Coating bacteriological dishes with fibronectin permits spreading and growth of human diploid fibroblasts

The ability of human diploid fibroblasts to spread and grow has been compared in control and fibronectin-coated bacteriological dishes and tissue culture dishes. After 6 hr in culture, complete cell spreading occurred in tissue culture dishes, but no cell spreading was observed in bacteriological dishes. If however, the bacteriological dishes were pre-coated with fibronectin, cell spreading occurred similarly to that observed in the tissue culture dishes. Rates of cell growth were found to be similar in control tissue culture dishes and in fibronectin-coated tissue culture and bacteriological dishes. After 72 hours, the cell yield was about 490% of the starting cell number. On the other hand, in control bacteriological dishes there was much less cell growth. After 72 hours, the cell yield was only about 170% of the starting cell number.     

Morphological changes in a human scirrhous gastric carcinoma cell line (KATO-III) when cultured in collagen-coated dishes

The morphological differences between cells of a human scirrhous gastric carcinoma cell line (KATO-III) cultured in plastic dishes and in collagen-coated dishes were examined by phase-contrast and electron microscopy. When KATO-III cells were inoculated into plastic dishes, a few cells became attached to the surface of the dishes and the rest remained in suspension. However, when they were inoculated into collagen-coated dishes, they all remained in suspension. In both types of dish, most of the cells in suspension were single although a few were in clusters. The cells in suspension in collagen-coated dishes differed in morphology from those in the plastic dishes. They had abundant cytoplasm, well-developed Golgi complexes, and many microvillus-like cell protrusions. Moreover, they had hemidesmosome-like and desmosome-like structures on their surface and an increased amount of intracytoplasmic desmosome-like structures. The cells in clusters in the collagen-coated dishes were closely connected by junctional complexes, such as tight junctions, desmosomes and interdigitations, whereas those in plastic dishes were linked only by desmosomes. These results suggest that collagen affects the morphology of human scirrhous carcinoma cells.     

Impact of food source on survival of red flour beetles and confused flour beetles (Coleoptera: Tenebrionidae) exposed to diatomaceous earth

A series of experiments was conducted to determine the effect of a flour food source on survival of red flour beetle, Tribolium castaneum (Herbst), and confused flour beetle, Tribolium confusum (DuVal), exposed to the labeled rate (0.5 mg/cm2) of Protect-It, a marine formulation of diatomaceous earth. Beetles were exposed at 27 degrees C, and 40, 57, and 75% RH in 62-cm2 petri dishes. When beetles were exposed for 1 or 2 d in dishes with the labeled rate (0.5 mg/cm2, or 31 mg per dish) of diatomaceous earth or in dishes containing flour at varying levels from 0 to 200 mg mixed with the labeled rate of diatomaceous earth, survival of both species increased as the amount of flour increased, and quickly plateaued at levels approaching 100%. In a second set of experiments, beetles were transferred to dishes containing flour at varying levels from 0 to 200 mg after they were exposed for 1 or 2 d in dishes with the labeled rate of diatomaceous earth alone. There were no significant differences in beetle survival among the levels of flour, however, survival in dishes with flour was usually greater than survival in dishes with diatomaceous earth alone. In a third test, beetles were exposed for 1, 2, and 3 d in dishes with either the labeled rate of diatomaceous earth alone (clean dishes), dishes with diatomaceous earth and empty straws, or dishes with diatomaceous earth and approximately 300 mg of flour packed in the straws. Survival was not significantly different between clean dishes or dishes with straws, but survival in dishes containing the straws with flour was usually 100%, regardless of exposure interval. In all experiments, confused flour beetles were less susceptible to diatomaceous earth than red flour beetles. In addition, survival was negatively related to exposure interval and positively related to relative humidity.     

Morphological changes in a human scirrhous gastric carcinoma cell line (KATO-III) when cultured in collagen-coated dishes

The morphological differences between cells of a human scirrhous gastric carcinoma cell line (KATO-III) cultured in plastic dishes and in collagen-coated dishes were examined by phase-contrast and electron microscopy. When KATO-III cells were inoculated into plastic dishes, a few cells became attached to the surface of the dishes and the rest remained in suspension. However, when they were inoculated into collagen-coated dishes, they all remained in suspension. In both types of dish, most of the cells in suspension were single although a few were in clusters. The cells in suspension in collagen-coated dishes differed in morphology from those in the plastic dishes. They had abundant cytoplasm, well-developed Golgi complexes, and many microvillus-like cell protrusions. Moreover, they had hemidesmosome-like and desmosome-like structures on their surface and an increased amount of intracytoplasmic desmosome-like structures. The cells in clusters in the collagen-coated dishes were closely connected by junctional complexes, such as tight junctions, desmosomes and interdigitations, whereas those in plastic dishes were linked only by desmosomes. These results suggest that collagen affects the morphology of human scirrhous carcinoma cells.     

Cell-to-substratum adhesion and guidance of axonal elongation

The behavior of axonal growth cones on surfaces with patterned variations in substratum was observed. Cells from sensory ganglia of 8-day-old chicken embryos were cultured on plastic petri dishes, plastic tissue culture dishes, and polyornithine-coated tissue culture dishes, all of which contained gridlike patterns of palladium (Pd) deposition.The results indicated that growth cones elongated on the Pd-shadowed areas vs areas lacking Pd deposits depending on the relative adhesivity of the growth cones to the substrata. In petri dishes, growth cones stay on the Pd; in tissue culture dishes, they cross from one surface to the other; and in polyornithine-coated dishes, they elongate for great distances on the Pd-free areas. Analyses of time-lapse movies showed that, on Pd-shadowed polyornithine dishes, growth cones often approach the Pd-coated areas and microspikes touch the Pd surface. Yet, the axon tip continues to elongate on the Pd-free polyornithine surface.The conclusion is offered that interactions between microspikes and the substratum adjacent to the growth cone are important determinants of the directions and pathways of axonal elongation.     

Induction of hypoxia in glass versus Permanox petri dishes

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.     

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.     

Application of robotics and image processing to automated colony picking and arraying.

We describe a system that applies image processing and robotic techniques to automatically pick individual colonies from square petri dishes and array them in 96-well microtiter plates. Digital images of the colony distribution in the dishes are acquired using a video camera and frame buffer. Commercial image processing software is used to identify individual colonies and determine their locations. A Hewlett-Packard Microassay System robot reads the resulting coordinate file for each dish, picks cells from each identified colony and transfers the cells into a microtiter plate well. A disposable pipet tip is used as the sterile implement for colony picking. Custom holders position the dishes accurately and provide common coordinate systems for imaging and picking. The system is calibrated to account for the depth of agar in the dishes. The robot can process up to 10 dishes and 20 plates (1920 colonies) in a single run. It has successfully arrayed a cosmid library of the S. pombe genome consisting of approximately 6000 colonies in 30 petri dishes in about 40 hours of robot time. Future enhancements to the system are discussed.     

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Machine for Automatic Bacteriological Pour Plate Preparation

A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated.     

A simple method for determining the sensitivity of micro-organisms to serial dilutions of antibiotics

Summary At this laboratory the routine sensitivity tests of micro-organisms against sulfathiazol and antibiotics have been performed in plastic dishes for half a year. The dishes are cleaned and sterilized in the same way as bacteriological glassware. The use of these dishes offers some advantages over other conventional dilution methods employing tubes or Petri dishes with solid media: the sensitivity to serial dilutions of one antibiotic may be measured in one and the same plate, and in this way a considerable amount of glassware and of space in the incubator may be saved; the readings are easily done and distinct. It has been demonstrated that sensitivity tests with two standard strains against sulfathiazol and various antibiotics may be reproduced with the same endpoints.     

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set.
In the present experiment, 4 dishes were prepared under the same conditions. From each of the dishes, 20 fields were selected, and the numbers of muscle cells *** were counted and separately recorded. Since the purpose was to compare two groups of dishes, the design was a simple case of a nested one. From the experiment, the type of distribution seemed approximately a long-normal distribution with constancy of variance (homoscedasticity). Since the distribution of the cells in dishes belonging to the same group could be considered to be the same, the numbers from each dish could be pooled within a group. Therefore, if the test criteria for Student's t test and the sensitivity to descriminate the ratio of the number of groups are given, the number of fields to be observed per dish times that of dishes can be uniquely determined. This method can be applied for the same purpose to other kinds of cells with log-normal distribution.     

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.     

In vitro mineralization by mesenchymal stem cells cultured on titanium scaffolds

Titanium has been utilized in the field of orthopaedic and dental reconstructive surgery, but mineralization through osteogenic differentiation of osteogenic cells on titanium surfaces has not been fully investigated. Here we cultured rat mesenchymal stem cells (MSCs) on the surfaces of titanium dishes in osteogenic media containing calcein which is a calcium-binding fluorescence dye. On titanium dishes, MSCs showed high viability to adhere to the surfaces and excellent proliferation. At day 14 of culture, MSCs differentiated into osteoblasts to form mineralized matrices on titanium dishes as well as tissue culture polystyrene (TCPS) dishes which are widely recognized as optimal culture substrates. Calcein was incorporated into the bone minerals fabricated by MSCs cultured on both substrates to show green emission under fluorescence microscopy. The fluorescence intensity was quantified with an image analyser during culture periods. These results indicate that the surfaces of titanium showed a high adhesion/proliferation potential to MSCs and that the titanium effectively supported the osteogenic differentiation of MSCs comparable to TCPS dishes. Therefore, the titanium is an effective scaffold that is applicable in bone reconstruction surgery.     

Bubble Contact Angle Method for Evaluating Substratum Interfacial Characteristics and Its Relevance to Bacterial Attachment

A bubble contact angle method was used to determine interfacial free-energy characteristics of polystyrene substrata in the presence and absence of potential surface-conditioning proteins (bovine glycoprotein, bovine serum albumin, fatty acid-free bovine serum albumin), a bacterial culture supernatant, and a bacterial exopolymer. Clean petri dish substrata gave a contact angle of 90°, but tissue culture dish substrata were more hydrophilic, giving an angle of 29° or less. Bubble contact angles at the surfaces exposed to the macromolecular solutions varied with the composition and concentration of the solution. Modification by pronase enzymes of the conditioning effect of proteins depended on the nature of both the substratum and the protein, as well as the time of addition of the enzyme relative to the conditioning of the substratum. The effects of dissolved and substratum-adsorbed proteins on the attachment of Pseudomonas sp. strain NCMB 2021 to petri dishes and tissue culture dishes were consistent with changes in bubble contact angles (except when proteins were adsorbed to tissue culture dishes before attachment) as were alterations in protein-induced inhibition of bacterial attachment to petri dishes by treatment with pronase. Differences between the attachment of pseudomonads to petri dishes and tissue culture dishes suggested that different mechanisms of adhesion are involved at the surfaces of these two substrata.     

Spatial Patterns of Entomopathogenic Nematodes in Microcosms: Implications for Laboratory Experiments

Laboratory microcosms were used to: i) measure the effects of soil moisture on survival of Steinernema riobravis and ii) investigate the suitability of using microcosms to study motility and survival of these nematodes. Nematodes recovered from soil contained in petri dishes declined by more than 95% during 7 days, whereas nematodes recovered from the inner surfaces of dishes increased 35-fold. After 7 days in dishes, >20 times as many nematodes were recovered from dish surfaces than from soil. Nematodes exhibited a negative geotropism; greater numbers of nematodes were recovered from the lid surfaces than from the surfaces of dishes. Survivorship of nematodes in soil in plastic centrifuge tubes was somewhat greater than in petri dishes, and fewer nematodes ascended above the soil line in tubes than dishes. Downward migration of nematodes was inversely related to soil column diameter, possibly due to relatively unimpeded movement along container surfaces. An assay was developed by which nematodes were rinsed from the inner surfaces of centrifuge tubes into the soil. The resulting slurry was then processed on Baermann trays to recover motile nematodes. Nematode survival in soil in centrifuge tubes was higher at soil moistures between 2-4% than at lower (0.5-1.0%) and higher (4.0-12.0%) moisture levels. Survival of S. riobravis may be enhanced by quiescence induced by moisture deficits.     

Response of female Lutzomyia longipalpis to host odour kairomones from human skin

Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) is the vector of Leishmania chagasi, the aetiologic agent of visceral leishmaniasis in the New World. In the present study, the response of female sandflies from Jacobina, Brazil, to human odours from six different volunteers was investigated. Glass Petri dishes were handled by different volunteers and then exposed to female sandflies. There was a significant difference between subjects in that some individuals were more attractive or less repellent to sandflies. Response of flies to handled Petri dishes was higher during the first minutes of observation, suggesting the presence of volatile compounds in hand odours. Extracts of glass Petri dishes that had been handled by the volunteers were made with organic solvents such as acetone, methanol, pentane and ether. These were then concentrated and tested for sandfly response. Only extracts carried out with non-polar solvents such as pentane and ether were able to transfer odours from handled glass Petri dishes onto clean dishes. The attractivity of male and female human subjects was monitored for 80 days, and minor fluctuations in attractiveness were observed.     

Detrimental Effect of Water Submersion of Stools on Development of Strongyloides stercoralis

Strongyloidiasis is prevalent in Thailand, yet its prevalence in the south is lower than in other parts of the country. This might be due to the long rainy season in the south resulting in stool submersion in water inhibiting worm development. In this study, the effect of water submersion of fecal samples on development of Strongyloides stercoralis was investigated. Ten ml of a 1∶5 fecal suspension were placed in 15-ml tubes, 35-mm dishes, and 90-mm dishes producing the depths of 80 mm, 11 mm and 2 mm-suspensions, respectively. The worm development was followed at 1/6, 4, 6, 8, 10, 12, 14, 16, 24, and 36 h, by determining the number of filariform larva (FL) generated from agar-plate cultures (APC). Fecal suspensions kept in tubes and 35-mm dishes showed a decline in FL yield relative to incubation time and reached zero production 14 h after incubation. In contrast, the number of FL generated from the suspension kept in 90-mm dishes remained stable up to 36 h. Cumulatively, all tubes and 35-mm dishes became negative in APC after 14 h while 90-mm dishes remained APC-positive up to 36 h. Adding more water or stool suspension to dishes resulted in a decreased number of FL. Mechanical aeration of the suspensions in tubes restored an almost normal FL yield. It appears that the atmospheric air plays a significant role in growth and development of S. stercoralis in the environment and may be one of factors which contribute to a lower prevalence of human strongyloidiasis in the south of Thailand.     

Factors influencing the growth and respiration of rat cerebral astrocytes in primary culture

Cell densities and respiratory rates of astrocytes from neonatal rat brain grown in primary culture were determined after 20–30 days in vitro. Cells grown in flasks reached lower densities (g DNA/cm²) and higher protein: DNA ratios than cells grown in petri dishes. Respiratory rates were lower for cells grown in flasks compared to cells grown in dishes. The pH of the medium in flasks fell below 6.9 between feedings while the pH of the medium in dishes remained at about 7.2. Cells grown in dishes with the medium pH adjusted to 6.8 also showed lower final cell densities, higher protein: DNA ratios, and lower respiratory rates, compared to cells grown under similar conditions at pH 7.5. Intermediate values of each parameter were found in cells grown at pH 7.5 for one week and then at 6.8 for 20 days. We conclude that the effects of ambient pH account for the differences in growth characteristics and respiratory rates of astrocytes grown in dishes versus those grown in flasks.