2 , and the expected viability Transfer the cell suspension into prepared culture vessels by inoculating the appropriate volume of the cell suspension. For routine subculture a split ratio is used (1:2 or 1:3 etc.). This is calculated as the ratio of surface areas; for example, a 75-cm 2 has a split ratio of 1 into 3 (1:3), i.e. three 75 cm 2 Roux flasks or only one 225 cm 2 flasks
Applied Veterinary Virology: The isolation and identification of viruses using cell cultures 23 | P a g e Place the inoculated vessels in an incubator at the correct temperature. If a CO 2 incubator is used leave culture flask caps loose Check the cultures after a few hours for cell attachment and pH. If the cultures are too acid (yellow), or alkaline (purple), the CO 2 concentration must be immediately checked and adjusted. To prevent the loss of cultures due to a failure of the supply of CO 2 keep the flask saps tightened after cultures have equilibrated, i.e. 1 – 2 h. The duration between sub-cultures will depend on the incubation temperature and cell type. The majority of mammalian cell lines require sub-culturing every 3 - 7 days. If the duration is longer than 5 days, change the medium of the cultures every 3 - 4 days. Quantification of cells To measure performance, to achieve reproducibility, or to make comparative studies, a means of quantifying the cell population is needed. Classically, direct count of cell numbers using a microscopic counting chamber (haemocytometer), usually in conjunction with a vital dye (e.g. trypan blue) to distinguish viable and non-viable cells, is used. However, all vital stains are subjective and cannot give absolute values, and by counting cell numbers no differences in cell size/mass are taking into account. The method is simple, quick and cheap, and requires only a small fraction of the total cells from a cell suspension. Automation of cell counting is possible with electronic counters, especially for non-clumping single suspension cells. Other methods for total cell numbers include: • counting cell nuclei after dissolving the cytoplasm. This is particularly useful for large clumps of cells where cells are inaccessible (e.g. in matrices), or where cells are difficult to trypsinize off substrates (e.g. micro-carriers) • if cell mass, rather than number is the important factor then a cell constituent has to be measured • an indirect measurement can be used e.g. glucose or oxygen consumption rates. Other metabolites include lactic and pyruvic acid and carbon dioxide. There are reservations about these methods as these metabolic rates are not constant throughout the growth cycle of a specific cell and may also be influenced by changes in the culture, which is the subject of the investigation. These biochemical methods are best used over a time-course in culture so that successive readings will show a definite trend in the culture dynamics, e.g. stationary, growing or dying. A combination of one or two methods is the ideal.
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- Test, representative, Elastase, Cell culture, Earle, Applied Veterinary Virology