Sterile medium

The contents of the seed fermenter were used to inoculate 1,500 liters of steam sterilized medium of the following composition in tap water. 

The pH of the medium thus prepared is about 6.8. An 8 ml portion Is measured into an 8 inch 8rewer tube and sterilized at 120°C for 20 minutes. The sterilized medium is than inoculated with 0.5 ml of an aqueous spore suspension of a strain of S. aureofaciens capable of producing chlorodemethyltetracycline, such as S-604, containing approximately 40-60 million spores per milliliter. The inoculated medium is incubated for 24 hours at 28°C on a reciprocating shaker operated at 110 cycles per minute. 

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 ... 

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). 

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 ... 

The seed flash was incubated for 24 hours at 32°C on a reciprocating shaker after which it was used as an inoculum for a 20 liter seed fermenter in the amount of approximately 5%. the 20 liter seed fermenter contained a sterile medium consisting of ... 

A spore suspension obtained upon washing a culture oi Streptomyces distallicus is added to 3,000 ml of a sterile medium consisting of the following ... 

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. 

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. 

Figure 1. Open circuit potential vs. time in sterile medium and in medium with bacteria (a) platinum, (b) 304L stainless steel. (Reprinted from Ref 1 with permission from NACE International.)...

Tomato cell suspensions in modified Murashige and Skoog medium (12) were assayed for change in cell volume at varying concentrations of polar and nonpolar extracts. Twenty-five ml erlenmeyer flasks (5 reps/treatment) containing 10 ml of sterile medium, tomato cell suspension, and extract were allowed to settle for 20 min in sidearm test tube attachments. Relative height of cell volume fraction to medium was recorded each day for 11 days. Cell suspensions were incubated in the dark at 27 C on an orbital shaker at 125 rpm. The doubling time of control cell suspensions were approximately 2.25 days. 

Inoculate the sterilized medium with Claviceps purpurea under sterile conditions, stopper with sterilized cotton and incubate for two weeks periodically testing and maintaining pH 4. After two week a surface culture will be seen on the medium. Large-scale production of the fungus can now begin. 

The concentrated sterilized medium was pumped into the Wavebag with a peristaltic pump through autoclaved silicone tubes. Then distilled water (17.6 L) was pumped in through a disposable, sterile microfiltration capsule. Rocking was started at an angle of 10.5° at 36-37 rpm as well as the temperature regulation, set-point 28 °C. 

Figure 7. Effects of amylolytic bacteria of the FTIR spectrum of starch/P / AA plastic films (a) film incubated for 24 hours in sterile medium (b) film incubated for 28 days in sterile medium and (c) film incubated for 28 days in medium inoculated with LD76.

In subsequent loaded chamber heat penetration studies, penetration thermocouples should be positioned within the container at the previously determined cold spot. The temperature profile of the container should remain constant among different sterilizing chambers, utilizing steam heat as the sterilizing medium. 

The intent of this study is to demonstrate the temperature uniformity and stability of the sterilizing medium throughout the sterilizer. Temperature distribution studies should be conducted on both empty and loaded chambers with maximum and minimum load configurations. Temperature uniformity may be influenced by the type, size, design, and installation of the sterilizer. The manufacturer of the vessel, based on the variables mentioned, should determine a satisfactory empty chamber temperature uniformity. 

To confirm that product safety considerations have been addressed, review of tunnel construction and operation materials for product contact potential or suitability shall be documented. Tunnel construction materials, which contact the sterilization medium, shall be identified. This would include ... 

Heating characteristics of the sterilizer, product carrier system, and the sterilization medium employed... 

To evaluate the heating characteristics of the tunnel, carrier system, and sterilization medium employed under loaded conditions... 

To evaluate the heating characteristics of items within the tunnel when subjected to the sterilization medium... 

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