Sterilization culture media

Fig. 5. Fermentative production of amino acids (140). A, pure culture B, inoculation C, boiler D, air compressor E, air filter F, seed tank G, ammonia water for pH control H, fermenter I, sterilizer , culture media K, preparation tank L, centrifugal separator M, ion-exchange column N, crystallizing...

Naturally, the size ofthe membrane pores should depend on the size of microbes to be filtered. For example, membranes with 0.2 pm pores are often used to sterilize culture media, while membranes with 0.45 pm pores are often used for the removal of microbes from culture products. It should be noted here that the so-called pore size of a membrane is the size of the largest pores on the membrane surface. The sizes of the pores on the surface are not uniform moreover, the pore sizes usually decrease with increasing depth from the membrane surface. Such a pore size gradient will increase the total filter capacity, as smaller microbes and particles are captured within the inner pores of the membrane. 

In finished product manufacturing areas, production personnel should be evaluated twice a year for their ability to maintain the sterility of the product by undergoing media fills where each employee manipulates sterile filling equipment and fills 300+ vials aseptically with sterile culture media. Additionally, personnel should be monitored daily for levels of contamination by ROD AC contact plates on fingers and other parts of the sterile gown. This requirement is becoming standard practice for bulk manufacturing personnel. 

The carriers containing the biological indicators are retrieved following exposure, transferred aseptically into sterile culture media, and incubated for the required length of time, usually two to seven days. Some unexposed indicators are also incubated to prove that the spores were viable. If no growth is observed while the viability control displays the required growth, the conclusion is made that the sterilization cycle was successful. In order for the biological indicator concept to work, it is necessary to place the indicators into that portion of the sterilizer load considered the most difficult for the sterilant to reach. The lot of products is quarantined to prevent dissemination in case of incomplete sterilization. 

The culture is incubated at a temperature of 28°C in the reactor for 60 hours with mechanicai agitation and constant aeration. The resulting broth is seeded into 600 liters of a sterile culture medium contained in a metal fermenting vat 1,800 liters in capacity and prepared according to the following formulation ... 

Fig. 1.3 Pasteur s swan-necked flasks in the first flask, the unbroken neck hinders contamination if the neck is broken off as in the second flask, the sterile culture medium is invaded by microorganisms. After Pasteur (1862)...

Axenically dissolve the allelochemical in sterilized culture medium... 

The culture medium was prepared by dissolving all the compounds for the culture medium except L-threonine in an Erlenmeyer flask containing 5 L of water. The pH was adjusted to 5.5 with NaOH and then sterilized for 20 min at 120 °C. L-Threonine was filtered (syringe sterile filter Sartorius Minisart 0.2 pm) and was added to the sterilized culture medium. 

Prepare a solution of each additive as specified by the manufacturer and/or as discussed below, filter-sterilize the solution and add to the sterile culture medium. 

One method that may be used in evaluating the aseptic system is to process under routine conditions, the equivalent of a representative lot of containers filling them with suitable sterile culture medium including the entire lot, and examining for the presence of contamination. Records of evaluation and results are maintained. 

Aseptic work or a significant modification thereof must be validated by performing simulations of the aseptic process with a suitable sterile culture medium. The number of filled containers must be sufficiently large to offer information on the risk of infection. 

The eggs should be broken and the contents homogenised aseptically. One volume of homogenate should be added to five volumes of sterile culture medium to dilute the enzyme lysozyme, which dissolves bacterial cell walls. The preparation is then tested for Salmonella,... 

Substance mixtures were provided by mixing the substances in a ratio of 1 1, and 3 ml of the 2% solutions were added to 3 ml of sterile culture medium and 1 2 ratio dilution rows were prepared. Dilution steps were 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.0313% and 0.0156%. The inoculation was done with an overnight culture oiE. coli, washed with 200 pi PBS, and using an OD of 600 nm with 0.2 0.01 accuracy. 

After 24 hours of growth at 28°C under stirring (220 revolutions per minute) and aeration (1.65 m /hr), 1.8 liters of the obtained culture is removed under sterile conditions and transferred with 28 liters of the same sterilized nutrient medium into a fermenter of the same size. 

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]

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. 

In order to disprove this theory, Pasteur used swan-necked flasks unheated air could now enter the sterile culture solution. But in this case, the microorganisms in the air were deposited in the long S-shaped neck and did not enter the culture medium. If, however, the neck of a flask was broken off, they could enter the solution and multiply. 

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