Surface area and anchorage-dependent growth of Chinese hamster ovary cells.

Cell growth can be affected by many factors. For study of the cell responses under various environmental changes, the traditional method using batch cultures may not provide precise answers. It sometimes even leads to misapprehension. A flow system was therefore designed to eliminate the variations in chemical environment during the cell growth. An anchorage Chinese hamster ovary (CHO) cell line was chosen for study under this system. With the help of time-lapse video recording and image analysis, it was found that the exposed surface area (Ae) of Chinese hamster ovary (CHO) cells was significantly affected by the serum concentration (S) in the medium. Cells appeared to be larger when cultivated in medium of low serum content. Comparing the growth rates of cells on different substrata, we have found that, although they were different at a given serum concentration, the growth curves could be correlated to a Monod type equation by replacing the substrate term with AeS. Furthermore, when we studied the effect of density-dependent inhibition of cell growth, we found that there exists a minimum value of AeS required for stimulating cell proliferation. Once AeS falls below this value, cell proliferation stops. These observations seemed to indicate that the interaction between serum factors and cell surface, i.e., AeS, is an important parameter of the growth of CHO cells.

Microbiological evaluation of bottled uncarbonated mineral water in Taiwan.

The microbiological quality of 136 samples of bottled uncarbonated mineral water, including 88 domestic and 48 imported samples, was investigated. The numbers of samples with heterotrophic plate count (HPC) over the maximum level legally permitted in Taiwan (200 colony forming units ml-1) were 45 (51.1%) and 29 (60.4%) for domestic and imported samples, respectively. Coliforms and faecal streptococci were not detectable in the samples tested. Two of the domestic samples were contaminated with Aeromonas hydrophila and four with Pseudomonas aeruginosa. Bacteria isolated from water samples were identified as species of Pseudomonas, Aeromonas, Flavobacterium, Pasteurella, Xanthomonas, and Staphylococcus. Mold and yeast were detected in 38.6% and 18.8% of domestic and imported samples, respectively. The HPC of bottled mineral water stored at 25 degrees C increased quickly to 10(4)-10(5) colony forming units ml-1. In inoculation studies both A. hydrophila and Escherichia coli grew very well and mutualistic growth for both organisms was observed in mineral water at 25 degrees C.

Incidence and toxigenicity of Aeromonas hydrophila in seafood.

Three selective media, Oxoid Aeromonas agar (OA), blood ampicillin agar (BA) and starch ampicillin agar (SA) were used to evaluate the presence of Aeromonas hydrophila in 66 samples of oyster, shrimp, fish and surimi products. Oyster had the highest incidence, with 50% positive, whilst no A. hydrophila was found in the surimi. Of the three selective media, BA displayed the highest recovery rate of A. hydrophila from seafood. Forty-eight isolates from this survey were tested for their capability to produce hemolysin and cytotoxin. Hemolysin was produced by 79.2% of the isolates and cytotoxin was produced by 91.7% of the isolates in brain heart infusion broth. One of the toxin-producing isolates from oyster, strain 8-169, was further tested for growth and toxin production in oyster, shrimp and fish at various temperatures. This particular isolate grew best and had highest toxin production in oyster. Hemolysin and cytotoxin were produced earlier at 28 degrees C than at 37 degrees C, and titers of hemolysin were also higher at 28 degrees C. At 5 degrees C, it was able to grow and produce hemolysin in oyster.

Effects of temperature, medium composition, pH, salt and dissolved oxygen on haemolysin and cytotoxin production by Aeromonas hydrophila isolated from oyster.

The effects of temperature, medium composition, pH, salt content and dissolved oxygen (DO) on the production of haemolysin and cytotoxin by one strain of Aeromonas hydrophila isolated from oyster were investigated. Four media were tested: brain heart infusion broth (BHIB), casamino acid-yeast extract broth (CAYEB), nutrient broth (NB), and trypticase soy broth (TSB). BHIB was the best for toxin production even though the growth rates for Aeromonas hydrophila in all of these media were quite similar. Aeromonas hydrophila could produce haemolysin and cytotoxin at 37, 28 and 5 degrees C; however, the toxins were produced faster and were more stable at 28 degrees C than at 37 degrees C. Although Aeromonas hydrophila itself is tolerant to 5% (w/v) salt in BHIB and a pH range of pH 5.5 to 10.0, the production of haemolysin and cytotoxin was apparently decreased in the presence of 1-5% (w/v) NaCl or when the pH of the medium was greater or less than 7.2. The DO values in the culture medium during the stationary growth phase also seemed to affect toxin production; greater quantities of toxins were produced when the DO values were higher.

Antibacterial activity of shrimp chitosan against Escherichia coli.

The effects of cell age, reaction temperature, pH value, and salts on the inhibitory activity of shrimp chitosan (98% deacetylated) against Escherichia coli were investigated. The age of a bacterial culture affected its susceptibility to chitosan, with cells in the late exponential phase being most sensitive to chitosan. Higher temperature (25 and 37 degrees C) and acidic pH increased the bactericidal effects of chitosan. Sodium ions (100 mM Na+) might complex with chitosan and accordingly reduce chitosan's activity against E. coli. Divalent cations at concentrations of 10 and 25 mM reduced the antibacterial activity of chitosan, in the order of Ba2+ > Ca2+ > Mg2+. Chitosan also caused leakage of glucose and lactate dehydrogenase from E. coli cells. These data support the hypothesis that the mechanism of chitosan antibacterial action involves a cross-linkage between the polycations of chitosan and the anions on the bacterial surface that changes the membrane permeability.

Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation.

The antibacterial effects of sulfonated and sulfobenzoyl chitosans were evaluated and compared with that of 69% deacetylated chitosan (DD69 chitosan). Minimal inhibitory concentrations of sulfonated chitosan (SC1, 0.63% sulfur content) against Shigella dysenteriae, Aeromonas hydrophila, Salmonella typhimurium, and Bacillus cereus were found to be lower than those of DD69 chitosan. A high sulfur content in sulfonated chitosan adversely influenced its antibacterial effect. Sulfobenzoyl chitosan (SBC) has excellent water solubility and an antibacterial effect comparable to that of SC1. SBC at 1,000 and 2,000 ppm extended the shelf life of oysters at 5 degrees C by 4 days at the former or by 7 days at least at the latter concentration. The growth of coliforms and Pseudomonas, Aeromonas, and Vibrio species on oysters was retarded by the addition of DD69 chitosan or SBC.

Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation.

The antibacterial activity of a chitooligosaccharide mixture prepared by digestion of shrimp chitosan with cellulase at 50 degrees C for 14 h was evaluated. Sugars with 1 to 8 degrees of polymer (DP) were found in this chitooligosaccharide mixture, and the weight percentage of sugars with DP > or = 6 was 44.3%. Minimal lethal concentrations of this mixture against Aeromonas hydrophila, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Shigella dysenteriae, Staphylococcus aureus, Vibrio cholerae, and Vibrio parahaemolyticus in nutrient broth were 5 to 29 ppm, which were much lower than those of the chitosan reactant (50 to 1,000 ppm). The antibacterial activity of this mixture in the sterilized milk against E. coli O157, L. monocytogenes, Salmonella Typhimurium, and S. aureus was much stronger at 4 degrees C than at 37 degrees C. When raw milk was supplemented with either 0.24% or 0.48% (wt/vol) of this oligosaccharide mixture and stored at 4 degrees C for 12 days, its mesophilic and psychrotrophic counts were reduced by at least 3 log cycles, and there was very little change in pH. In addition, this mixture retarded the growth of Salmonella species and caused quicker reduction of Staphylococcus species in raw milk. Accordingly, the shelf life of raw milk at 4 degrees C was extended by at least 4 days.

Enzyme-linked immunosorbent assay for detection of molds in cheese and yogurt.

An enzyme-linked immunosorbent assay was developed for the detection of molds in dairy products. New Zealand White female rabbits were immunized with .45 mg of partially purified extracellular antigen from freeze-dried culture filtrates of Aspergillus versicolor, Cladosporium herbarum, Geotrichum candidum, Mucor circinelloides, and Penicillium chrysogenum. Blood was drawn at various intervals, and antibodies were separated and purified. Antibody-peroxidase conjugates were prepared with the following ratios being the optimum ones: A. versicolor 10:20; C. herbarum 5:10; G. candidum 1:10; M. circinelloides 5:5; and P. chrysogenum 10:10. The assays were sensitive within a range of 1 ng to 1 microgram/ml, depending on the mold used. Inhibition tests were done for each mold with concentrations of 0 to 5000 micrograms/ml of antigen. The enzyme-linked immunosorbent assay tests for Cladosporium, Geotrichum, and Mucor were only inhibited by antigens from other species of the same genus; whereas there was crossreaction between antibodies and antigens of species of Penicillium and of Aspergillus. Citrate buffer was best for extracting the mold from cheese and yogurt. The extract was adjusted to pH 7.2 and ELISA was performed. Results showed that these molds can be detected in Cheddar and cottage cheeses and yogurt within 2 d, which is before mold growth is visible in these products.

Partial purification and characterization of mold antigens commonly found in foods.

Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.

Some considerations for optimization of desorption chromatography.

The effect of isotherm parameters of a displacer on the efficiency of desorption Chromatography has been investigated numerically. A general nonlinear multicomponent rate equation model with Langmuir isotherm was used in this study. It was found that the best displacer in this kind of operation is usually not the one that is more strongly adsorbed than the adsorbates when the operation is to displace and concentrate the adsorbates from a saturated or partially saturated column and to minimize the amount of displacer used. The desorption Chromatography is different from the classical displacement development in both operational purpose and the requirement for the displacer. The desorption Chromatography in industrial practice was also analyzed and discussed for the case in which the displacer is introduced in either the same or the reverse flow direction after an incomplete frontal adsorption operation.

Monitoring and modeling density-dependent growth of anchorage-dependent cells.

The density-dependent growth of Chinese hamster ovary (CHO) cells was monitored on-line by using an inverted microscope. A flow system was employed for cell cultivation so that nutrient concentration could be maintained and metabolic wastes were removed. With the help of video image analysis, local cells density could be accurately calculated and cell motility and exposed cell surface area could be estimated. A computer program which accounted for change of sell size and translocation of cells was developed to stimulate cell growth. The stimulated results of the population dynamics and the variations in cell size showed good agreement with our experimental observations, Cell motility and initial cell distribution on the substratum were found to have strong effect on cell growth.

Thymic hyperplasia in a patient with Graves' disease.

Hyperplastic changes of the thymus may be found in patients with Graves' disease. However, this rarely presents as an anterior mediastinal mass, particularly among adults. In this report, we describe a 32-year old woman with Graves' disease and hyperthyroidism. During medical evaluation and treatment for her hyperthyroidism, a large anterior mediastinal mass was incidentally discovered. A cytological study of the lesion via computed tomogram-guided fine needle biopsy could not make a definitive diagnosis and suggested the possibility of a thymoma, which led to a surgical exploration. However, the final pathological diagnosis of the surgically removed tissue was thymic hyperplasia. The relationship between Graves' disease and thymic changes is discussed.