Aseptic medium

A biosynthetic study of the 18-carbon chain skeleton of broussonetines was reported [38]. To verify the biosynthetic route of these alkaloids, the plant was grown on an aseptic medium and the enriched l3C of the isolated alkaloids was analyzed by NMR after feeding with [l-13C]glucose. The labeling pattern of broussonetine J (27) obtained... 

After 55 hours of fermentation, the contents of the round flask is transferred under aseptic conditions into a metal reactor of about 100 liters capacity containing 60 liters of sterile medium prepared as follows ... 

The mixture of broth and mycelium thus formed was then transferred under aseptic conditions to a 3-iiter fermentor containing 2 00 ml of a sterile fermentation medium having the following composition 60 g Cerelose (dextrose hydrate), 18 g soybean meal, 5 g distillers solubles, 12 g cornmeal and tap water in a sufficient amount for a 1,000-ml total volume, adjusted to pH 7.0 to 7.2 with potassium hydroxide. 

After seeding the nutrient medium with the preformed inoculum previously described, the mixture was subjected to agitation and aeration under aseptic conditions for 72 hours at 27°C to 28°C for the first 24 hours, then at 25°C to 26°C for the next 48 hours during this period, the pH was in the range of 6.4 to 6.8. Aeration was accomplished by cultivation under submerged conditions at an air flow rate of one volume of air per volume of medium per minute. After termination of the process, the mycelium was removed by filtration and the filtered broth found to contain 450 7of oleandomycin per ml of solution. 

Tank fermentation of Micromonospora inyoensis — Germination stage 1 Under aseptic conditions, add a lyophilized culture (or cells obtained from a slant culture) of M. inyoensis to a 300 ml shake flask containing 100 ml of the following sterile medium ... 

A spore sand culture containing Gibberella zeae (Gordon) NRRL-2830 was aseptically placed in a sterile tube containing 15 ml of Czapek s-Dox solution and a small amount of agar. This medium was then incubated for about 168 hours at approximately 25°C. At the end of the incubation period, the medium was washed with 5 ml of steriledeionized water and transferred to a sterile tube containing 45 ml of Czapek s-Dox solution. The contents of the tube were then incubated for about 96 hours at about 25°C after which the material was available for use in inoculation of a fermentation medium. 

The factors chosen were variations in vessel size, the effect of non-aseptic operation and medium recycle, and variations in recovery and drying procedures. 

Aseptic harvesting is necessary to overcome the need for medium re-sterilisation before recycling. Sterilisation costs are high. If biomass can be recovered by an aseptic process, the medium can be recycled without re-sterilisation. This excludes centrifugation, which cannot be operated under aseptic conditions. 

Not all the nutrients required during fermentation are initially provided in the culture medium. Some are sterilized separately by batch or continuous sterilization and then added whilst the fermentation is in progress, usually via automatic systems that allow a preset programme of continuous or discrete aseptic additions. 

Biological indicators (Bis) for use in thermal, chemical or radiation sterilization processes consist of standardized bacterial spore preparations which are usually in the form either of suspensions in water or culture medium or of spores dried on paper, aluminium or plastic carriers. As with chentical indicators, they are usually placed in dummy packs located at strategic sites in the sterilizer. Alternatively, for gaseous sterihzation these may also be placed within a tubular hehx (Line-Pickerill) device. After the sterilization process, the aqueous suspensions or spores on carriers are aseptically transferred to an appropriate nutrient medium which is then incubated and periodically examined for signs of growth. Spores of Bacillus stearothermophilus in sealed ampoules of cultrrre medium are used for steam sterilization morritoring, and these may be incubated directly at 55°C this eliminates the need for an aseptic transfer. 

Aseptic transfers are also avoided by the use of self-contained units where the spore strip and nutrient medium are present in the same device ready for mixing after use. 

Clinical specimens must be collected aseptically and then promptly transported to the clinical laboratory in a properly labeled sterile container. Specimens collected on swabs or transported to the clinical laboratory in a transport medium are unacceptable for mycological study. An adequate quantity of clinical material is necessary if the information obtained in the laboratory is to be meaningful. 

A concentrated Ca-D-pantothenate solution (1 mg/ml) is prepared in distilled water and dilutions made as needed. Refrigerated solutions are stable for 6 months. Pantothenate is added at 5, 10, 20, 40, 60, 80, and 100 mpg/ml final concentrations the control flask consists of basal medium alone for estimation of carry-over error—i.e., the pantothenate activity of the inoculum. The details of aseptic technique have been discussed elsewhere (H18, H19). Growth is measured in optical density units with a Welch Densichron, equipped with a red-sensitive probe to minimize blank readings due to the color of the medium. 

Inoculum. Pleurotus sajor-caju was grown at 30°C on the above medium containing 1% glucose as a carbon source in 250 ml Erlynmeyer flasks on rotary shaker at 200 rpm for 60 h. The mycelial biomass thus produced was blended in a Waring blender for 1 min. under aseptic conditions in order to obtain an homogenous inoculum of well dispersed mycelial bits. The inoculum was used at the rate of 10% vol/vol. The inoculated experimental Erlynmeyer flasks were incubated at 30 C on a rotary shaker at 200 rpm for various intervals of time. 

The use of hydrocyclones for separating mammalian cells from the culture medium opens the possibility of using them to perform perfusion in bioreactors. As hydrocyclones have no moving parts, they are ideally suited for operation under aseptic conditions as required by the biotechnology industry. 

All handlings should be performed in a way as aseptic as possible. Prepare the culture medium according to the USP and fill the medium into sterile RODAC dishes to the brim to obtain a convex surface. Extreme care should be taken to prevent the formation of air bubbles and to prevent the medium from overflowing (if either occurs, these plates should be discarded). Allow the plates to solidify. Preincubate the plates at 55°C for 24 h and then at 25°C for 24 to 48 h. 

Once the medium has been processed, it is held for a period of time at least equal to that for aseptically produced materials. Any aseptic manipulations performed during and at the end of the hold period should be simulated as well (i.e., sampling, refiltration, hold times, and product recalculation). 

The containers are sealed and the medium-filled units are collected in sequentially numbered trays or boxes (notified to the filling time). It is preferable to use materials, components, and closures which have remained in the aseptic processing area for extended periods. 

Containers are hlled with medium, and stoppers are partially inserted. The containers are loaded into the lyophilizer. A partial vacuum is drawn on the chamber and this level is held for a predetermined time. The vacuum must not be so low as to permit the medium in the containers to boil out. The chamber is then vented and the stoppers are seated within the chamber. The stopper units are removed from the aseptic processing area and sealed. 

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