Filters sterilisation

The production-scale fermentation unit, with a projected annual capacity of over50,000 tonnes was fully commissioned in 1980. The bioreactor (Figure 4.8) is 60 m high, with a 7 m base diameter and working volume 1,500 m3. There are two downcomers and cooling bundles at the base. Initial sterilisation is with saturated steam at 140°C followed by displacement with heat sterilised water. Air and ammonia are filter sterilised as a mixture, methanol filter sterilised and other nutrients heat sterilised. Methanol is added through many nozzles, placed two per square metre. For start-up, 20 litres of inoculum is used and the system is operated as a batch culture for about 30 h. After this time the system is operated as a chemostat continuous culture, with methanol limitation, at 37°C and pH 6.7. Run lengths are normally 100 days, with contamination the usual cause of failure. 

Tyrosine, tryptophan and tetracyline are filter-sterilised to prevent decomposition by heat sterilisation. 

The advantages of reconstituting to 10 X are 1) there is a smaller volume to filter sterilise 2) the 10 X stock occupies less cold room space 3) it is more economical to buy bottles which reconstitute to 5 1 of 10 X stock (50 1 of 1 X medium) than smaller amounts. 

Fig. 8.1. Diagrammatic outline of the arrangement of vessels used in filter sterilisation. When filters of larger diameter are used the filter holder is not designed to hold any liquid and a dispensing pressure vessel must be used. However, with the 100 ml filter holders the dispensing pressure vessel is unnecessary if only small volumes are to be filtered. In this case the filtrate may be collected directly into a sterile reagent...

Certain items, such as balanced salt solutions and versene which are heat-stable, are generally sterilised by autoclaving, but the majority of organic materials used in cell culture are filter-sterilised. For heat-sterilised materials it is generally sufficient to rely on a sterilisation indicator which should be included with each batch of materials being sterilised. Often autoclave tape is sufficient if used on each packet, but for solutions and larger containers, e.g. cans of pipettes, it is recommended that a liquid indicator is included within the bottle or can. 

All these methods succeed in accumulating 80-90% of the cells at the Gl/S-interphase and are very suitable for obtaining populations of S-phase cells for study. They are readily scaled up to the level of roller bottles though at this scale the amount of thymidine required for a double block is considerable (0.5 g/roller bottle). Concentrated solutions of thymidine can be made up and sterilised by autoclaving but most of the other solutions should be filter sterilised. 

They may be combined aseptically, but as a glucose and glutamine also have to be added and these require filter sterilising, it is more convenient to sterilise after preparation of a 10 x stock ... 

Thermal treatment or filter sterilisation is advised when risk of bottle infection is high... 

Compressed air shall be dry and oil-free. All compressed gases that contact products, container/dosures or product contact surfaces shall be filter sterilised. 

Media supplements substances such as vitamins, amino acids, growth factors and antibiotics are added to media for a definite purpose, e.g., actidione (cyclohexamide) -an antibiotic added after filter sterilisation, L-(+)-arabinose,... 

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