Culture fluid

Independent Assays for Provings Virus Removal. Retrovimses and vimses can also be present in culture fluids of mammalian cell lines (15,24). Certainly the absence of vims can be difficult to prove. Model vimses, eg, NIH Rausher leukemia vims and NZB Xenotropic vims, were spiked into fluids being purified, and their removal subsequently vaUdated when subjected to the same purification sequence as used for the product. 

Following the discovery of penicillins, an extensive program for the screening of culture fluids and residual mycelial material commenced which resulted in the discovery of a large number of pyrazinones and related 1-hydroxy-2-pyrazinones with pronounced antibiotic character. Some examples are shown in Table 4. One of the earliest substances to be isolated, aspergillic acid (110 = OH, = Me, R = Et, R = R = H, R = Pr ), was found... 

Fig. 1. Preparative separation of the components of the concentrated culture fluid on the PVT-porous glass column. (/) fraction of purified rotaviruses, (2, 3) other components of the culture fluid [32]...

Fig. 2a-c. Electron micrographs of the chromatographic fractions (a) concentrated culture fluid containing rotaviruses ( x 30000), (b) purified rotaviruses (fraction 1, Fig. 1, x 50000), (c) other components of culture fluid (fraction 3, Fig. 1, x 30000) [32]... 

Fig. 5. Preparative separation of the components of concentrated culture fluid on the porous glass chemically modified by the copolymer of N-VP and N-HEAA. 30 ml of concentrated culture fluid was applied to the column (2.3x90 cm) equilibrated with 0.01 mol/1 phosphate buffer, pH 7.4 and eluted with this buffer at flow rate 300 ml/h. (1) — fraction of purified rotavirus, (2, 3) — other components of the culture fluid [51]...

The olivanic acids (general structure, Fig. 5.5D) are naturally-occurring /3-lactam antibiotics which have, with some difficulty, been isolated from culture fluids of Strep, olivaceus. They are broad-spectrum antibiotics and are potent inhibitors of various types of/3-lactamases. 

The product is extracted from the culture fluid by adsorption onto caibon or resins rather than by solvent. This illustrates an important general point that antibiotic manufacturing processes differ from one another much more in their product recovery stages than in their fermentation stages. Figure 7.4 illustrates a typical production ronte from inoculum to bulk antibiotic. 

Separation. The proeess by which the bacterial cells are separated from the culture fluid. Centrifugation using either a batch or continuous flow process is commonly used, but preeipitation of the cells by reducing the pH is an alternative. In the case of vaeeines prepared fiom eells, the fluid is discarded and the cells are resuspended in a saline mixture where vaeeines are made fiom a constituent of the fluid, the cells are discarded. 

Cultures from different times of growth were collected. Culture fluids were cleared by passing through glass fibre filter. After dialysis for 16-18 h against distilled water at 5°C, filtrates were assayed for enzyme activities and proteins. 

Preparation of enzyme. Culture fluids of three days on glucose 0.5% (w/v) and then four days on pectin 0.5% (w/v), cleared by passing through glass fibre filter, were used for the purification of PNL. A small quantity was remainder, dialyzed, and assayed for enzyme actitity and the remained was precipitated. 

Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel — filtration on Sefadex G — 75 was found to be 35,000. 

Brandi et al. [71] using culture fluid of Acidovorax delafieldii and cyclic 3HB oligomers were in agreement with the presence of endo-hydrolase activity of poly(3HB) depolymerases. Similar results were obtained by de Koning et al. [72] who demonstrated that covalently cross-linked poly(HAMCL) was hydrolyzed completely by P. fluorescens. It is assumed that most - if not all - extracellular poly(HA) depolymerases have endo- and exo-hydrolase activity. Depending on the depolymerase the hydrolysis products are only monomers, monomers and dimers, or a mixture of oligomers (mono- to trimers). 

Kulski JK, Pryce T. Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection. J. Clin. Microbiol. 1996 34 1985-1991. 

For AIC conditions, the apical surface of the epithelial cell layer is exposed to air after the nasal cells reached confluence on the Transwell insert, while the basolateral side is fed with culture fluid. Figure 9.3 shows TEER changes in epithelial cell layers cultured up to 20 days in LCC versus AIC methods [46], In AIC condition (initiated from day 3 after seeding), TEER peaked on day 5 and maintained above the TEER values observed for LCC counterparts. By contrast, TEER observed for LCC conditions peaked on day 2 and declined toward zero by day 15. These data indicate that human nasal epithelial cells at an air interface culture exhibit better electrophysiological characteristics than those cultured by the conventional liquid-covered conditions. 

Selenium, in the form of selenate or selenite, is toxic to D. desulfuricans (Tomei et al. 1995) and Wolinella succinogenes (Tomei et al. 1992) at elevated levels. At sublethal levels of 0.1-1. OmM selenite or 10 mM selenate, minimal levels of growth is observed with both D. desulfuricans and Wolinella succinogenes. With both selenate and selenite, colloidal elemental selenium (Se°) is produced inside the cell and released into the culture fluid after cell death. This reduction of Se(VI) and Se(IV) by these anaerobes is not coupled to growth and proceeds by mechanisms that have not yet been identified. Selenite and selenate reduction with formation of elemental selenium by these nonrespiratory processes serve to detoxify the environment for future bacteria and may be important for the geochemical cycle of selenium. 

Figure 2. Degradation (O) of -metltylated aqueous acetone spruce wood extract ( ) in presence of concentrated extracellular P, chrysosporium culture fluid at pH 3.0 containing 0.05 gL protein with 0.2 mM H2O2 during 1 h at 37 C. Sephadex LH20/DMF elution profiles adapted and redrawn from reference 13.

Figure 3. Polymerization of 1 mg mL alkali-isolated straw lignin (dotted line) by 0.8 units mL lignin peroxidase (as concentrated extracellular P, chrysosporium culture fluid) at pH 4.0 in the absence (broken line) and presence (solid line) of 1.2 /imole mL veratryl alcohol 0.54 /imole H2O2 added at 2 hourly intervals for 24 h. Sephadex G75/aqueous 0.5% (w/v) NaOH elution profiles adapted and redrawn from reference 32.

Nevertheless, the partial ( 22%) depolymerization of a C-methylated aqueous acetone extract from spruce wo, reported in the other of the first two accounts about lignin peroxidase (13), was subsequently confirmed (52). Yet extended qx)sure at pH 3.0 to extracellular P. chrysosporium culture fluid, or purified lignin peroxidase, with H2O2 failed to reduce the degree of polymerization below that initially achieved, even... 

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