Inoculum transfer

The medium is contained in a 1000 ml Pyrex Blake mottle. After inoculation the medium is incubated at 45°C with constant vigorous agitation, on a rotary shaker for 16 hours. During this period a vigorous growth of the organism ensues. The contents of two such incubated Blake bottles are pooled into a 500 ml Pyrex inoculum transfer bottle fitted with a tubulature at the bottom and containing 150 ml of sterile water. 

The entire contents of the inoculum transfer bottle are transferred to a 100-gallon stainless steel fermentor, which is prepared for it as follows ... 

Ten percent of the resulting inoculum is then transferred to a 250 ml Erlenmeyer flask containing 50 ml of the medium employed above and the flask agitated a further 72 hours under the same conditions. One ml of the resulting inoculum is then employed for the inoculation of 10 ml of an aqueous medium containing, per 1,000 ml, 30 grams extraction... 

Inoculum Preparation Stage Two batches of inoculum of about 50 gallons each are prepared by the following method A 25 ml inoculum (from the germination stage) is transferred to each of four 2-liter flasks, each containing 500 ml of the sterile medium utilized for germination. The flasks and contents are incubated for 5 days at 28°C on a rotary shaker (280 rpm, 2 inch stroke). 

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. 

A culture of Bacillus polymyxa in a tube with Trypticase soybean broth was incubated overnight at 25°C. 5 ml of this culture was transferred to 100 ml of the tank medium in a 500 ml Erlenmeyer flask which was incubated for 48 hours at room temperature. This 100 ml culture served as inoculum for one tank. During the course of fermentation the medium was aerated at the rate of 0.3 volume of air per volume of mash per minute. The temperature was maintained at about 27 C. Samples of mash were taken every 8 hours in order to determine pH and the presence of contaminants and spores. After 88 hours of fermentation the pH was about 6.3 and an assay using Escherichia coll showed the presence of 1,200 units of polymyxin per cubic centimeter. The polymyxin was extracted and purified by removing the mycelia, adsorbing the active principle on charcoal and eluting with acidic methanol. 

The medium used to produce the inoculum should be designed for rapid growth of the production organism without exopolysaccharide production. Production of the latter in the inoculum train can give rise to highly viscous cultures that are difficult to transfer from one vessel to another. 

Optimisation of biomass production would require a large inoculum, comprising 10% of each inoculum stage. However, this involves many transfers which increases the risk of contamination. 

The correct answer is seven. In practice, the number of inoculum steps does not usually exceed four to reduce the risk of contamination. This also reduces capital investment and production costs since fewer transfers require fewer vessels for development of the inoculum. 

Contamination of the production vessel leads to serious financial penalties and each step in the inoculum train is monitored for contamination. To reduce the risk of contamination during sampling it is usual to take a sample from the residue left in each vessel after its contents have been transferred to the next reactor. Since these contamination checks are retrospective, a heavy reliance is placed on the growth characteristics of the production organism. Kinetic variables such as growth rate and oxygen consumption rate are also used to assess the quality of the inoculum. 

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. 

The inoculum stage media are designed to provide the organism with all the nutrients that it requires. Adequate oxygen is provided in the form of sterile air and the temperature is controlled at the desired level. Principal criteria for transfer to the next stage in the progression are fieedom from contamination and growth to a pre-determined cell density. 

Production of Lignin Peroxidase. Medium for the inoculum was rich in yeast extract (25 g/1) and glucose (25 g/l) to promote maximal growth of the mycelia. The inoculum of Phanerochaete chrysosporium ATCC 24725 was first cultivated for 3 days at 30°C in five litres of medium divided in five shake flasks. The shake flask batches were transferred to a 100 litre bioreactor and cultivated again for 3 days at 30°C. The batches were stirred and aerated to obtain maximal growth of mycelia. 

The medium for enzyme production was completely synthetic and limited in carbon to prevent excessive growth of mycelia. It contained glucose 2 g/l plus ammonia nitrogen, vitamins and trace elements according to Leisola et al.(ii). The 100 1 inoculum was transferred into 8001 of medium in a 10001 bioreactor equipped with 12 rrfi of nylon wool sheets supported by steel wire screen (Figure 1). The medium was continuously stirred with the impeller (100-200rpm) and saturated with pure oxygen. The temperature was maintained at 37°C. 

Production. The inoculum grew vigorously in the rich yeast extract containing media and produced a thick viscous dispersion in the stirred tank bioreactor. No lignin peroxidase activity could be detected at this stage. When transferred to the 1000 1 production bioreactor, the mycelium of P.chrysosporium attached completely to the nylon wool sheets within a few hours after inoculation and the medium remained completely clear throughout the cultivation. The enzyme had to be harvested immediately after the maximal activity level was reached due to its... 

Jenner used pustular material from a cowpox lesion as an inoculum instead of similar material from a smallpox lesion. For this experiment, he inoculated cowpox lesion material from the hand of a milkmaid into the skin of an 8-year-old boy (Figure 1.1). The boy resisted variolation when challenged with smallpox about 6 weeks later. During another outbreak, several more inoculations were performed, and subsequent inoculations took place by arm-to-arm transfer of infectious material. Two years later, Jenner wrote The Inquiry (An inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the Western countries of England, particularly Gloucestershire, and known by the name of Cow Pox). By 1799, Jenner s observations had been confirmed by several other practitioners, and over 1000 people had received the cowpox vaccine. Within another 3 years, the practice of cowpox inoculations had spread across Europe to North America and Asia, utilizing... 

Inoculation Inoculation is the seeding of a culture vessel with the microbial material (inoculum). The inoculum is introduced with a metal wire or loop which is rapidly sterilized just before its use by heating it in a flame. Transfers of liquid culture are often made by using a sterilized pipette. The inoculation is usually done in a laminar flow hood to minimize the risk of contamination. It is important to know proper pipetting techniques for inoculating or sampling during cultivation. 

A 24-h culture of transformed E. coli (LB/amp broth 37°C, 100 rpm) was transferred onto the surface of LB/amp/IPTG agar and incubated at 37°C for 24 h. Isolated green fluorescent colonies (illuminated with a handheld long UV lamp, 360-395 nm Model UVL 4 UVP, Upland, CA) were picked and transferred to 25 mL of LB/amp broth in 250-mL Erlenm-eyer flasks (starter cultures). The starter cultures were incubated at 37°C and 100 rpm) until a cell density of 0.0054-0.026 absorbance units (104-10s CFU/mL) was obtained as measured by OD660 with a spectrophotometer (Beckman DU-600 Beckman Coulter, Fullerton, CA). An inoculum of... 

---