API ZYM Kit Research Articles

Viability and enzymatic activity of cryopreserved porcine heart valve.

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.

Influence du passage de Salmonella spp. et E. coli en eaux usees sur leur survie ulterieure en eau de mer

Sewage treatment plants are designed to purify wastewaters for given parameters, but their microbiological effectiveness is often poor without addition of a specific treatment (disinfection, filtration, etc.). Large amounts of enteric bacteria are thus released into the environment, in rivers and seawater. Is their subsequent survival in these hostile environments influenced by their previous stay in wastewaters? In order to assess this potential adaptation, we studied the behavior of E. coli, S. typhimurium and S. manhattan in seawater microcosms, with and without a previous 24 to 72 h stay in raw and treated wastewaters. The seawater and wastewater media were 0.22 μm filtered and autoclaved. The protocol followed is outlined in Fig. 1. At each step, bacteria were harvested by filtration and re-suspended in the following medium. All microcosms were maintained at 20°C, in the dark. Seven experiments were carried out, testing seven different wastewaters. The survival of the inoculated bacteria in seawater during 6 days was monitored by plate counts on trypticsoy agar and by acridine orange epifluorescence counts (Hobbie et al., 1977). In two experiments, the bacterial enzymatic activity was assessed before and after the stay in seawater, using APIZYM kits. S. typhimurium's survival in seawater was increased when cells had previously spent 24 h in raw wastewaters, and 24 and 48 h in treated wastewaters (Fig. 3). The culturable count was about 2 log units higher after 6 days. The same phenomenon was observed for S. manhattan tested in two raw wastewaters (Fig. 4). For this strain, with a concentrated inoculum (7 × 10 6/ml), survival was quite the same, with or without a previous stay in raw sewage. An adaptation of E. coli while in wastewater to subsequent seawater stress seemed less obvious (Fig. 5). While monitoring the behavior of enteric bacteria in seawater with different previous conditions, we assessed the ratio of culturable cells in the total population. Thus, loss of culturability of tested strains was smaller when bacteria had been put in wastewaters before seawater (Table 1). This could show that the level of cellular metabolism maintained was higher in this case. This hypothesis was confirmed by studying the enzymatic activity of S. typhimurium before and after 6 days in seawater (Fig. 6). A previous stay in sewage could preserve or even induce some enzymatic activity which was then maintained during the entire exposition to seawater. This suggests an active metabolism enabling new protein synthesis. In this study, we showed that Enterobacteriaceae could adapt to seawater stress during their stay in sewage and sewage treatment plants. The actual mechanism is not yet clear. It could be osmoprotection, modification of cellular metabolism, or another. Very few studies have dealt with this topic. Munro et al. (1987, 1989) observed that a previous culture in a saline medium or on a mixed wastewater/seawater agar could allow better E. coli survival in seawater. The osmoregulation mechanisms induced by high osmolarity are probably set up during this phase. This hypothesis cannot explain our results, since the wastewaters we used had very low osmolarity. It seems that adaptation to drastic conditions occurs during the stay in wastewater (lower temperature, lack of easily assimilated substrates, etc.) allowing for better survival in more drastic seawater conditions. This conclusion may be of great importance in assessing the sanitary quality of coastal areas used for bathing and shellfishing activities.

Peptidase activity and the ability of wine yeasts to utilise grape must proteins as sole nitrogen source

Abstract Peptidase activity of 14 Saccharomyces cerevisiae wine strains was tested with API ZYM kits LRA ZYM AP‐III and LRA ZYM AP‐IV, each containing 10 different enzymatic substrates. The ability of the yeast strains to utilise grape must proteins directly as sole nitrogen source was also examined by growth and fermentation tests in synthetic nutrient medium without additional nitrogen compounds, using a washed alcoholic precipitate of grape must. These observations strongly support the existence of an extracellular grape must protein hydrolytic enzymatic system in wine yeasts. Free amino acid content evolution in clarified and unclarified Chardonnay and Cortese pressed and free‐run juices, with or without growing yeast cells, was investigated by high‐performance liquid chromatography. Clarification does not seem to affect the grape amino acid contents. These remained almost constant from 0 to 8 h of contact with suspended solids when no growing yeast cells were detected. No grape proteolytic activity o...

Description of Two Morphotypes of Peptostreptococcus micros

Peptostreptococcus micros is often isolated from abscesses in several parts of the human body. The oral cavity is considered the natural habitat for the species, which has been implicated as a periodontal pathogen. In plaque samples from periodontitis patients we observed the presence of a rough morphotype of P. micros in addition to the previously recognized smooth morphotype. The rough morphotype has not been described previously. Both morphotypes are frequently isolated simultaneously from the same patient. In this paper strains of both morphotypes are described. The smooth morphotype, represented by the type strain, grew as small, dome-shaped, bright white, nonhemolytic colonies. The rough morphotype grew as equally white dry colonies which were hemolytic and had wrinkled edges. DNA-DNA reassociation studies revealed homology at the species level between the two morphotypes; in addition, no differences in physiological characteristics were observed when the organisms were tested with API-32A and API-ZYM kits. The rough cells had long, thin fibrillar structures outside the cell envelope when they were stained negatively for electron microscopy. In the smooth morphotype these structures were not present. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole-cell extracts were different for the two morphotypes. In xylene-water phase partition studies, the smooth morphotype was found to be hydrophobic, whereas the rough morphotype was found to be relatively hydrophilic. The distinct morphotypes were stable on blood agar; however, the rough morphotype changed to a nonfibrillar type with a smooth colony morphology after repeated subculturing in broth.

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.

Aeromonas hydrophila in acute diarrheal disease: detection of enterotoxin and biotyping of strains

Eleven isolates of Aeromonas species from human stool cultures were found to produce enterotoxin activity as determined by assay of culture filtrates in rabbit intestinal loops and rabbit skin and on adrenal Y1 cells. Hemolysin(s) and a cytotoxic protein were found to interfere in all three assay systems but could be inactivated upon heating at 56 degrees C or by specific antihemolysin. Biotyping of each isolate was performed with a conventional test system and with API 50E and APIZYM kit systems (Analytab, Inc.). No single test of the more than 70 biochemical reactions investigated was found to correlate with enterotoxigenicity in the strains of Aeromonas hydrophila examined. All strains were found to belong to ideal phenotypes of A. hydrophila, but each strain possessed its own biochemical profile.