Small scale cultures

For those interested in cloning reference should be made to Chapter 7. A small scale perfusion vessel is considered in 3.4.3. For many biochemical studies involving incubation of cells with radioisotopes in the presence of drugs, anti-metabolites, hormones etc. small numbers of cells are required and these may conveniently be grown on the bottoms of glass scintillation vials or in the wells of a 6 or 24 well TC plate or even in the wells of a microtitre plate (see Table 3.1). This last method enables 96 replicate cultures to be handled simultaneously but the maximum volume that each well will hold is 0.25 ml. 

Coverslips are used to greatest advantage when placed in the 24 wells of a tissue culture plate. Each well with its coverslip requires 0.5 ml of medium and can be seeded initially with around 20,000 cells. Alternatively, up to 8 separate cultures can be grown on a 

Scintillation vials require 1 ml medium and, although more difficult to handle in large quantities, offer the advantage that at the end of the experiment the labelled cells require fewer manipulations prior to estimation of the extent of radioactive incorporation. Alternatively several coverslips may be placed in a 5 cm plastic Petri dish prior to seeding cells. 

This has the advantage that each coverslip culture is maintained under identical condition. 

The plastic containers are bought in sterile wraps from commercial suppliers, e.g. Nunc, Falcon, Linbro, Coming, Costar (see Appendix 3). 

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Bushell ME, Dunstan GL, Wilson GC, Effect of small scale culture vessel type on hyphal fragment size and erythromycin production in Saccharopolyspora ery-threae, Biotechnol Lett 19 849—852, 1997. 

To develop a process for production of the biocatalyst, small scale cultures (5mL to 5L) are used. Initially, tubes or shake flasks are commonly apphed, especially when a large number of values for a given variable such as media composition, temperatnre and pH are evaluated. As development progresses and quantitative data are required, more controlled conditions than those provided by Erlemneyer flasks are necessary. Commoidy,... 

For the subculture of adherent cells, removal of culture medium and the detachment of cells from the monolayer are necessary. This detachment is usually performed with trypsin, but other proteases, such as pronase, dispase, and collagenase, can be employed. In general, a chelating agent, such as EDTA, is also added to capture the Ca2+ ions involved in the cell adhesion process. Some cell lines bind weakly to surfaces and, in small-scale cultures, can be removed mechanically by gently tapping or hitting the culture flask by hand. 

Inoculum propagation and small-scale culture systems... 

Small-scale trials are conducted to determine the growth and production characteristics of a cell line as a function of the culture environment once a suitable host organism is selected. A small-scale culture typically requires shake flasks with capacities in the... 

Over-expression in P. pastoris GS115. P. pastoris was transformed with pPIC3.5K/fae-1 vector. Transformant were grown as small-scale cultures in... 

The host cells containing the vector are grown in small-scale culture to select only for the correct clone that contains the desired gene and is able to express the best yields of the protein (32). The selected cloned cells (or cell bank cells) are used as inoculum first for a small-scale cell culture/fermentation, which is then followed by larger fermentations in bioreactors. The medium is carefully controlled to enhance cell reproduction and protein synthesis. The host cells divide, and the vectors within the hosts multiply. The host produces its natural proteins along with the desired protein, which may be secreted into the growth medium. The protein of interest can then be isolated from the fermentation, purified, and formulated to give a potential rDNA-produced pharmaceutical. 

More detailed information on methods for small-scale culture maintenance, preservation and on the different media can be found in Andersen (2005) as well as on the websites of some algal culture collections (Table 4.2). Additional information including an historical context is given by Stein... 

Extraction of proteins from small-scale cultures of E. coli cells can be done in several ways, including by the sonication, freeze and thaw procedure (14), or using a detergent-based chemical lysis. Below we present protocols for protein extraction by sonication, and chemical lysis using the detergent-based reagent, BugBuster . 

With the development of the aquaculture industry and the need by hatcheries to rear larval and juvenile aquaculture animals came the need for production of microalgae as live feeds. While some hatcheries grow microalgae in multiples of relatively small scale culture containers, for example 20 litre carboys, it is more common for miCToalgal cultures to be grown in disposable plastic bags (200-1000 litres is typical) that are either supported vertically (Plate 9.1b) in metal frames or lie horizontally. 

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