Inoculation volumes

An inoculum for an industrial scale fermerrtation is prepared from such preserved strains by inoculating the stock in a small volume (e.g. 5mL) and performing stepwise increases of the culture volume in order to reach the proper inoculation volume (typically 1-10%) of the production fermenter. 

Inocula were prepared by adding a loopful of cells from the stock culture to 100 mL of Difco nutrient broth and incubating at 30°C for 16 h on a rotatory shaker at 150 rpm. Inoculation volumes corresponding to about 0.04 g/L of exponential-phase cells were used. 

This study showed that the bacterial cellulose derived fix)m coconut and pineapple juices can be converted efficiently to bacterial cellulose by the supplementation of yeast extract and ethanol under static fermentation conditions at 30 °C. Bacterial celluloses produced from all strains are growth associated products. Coconut juice seems to be a better substrate than pineapple juice. In view of energy consumption, the productivity of BC on this medium is high, which makes the production costs lower than expected. It is also clear that different A. xylinum strains produce different BC content levels under the same inoculation volumes and under static cultivation conditions. These results suggest that bacterial cellulose pellicles of all strains appear to be easily applied to use in many applications such as food, paper, and textile industries, without requiring additional steps of decolorization and purification. Furthermore, the properties of cellulose, in tenns of crystallinity, high water-absorption capacity, and mechanical strength of the reported strains, have additional applications in cosmetics and medicine. 

It should be noted, however, that compared with properly prepared starters where population densities may exceed 10 CFU/mL, cell density in wine undergoing MLF is considerably less. Thus, much larger inoculation volumes (10-25% v/v) may be required to reach levels of 10 CFU/mL (Firme et al., 1994). 

Figure 5 Quantitative analysis of tripeptide proteasome inhibitor Z-LLL on rAAV-2 transduction in mouse lung. 1x10 viral particles of AV2.RSVluciferase virus was delivered into the mouse lung by nasal inhalation. To evaluate the effects of proteasome inhibitor on gene transfer, 400 (jM Z-LLL (final concentration, representing 1% viral inoculation volume) was coadministrated at the time of viral infection. The entire lung was harvested at 8 months postinfection and luciferase activity was evaluated using a previously described protocol (12). The data represents the mean ( SEM) from four independent mice in each group.

Mix 6 2 ml. (6 4 g.) of pure ethyl acetoacetate and 5 ml. of pure phenylhydrazine in an evaporating-basin of about 75 ml. capacity, add 0 5 ml. of acetic acid and then heat the mixture on a briskly boiling water-bath (preferably in a fume-cupboard) for I hour, occasionally stirring the mixture with a short glass rod. Then allow the heavy yellow syrup to cool somewhat, add 30-40 ml. of ether, and stir the mixture vigorously the syrup may now dissolve and the solution shortly afterwards deposit the crystalline pyrazolone, or at lower temperatures the syrup may solidify directly. Note. If the laboratory has been inoculated by previous preparations, the syrup may solidify whilst still on the water-bath in this case the solid product when cold must be chipped out of the basin, and ground in a mortar with the ether.) Now filter the product at the pump, and wash the solid material thoroughly with ether. Recrystallise the product from a small quantity of a mixture of equal volumes of water and ethanol. The methyl-phenyl-pyrazolone is obtained... 

The culture broth is sterilized by heating it under pressure at about 120°C for about 30 minutes. The broth is cooled and the above inoculant culture is added aseptically. The organism is grown in the broth for 4 days at a temperature of 26°C. During the growth period the broth is stirred and sterile air is blown through the broth at a rate of about 0.5 volume of air per volume of broth per minute. At the end of the growth period the broth shows an antibiotic activity equivalent to about 150 meg of erythromycin per ml of broth. 

Fluoro-l 13,17ot-Dihydroxy-21-Acetoxy-1,4-Pregnadiene-3,20-Dione A medium consisting of 1% dextrose hydrate, 2% cornsteep liquor of 60% solids and Kalamazoo tap water was adjusted to pH 4.9 with sodium hydroxide. The medium was steam sterilized at 15 pounds pressure for 30 minutes, cooled, and then inoculated with a 24-hour growth, from spores, of Septomyxa affinis, ATCC 6737. The medium was agitated, sparged with sterile air at the rate of one-tenth volume of air per volume of medium per minute. At the end of 24 hours of fermentation at room temperature, the pH was about 7.4. 

This was inoculated with a spore suspension of P. patu/um (1 liter containing 3-5 x 10 spores/ml) and grown at 25°C in 100 gallon tank. The inoculum is transferred at 40 hours or when the mycelial volume (after spinning 10 minutes at 3,000 rpm) exceeds 25%. The fermentation is conducted as near to the ideal pH curve as possible by addition of crude glucose, according to U.S. Patent 3,069,328. 

The fermenters are inoculated with 7.5% by volume of a 24-hour old culture of Aspergillus sderotiorum Huber grown at 28°C in 50 ml aliquots of the above described soybean-glucose medium contained in 300 ml Erlenmeyer flasks, placed on a shaker rotating at approximately 230 rpm. The inoculated fermenters are agitated at 1,380 rpm and each aerated with 1 liter of air per minute and at a temperature of 28°C for 47 hours. A silicone antifoam is added when required. At the end of the 47-hour period, the pH of the fermentation broth rose to 6.8 to 6.9. Sulfuric acid is then added with sterile precautions to restore the pH to 6.5. 

The above sterilized medium was inoculated with 11 liters of seed inoculum having a bacterial count of approximately 20 billion per cc. The tank was fermented at 37°C without pH adjustment, aeration, or other modification for 14 hours at the end of which time 320 cc of 50% dextrose was added. After this the pH was adjusted to 7.0 at 15 minute intervals with 5.0 N sodium hydroxide. The volume of sodium hydroxide required for neutralization was noted and 115% of this volume of 50% dextrose solution added after each pH adjustment. At the end of about 8 hours the bacterial count had ceased to increase and the fermentation was terminated. At this time the fermentation medium contained approximately 1,000 units of streptokinase per cc. 

A small fermentation tank (5,000 parts by volume capacity) was charged with 3,000 parts by volume of a culture medium (pH 6.0) comprising 3% glucose, 1 % polypepton, 0.5% yeast extract and 0.5% malt extract. The medium was sterilized by heating in a conventional manner and cooled. This medium was inoculated with 150 parts by volume of a pre[Pg.1565]

A 3 litre bioreactor with a working volume of 2 litre is inoculated with the three shaking flasks. The pH is maintained at 5.5 by automatic titration with 5mol l 1 NH4OH and the temperature is held at 37°C. 

It is very simple to perform batch fermentation in a small flask with a volume of say 200 ml. Now our target is to use a 2 litre B. Braun fermenter. All accessories are shown in Figure 10.5. The fermentation vessel only, as shown in Figure 10.6, with about 250 ml of media without any accessories but with some silicon tubing attached with a filter for ventilation is autoclaved at a 131 °C for 10 minutes at 15psig.9 After that, the system is handled with special care and all accessories attached. Media is separately sterilised and pumped into the vessel. Inoculum is transferred and the batch experiment is started right after the inoculation of seed culture. An initial sample is withdrawn for analysis. 

All of the above processes are operated as batch fermentations, in which a volume of sterile medium in a vessel is inoculated. The broth is fermented for a defined period. The tank is then emptied and the products are separated to obtain the antibiotic. The vessel is then recharged for batch operation with medium and the sequence repeated, as often as required. Continuous fermentation is not common practice in the antibiotics industry. The antibiotic concentration will rarely exceed 20gT 1 and may be as low as 0.5g-l 1. 

The inoculate was prepared in 250 ml flasks containing 100 ml of growth medium, which is inoculated with 10 ml of spore suspension. The mixture was shaken at 250 rpm and the temperature was controlled at 26 °C for 48 h. Then, 110 ml of resulting mycelia suspension is used to inoculate a 1000 ml broth in the airlift fermenter. The sterilised media are slowly pumped into the bioreactor at a flow rate of about lOOmlh-1 until 2 1 working volume is fully utilised. Aeration rates of 0.5, 1 and 2vvm (1,2 and 4 1 air/min) are used.6,7 Samples were taken at 24 hour intervals and evaluated for biomass, sugars and antibiotic concentrations. 

Batch fermentation means the cultivation of microorganisms, where the sterile growth medium in desired volume is inoculated with the microorganisms into the bioreactor and no additional growth medium is added during the fermentation. The product will be harvested at the end of the process. Typically, PHA s production is performed using batch fermentation because of low cost for investment and no special control. In addition, sterilization of the feed stock is easier than other fermentation processes, and operation is flexible. 

The quality control of both diphtheria and tetanus vaccines requires that the products are tested for the presence of free toxin, that is for specific toxicity due to inadequate detoxification with formalin, at the final-product stage. By this stage, however, the toxoid concentrates used in the preparation of the vaccines have been much diluted and, as the volume ofvaccine that can be inoculated into the test animals (guinea-pigs)... 

Growth of the Helianthus annuus 1805 cell suspension and biosynthesis of extracellular polysaccharides. A particular characteristic of plant cell suspensions is the requirement for a high inoculation density in order to initiate growth. This is due to one of their special features in order that their growth be initiated when transferred into the new medium, they need certmn growth factors which are released and secreted into the medium by the cells themselves. Consequently, to ensure the growth of plant cell suspensions, a certain volume (in which plant cells have to be present at above certain densities) has to be used to import the necessary quantity of these substances (17). 

H.annuus 1805 cell suspension was cultivated on a shaker (11.6 rad/s) at 26-28 °C in the dark in 1/5 net volume flasks. Duration of growth was different from 5 days for obtaining the inoculum to 10 days for studying the course of growth, the course of changes in the cell walls and the courses of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase as well. For inoculation 20 % (v/v) five-day cell suspension, containing 9 g/L dry cell biomass, was used. 

Hestrin, Avineri-Shapiro and Aschner9 report that levan formation by means of B. subtilis is greater if cultural products other than levan are continually removed by dilution or dialysis. The organism was inoculated into a phosphate-buffered solution contained in a cellophane bag suspended in a large volume of sucrose peptone solution. 

The upstream processing element of the manufacture of a batch of biopharmaceutical product begins with the removal of a single ampoule of the working cell bank. This vial is used to inoculate a small volume of sterile media, with subsequent incubation under appropriate conditions. This describes the growth of laboratory-scale starter cultures of the producer cell line. This starter culture is, in turn, used to inoculate a production-scale starter culture that is used to inoculate the production-scale bioreactor (Figure 5.7). The media composition and fermentation conditions required to... 

Procedure Distribute into identical test-tubes an equal volume of standard tetracycline solution and the sample to be examined (having presumed equal concentrations) and add to each tube an equal volume of inoculated nutrient medium (for instance 1 ml of the solution and 9 ml of the medium). Prepare at the same time two control tubes without the chlortetracycline, one containing the inoculated medium and the other identical with it but treated immediately with 0.5 ml of formaldehyde solution. These tubes are used to set the optical apparatus employed to measure the growth. 

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