Method of determining the antibiotic sensitivity of anaerobic microorganisms using standard disks

Three variants of the procedure for determination of antibiotic sensitivity in anaerobic microorganisms with the use of standard paper discs were developed. According to the first variant the solid nutrient medium is melted at 46 degrees C and mixed with the culture of the microbe being tested. The mixture is added to the cover of a Petri dish. When the medium becomes solid, antibiotic sensitivity discs are placed onto the agar surface. After that one more layer of the medium is added. The medium is allowed to solidify and some more medium is poured near the cover edge. Immediately after that the Petri dish is placed with its flat surface onto the agar layer in its cover. According to the first and second variants the mixture of the medium and culture is added to a Petri dish and immediately a transparent gas-proof polymer film of the dish size is placed onto the agar surface. Previously antibiotic paper discs or solutions are fixed on the films. THe incubation temperature for all three variants is 37 degrees C. The procedure allows one to observe the culture growth and to obtain the results earlier than in case the culture is incubated in an aerostate. The procedure is simple and saves labor and time.     

Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO₂ incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/day. Depending on the lid membrane thickness, pH drifts at ambient CO₂ levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm² through PDMS lids as compared with 18.69 g/day/dm² with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Supplementary material pertaining to this article is available on the Journal of Biosciences Website at     

The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata

This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes. Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year. Biofilms were developed over 1, 3, 8, and 14 d. Controls were clean glass and polystyrene Petri dishes. Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d. Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached. The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces. Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA). There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA). These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels.     

Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts

Chick embryo fibroblasts were plated on Petri dishes that had not been treated for use in tissue culture (bacteriological dishes). On these dishes the cells grow at the same exponential rate as cells plated on tissue culture dishes, but their growth becomes inhibited sooner after plating, and therefore at a lower cell number per dish. The inhibition of cell growth on bacteriological dishes is correlated with the formation of cell clumps. Clump formation is reversible by mechanical transfer of the clumps to a tissue culture dish: the cells migrate out of the clumps, form a monolayer, and cell growth resumes.Clump formation was studied by time-lapse cinematography, and was found to be due to reduced adhesion of the cells to the bacteriological dish surface. This reduced adhesiveness of the substratum is due to a lower number of negatively-charged residues on the bacteriological dish surface, which can be measured by the binding of crystal violet. The number of negatively-charged residues, and therefore the adhesiveness of the substratum can be altered by treatment of the dishes with sulfuric acid. Serum components of the medium were found to affect cell adhesion to the bacteriological dishes, consequently altering the efficiency of cell attachment, the extent of cell growth and the pattern of clump formation.The cells in clumps were compared with those in confluent monolayers on tissue culture dishes. Growth-inhibited cells on both types of dish were found to be equally viable. Cells in clumps on bacteriological dishes were found to be inhibited in the G1 phase of the cell cycle, as are cells in density-inhibited monolayers. Infection by the oncogenic virus, Rous sarcoma virus, can release the cells from growth-inhibition on both types of dish. Cell-induced alterations of the medium are not involved in the growth inhibition of cells on bacteriological dishes.     

Numerical modeling and dosimetry of the 35 mm Petri dish under 46 GHz millimeter wave exposure

Experimental studies on effects of millimeter wave (MMW) exposure on cells cultured in Petri dishes have attracted interest in recent decades. To improve the quantification of the biological responses toward the MMW energy, an accurate and precise MMW dosimetry is to be provided. By using the finite difference time domain (FDTD) method, the numerical dosimetry is performed for a typical 35 mm Petri dish under 46 GHz continuous MMW exposure from an irradiator of a specified power pattern. With the aim of building a precise model, the meniscus at the interface between the culture solution and the Petri dish sidewall is taken into account, followed by the modeling of smooth edges of the Petri dish. The trilinear interpolation is introduced to assist the FDTD method to obtain a more precise dosimetric assessment. The specific absorption rate (SAR) distributions in the cornea cells covered by culture solution in the Petri dish are calculated and compared to display the effects of using Petri dish models of various precision and the trilinear interpolation on dosimetry results. In addition, the SAR distribution in the cells is analyzed to study its homogeneity. The results indicate that the precise Petri dish model and the application of the trilinear interpolation are helpful in improving the precision of numerical dosimetry. It is also revealed that the inhomogeneity of the SAR distribution is well beyond neglect, which deserves cautious consideration in experiments investigating MMW effects on cells in vitro.     

Three-dimensional culture of bovine chondrocytes in rotating-wall vessels

Summary The Rotating-Wall Vessel (RWV) was used to culture chondrocytes for 36 d to observe the influence of low-shear and quiescent culture conditions allowing three-dimensional freedom on growth, differentiation, and extracellular matrix formation. Chondrocytes were freshly isolated from bovine cartilage and placed into the RWV with Cytodex-3 microcarriers. Nonadherent petri dishes were initiated with microcarriers as representative of standard culture conditions. In the RWV, large three-dimensional aggregates (5–7 mm) were formed in suspension. In addition, a large sheet of matrix adhered to the oxygenator core and vessel endcaps. Petri dish culture resulted in the formation of sheets of chondrocytes with no matrix production. Immunocytochemical analyses on histologic sections of tissue obtained from the RWV and the petri dish controls were performed with antibodies against fibronectin, collagen II, chondroitin-4-sulfate, chondroitin-6-sulfate, and vimentin. Results demonstrated increased signal in the RWV material while the petri dishes demonstrated a slight decrease in signal. In addition, differentiated chondrocytes were observed in sections of RWV material through 36 d, while few were observed in the sections of petri dish material. These results indicate that the unique conditions provided by the RWV afford access to cellular processes that signify the initiation of differentiation as well as production of normal matrix material.     

A simple model for the study of effects of the extracellular matrix on the cell morphology in vitro

In the present work, a simple technique is proposed to study the effects of native extracellular matrix (ECM) of one cell type on the properties of other cell types. It is based on a procedure in which, after cells of one type are removed from the substrate, cells of another type are seeded on the same substrate. To obtain preparations of native ECM, cells were removed from the substrate by 0.02% EDTA only, without any proteolytic enzymes. Cells were placed on coverslips in standard Petri dishes and incubated in a culture medium for a time sufficient for adhesion and spreading, but not long enough to undergo mitosis. Up to four coverslips per Petri dish can be incubated, and various combinations of ECM and cell types can be used in one dish. It is important, therefore, that the different "ECM-cell" combinations are present in the same culture medium. For evaluation of ECM effects, the area occupied by the cell on a substrate and the perimeter of the cell were measured, and frequencies of cell distribution were calculated according to these parameters.     

Cylindrical waveguide applicator for in vitro exposure of cell culture samples to 1.9-GHz radiofrequency fields

An applicator for in vitro cell culture exposure was developed based on a circularly polarized, cylindrical waveguide for the 1.9-GHz frequency band used by Personal Communications Services (PCS) in Canada. The applicator consists of two coaxial Petri dishes that sit on the open end of the cylindrical waveguide. The inner 60-mm Petri dish contains the cell culture while the outer 150-mm dish contains coolant water, which is circulated from a pump. A dosimetric evaluation was made using thermometric and E-field probe techniques. The latter allowed the entire inner dish to be scanned to determine the range of specific absorption rates (SARs) pertinent to the expected position of the cells. A representative SAR rate (SAR per unit of input power) of 8.6 +/- 2.1 W/kg/W (95th percentile) was determined 1 mm from the bottom, for a 10 ml sample volume of standard medium. Evaluation of the cooling system demonstrated that following an initial 0.3 degrees C temperature increase, a constant temperature was maintained for 24 h when the waveguide was energized to achieve an average sample SAR of 10 W/kg. These properties enable both acute and sub-acute in vitro bio-effect studies to be performed on a variety of cell culture samples.     

Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

Detection and isolation of radionuclide-accumulating bacteria by autoradiography

A simple and rapid method for the detection and isolation of radionuclide-accumulating microorganisms is described. Water samples are mixed with a radioisotope solution and peptone agar in Petri dishes and incubated at 25 C. After bacterial colonies have appeared the agar is removed from the dish and placed upside down on X-ray film covered with thin plastic foil. Black spots on the developed radiogram reveal which surface colonies contain radionuclide-accumulating bacteria. From these colonies pure cultures are obtained by standard methods. So far, bacterial strains have been isolated accumulating one of the following nuclides: cobalt-60, strontium-89, ruthenium-106, iodine-131, cesium-134, cerium-144.     

Technique for ecological studies of seed germination in relation to soil water potential

Summary Seeds were germinated in soils of known matric potential (ψ_m) achieved by adding the requisite amount of water to air-dry soil and mixing for several days. The quantity of water was derived from calibration curves of water content against ψ_m by use of pressureplate extraction equipment. Soils were transferred to plastic Petri dishes in which seeds were sown. Variations of the technique permitted germination counting through the transparent dish lid, or by opening the dish and either resealing or discarding the replicate dish. Measurements of ethylene and carbon dioxide in the soil atmosphere suggest that neither gas accumulated to a level which could interfere with interpretation of results. Some species showed sensitivity of germination to water potential which was correlated with the relative wetness of their habitats.     

Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants

Summary A method of clonal germplams preservation utilizing dehydrated somatic embryos and cool temperature storage conditions was demonstrated. Somatic embryos of grapevine (Vitis vinifera L) Autumn Seedless and Chardonnay were produced from suspension cultures. After washing twice with sterile water mature somatic embryos were blot-dried and placed on sterile filter paper in an open Petri dish in a laminar flow hood until they reached about 25% of their initial weight. Approximately 300 dried embryos were placed in each sterile 90×15 mm Petri dish, which was tightly sealed with two layers of Parafilm^TM. Sealed dishes were stored in the dark at 4°C in a standard refrigerator. Samples of 25–60 individual dehydrated somatic embryos were periodically tested for viability by placing them on solidified MS medium for germination and plant regeneration. After 42 mo. of dehydrated storage, 90% of the somatic embryos regenerated into plants. To further test utility, of this storage method dehydrated embryos stored for 12 and 26 mo. were shipped from Florida to Washington where 75 and 87.5% regenerated into plants, respectively. Cool temperature storage of dehydrated somatic embryos is a simple and inexpensive method of clonal, germplasm preservation when compared to alternatives such as cryopreservation.     

Surface modification of polystyrene Petri dishes by plasma polymerized 4,7,10-trioxa-1,13-tridecanediamine for enhanced culturing and migration of bovine aortic endothelial cells

Abstract

Plasma surface modification is an effective method for changing material properties to control cell behavior on a surface. This study investigates the efficiency of a plasma polymerized 4,7,10-trioxa-1,13-tridecanediamine (ppTTDDA) film coated on a polystyrene (PS) Petri dish, which is a biocompatible surface with carbon- and oxygen-based chemical species. The adhesion, proliferation, and migration properties of bovine aortic endothelial cells (BAECs) were profoundly enhanced in the ppTTDDA-coated PS Petri dishes without extracellular matrix (ECM) proteins, when compared with the uncoated PS Petri dishes. These observations indicate that ppTTDDA-coated PS Petri dishes can directly interact with cells, regardless of cell adhesion molecules. The increased cell affinity was attributed to the high concentration of carboxyl group on the surface of the ppTTDDA film. Such a carboxyl surface showed an excellent ability to promote culturing of BAECs. Plasma surface modification techniques are effective in improving biocompatibility and provide a surface environment for cell culture.     

A New Method for Collecting Sessile Ciliates in Plastic Petri Dishes With Tight-Fitting Lids

SYNOPSIS. Large numbers of sessile ciliates were successfully collected in plastic petri dishes with tight-fitting lids, transported in the water-filled dishes without disturbance. Each species within the transparent dishes was identified with a dissecting microscope and the position on the dish surface of each sessile individual was located and recorded on graph paper for further quantitative comparisons. This method was used for numerous experiments on the ecology and behavior of sessile ciliates and their responses to toxins.     

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley

We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.     

It is Time for a Change:Petri Dishes Weaken Cells

We wish to draw the attention to a potential deficiency in the biocompatibility of polystyrene cell culturc dishes which is caused by a softening of the material under relevant culture conditions.The finding confirms the central hypothesis of our previous model study.In it we assumed a local increase in pH at the interface between the hydrophilic polymer and liquid.The finding is of considerable biological interest.Polystyrene tissue culture dishes are now in use for 50 years.To the best of our knowledge their biocompatibility has never been challenged.Here we report the first experimental proof that exposure to water softens the surface of polystyrene Petri dishes.We expect that our results will stimulate the development ofa new generation of cell culture devices,including Petri dishes and culture flasks,and the establishment of improved biomimetic settings for tissuc engineering and stem cell research.New non-swelling biomaterials or nanocoatings designed to reduce the swelling of polymer culture dishes could improve cell performance.The need for further study is clear.     

A circadian rhythm of population behavior in Gonyaulax polyedra

Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation. Shape and size of the aggregations depend on container type, light intensity, and cell density. In Petri dishes, cells form oval "swarms"; within these, cells move downward in the highly dense center and rise up at the periphery. We have investigated the daily rhythm of this swarming activity in Petri dishes illuminated from the side, using time-lapse video recordings. At night, a "lawn" of cells forms at the bottom of the dish toward the light source (independent of light intensity). Before dawn, cells rise toward the surface and aggregate in swarms. The daily vertical migration occurs independent of light direction and intensity. The diurnal swarms, however, form every day at the same location within the dish, at a distance from the light that depends on light intensity, indicating a self-selection of light intensity. In constant light and temperature and with negligible vertical nutrient differences, all aspects of the rhythm continue to oscillate for up to 3 weeks, when the rhythm of the population becomes desynchronized. Under cycles of bright white-dim red light (WR), cell entrain to WR 10:10 but free run in WR 8:8 and shorter cycles, showing relative coordination (von Holst, 1939) to the driving light cycle. They also entrain to the 24-hr multiple of WR 6:6. Under nonentrained conditions, swarming activity is still influenced by light changes, and in spite of the apparent free run, the phasing of the averaged activity varies systematically with different T-cycle frequencies.     

Development of maize caryopses resulting from in-vitro pollination

Intact maize (Zea mays L.) ovaries were excised from unpollinated ears (pistillate inflorescences of field-grown plants and placed on defined, agar-based media in Petri dishes. Application of pollen to the end of silks (styles) positioned outside the Petri dish resulted in fertilization of 46% of the ovaries. The extent of subsequent kernel (caryopsis) development varied. After 40 days some kernels had only embryo development while others had embryo and variable endosperm development. About 5% of the initial ovaries developed into normal kernels; 60% of the kernels with some endosperm germinated under laboratory conditions, and 70% of the embryos excised from the embryo-only kernels germinated on culture media.Contribution from the Department of Agromy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA. Paper no. 9641 scientific journal series, Minnesota Agricultural Experiment Station     

A method for the immobilization of living Tetrahymena

The surfaces of plastic (polystyrene) Petri dishes from several suppliers were discovered to have the useful property of immobilizing cells of the ciliate Tetrahymena thermophilia upon contact in nutrient-free buffer (10 mM Tris, pH 7.4). The procedure works with cells in both logarithmic and stationary growth phase, so long as they are first transferred to nutrient-free buffer, and then added to dishes already containing buffer to a depth of 2–10 mm. Dish surfaces specially treated for tissue cultures are unsuitable for this purpose. Cells can be released from the dish surfaces by the simple addition of growth medium (1% proteose peptone). Immobilized cells are fully competent to complete conjugation or cell division. The technique offers promise for facilitating experiments requiring microinjection, microsurgery, or simply detailed observation of living protozoan cells.     

Adhesion, growth and detachment of cells on modified polystyrene surface

By adsorbing poly(N-isopropylacrylamide) (PNIPAAm) from an aqueous solution onto oxidised polystyrene without the need for grafting the polymer to the surface, we showed here that cells(CHO-K1) adhere and grow well at 37 °C and are detached by lowering the temperature to 10 °C without any other deleterious treatment. Both bacterial culture grade polystyrene Petri dishes and polystyrene beads (120 to 250μm diameters) commercially available used in static conditions of growth were tested with similar results. The contact angle of modified Petri dishes with a water droplet increases from 36 to 58° when the temperature is raised from 25 to 37 °C indicating change in hydrophilicity of the surface as a function of temperature. This revised version was published online in July 2006 with corrections to the Cover Date.