Viable counts

Count viable cells by Trypan blue exclusion and seed cells at the appropriate density in culture medium in untreated tissue flasks. 

The outlet of the compressed air supply point is opened and purged for 5 min. Adjust to a flow volume of about 30 1/min. Then the compressed air is passed through an air sampler equipped with a 0.22 pm filter. Sampling time should be about 5 min. This procedure should be performed three times. One filter should later be incubated for anaerobic, the other for aerobic viable count. Viable count should be < 200 CFU/m. ... 

Count viable lymphoid cells in a hemocytometer. Spleens from immune mice yield about 108 cells, from rats 3-5 x 108 cells, and the mesenteric nodes of rats up to 2 x 108 cells. 

Use 0.4% Trypan Blue to stain cells and count viable cells at seeding. Viability should be at least 95% at this stage. Culture cells in 50 mL media in Coming Spinner Flasks at 8% CO2, 37°C, in a humidified incubator (see Note 1). 

On the day of experiment, trypsinize the adherent cell cultures (see Note 1). After washing twice by centrifugation (5 min, 210 x g), resuspend the cells in the exposure serum-free culture medium, e.g., DMEM supplemented with 25 mM HEPES (DMEM-H). Count viable cells and adjust the cell density to a range of... 

In this guidance the FDA offers some direction for the demonstration of comparability. They request that the manufacturer provide evidence that the methods, facilities, and controls that were used to manufacture previous products conformed to cGMPs and to other applicable regulatory requirements. In addition they request the submission of validation summaries, as well as product characteristics such as total nucleated cell count, viable CD34 cell count, and number of colony-forming units. Stability data and information from the scientific literature can also be used. Clinical outcomes can be part of the comparability demonstration. 

Total cell count = viable cell count + dead cell count... 

Count viable cells mixing an aliquot of the cell suspension with trypan blue. Adjust the suspension to give a density of 1 x 106 cells/mL. 

The concentration of bacterial inoculum giving suitable results may be determined using direct microscopic count, viable counts, turbidity tubes, or spectrophotometric methods. These methods may not give the same result so one standard method should be used (see Chapter 5). Spectrophotometric methods may produce varying results between species due to differences in the size of the cells and hence their light scattering properties. Each species should therefore be checked. 

The total cells counted in this manner include both dead yeast and live yeast. The two must be differentiated in enology. In fact, counting viable microorganisms is preferable. When placed in a... 

Drocourt, J. L. Levesque, P. Method for counting viable cells by dual staining. PCX Int. Appl.WO 9855861, 1998 Chem. Abstr. 1998, 130, 35353. 

Keep the vials at 25 °C for 10-15 days and count viable flies that are otherwise lethal. 

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