples from field and/or trap cultures, belonging to five genera of AMF--Glomus, Gigaspora, Scutellospora, Acaulospora, and Entrophospora. Tillage had a significant influence on the sporulation of some species and non- Glomus AMF tended to be more abundant in the no-tilled soil. The community structure of AMF in the field soil was significantly affected by tillage treatment. However, no significant differences in AMF diversity were detected among different soil tillage treatments. AMF community composition in trap cultures was affected much more by the species of the trap plant than by the original tillage treatment of the field soil. The use of trap cultures for fungal diversity estimation in comparison with direct observation of field samples is discussed.     

Primary culture of bovine mammary acini on a collagen matrix

Lactating bovine mammary epithelial acini were isolated and primary culture on rat tail attached collagen gels are described. Acini rapidly attach to the gels and morphologically change little over days of culture under the culture conditions described herein. Cells release lactose, alpha-lactalbumin and alpha-s1 casein over a 6-day period. A new HPLC method for measuring lactose in mammary cell culture media is described. Comparisons of acini cultures with individual cell cultures show acini to be 1.5-5 times more productive than cells in secreting lactose and casein, respectively.     

Bacitracin increases size of parasporal crystals and spores in Bacillus thuringiensis

Summary The effect of bacitracin on Bacillus thuringiensis is described. When added after the end of the exponential phase, it produces a marked increase in the size of spores and parasporal crystals. The cultures to which the antibiotic was added are able to produce more crystal proteins than non-treated cultures. The protein composition of crystals from bacitracin-treated cultures is the same as that of crystals purified from control cultures.     

In vitro tests for the measurement of veterinary clostridial toxins, toxoids and antisera. I. Titration of Clostridium septicum toxins and antitoxins in cell culture

The assay of Clostridium septicum antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in vivo counterparts, permitting detection of substantially lower titres than is possible using in vivo indicators. It is suggested that cell culture indicators may prove useful for the titration of Cl septicum antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations.     

First principle-based models in plant suspension cell cultures: a review

In this work, the development and application of published models for describing the behavior of plant cell cultures is reviewed. The structure of each type of model is analyzed and the new tendencies for the modeling of biotechnological processes that can be applied in plant cell cultures are presented. This review is a tool for clarifying the main features that characterize each type of model in the field of plant cell cultures and can be used as a support on the selection of the more suitable model type, taking into account the purpose of specific research.     

A rapid assay for fusion of embryonic chick myoblasts

A rapid and sensitive assay for measuring myoblast fusion in suspension cultures of embryonic chick pectoral myoblasts is described. Fusion-competent cells are generated by growth in suspension using a low calcium medium. Fusion-promoting levels of calcium are added, and the suspensions incubated for 1–6 h. The cells are then trypsinized to disperse cellular aggregates and sized in a Coulter particle counter. This assay minimizes many of the artifacts inherent in measurements of fusion in monolayer cultures, and is designed for the rapid screening of agents for their effects on fusion.     

Rapid preparation of cyanobacterial DNA for real-time PCR analysis

AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.     

Lentiviruses are naturally resident in a latent form in long-term ovine fibroblast cultures.

Long-term ovine fibroblast cultures contain replicative-competent lentiviruses in a latent form. This in vitro phenomenon, never described previously for lentiviruses, was clearly demonstrated by activating the expression of latent viruses with various inducing cell treatments, some of which were efficient in inducing endogenous retroviruses or latent herpesviruses. Activated lentiviruses were highly lytic in ovine fibroblasts (type I), or they established persistent infections (type II) as described previously for field isolates from sheep with progressive pneumonia (Quérat et al., J. Virol. 52:671-678, 1984).     

The mouse as a model for heart morphogenesis in mammals: the origin of myocytes and studies with cardiac explants

The heart is one of the first organs to form during embryogenesis since its circulatory function is critical from early stages for embryo survival. In man, morphological events are affected by molecular perturbations, which can lead to a congenital heart defect. It is important therefore to understand not only the molecular signals, but also the morphological events, which govern myocardial cell identity. The study of transgenic mouse lines, Mlc1v-nlacZ-24 and Mlc3f-nlacZ-2, has led to the identification of a new precardiac territory, the anterior heart field, which has also been described recently in birds, and which contributes to the myocardium of the arterial pole of the heart. The use of explant cultures also indicates that pharyngeal mesoderm participates in the formation of the outflow tract and right ventricle and shows that the primitive heart tube has a predominantly left ventricular identity. We have also shown that Fgf-10 is expressed in the anterior heart field, where a role for FGF signaling in arterial pole morphogenesis is suggested by inhibitor experiments. Finally explant cultures have been employed to examine the acquisition of left-right atrial identity. The Mlc3f-nlacZ-2 line, which marks the right atrium, allowed us to determine the time window during which left-right signaling confers left-right atrial identity.