Determination the dilution rate

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How to determin the dilution rate?

One very important factor is the rate of sample dilution. The concentration of the cell suspension to be transferred to the measuring chip and assayed must be within the optimum measuring range of the EASYCOUNTER YC. In order to provide a statistically reliable result, the total cell count in the diluted cell suspension should be in the range of 1x10 5 to 1x10 7 cells/ml. optimal range - from 1х10 5 to 1х10 7 cells/ml measuring range - from 1х103 to 5х 10 7 cells/ml If cells concentration is in the range from 1x10 5 to 1x10 7 cells/ml, the initial sample is not diluted. If cell concentration is higher than 1x10 7 cells/ml the initial sample is diluted with distilled water. The rate of sample dilution for determination of dead cell count is 10 times lower than the rate of dilution for determination of total cell count. This is due to the fact that dead cells concentration in the sample is usually much lower than total cell concentration. Determination of dilution rate at: А. In case of unknown sample cell concentration 1. Take a 15 ml centrifuge tube containing 10 ml homogenized sample. 2. Place the tube in a centrifuge and balance the rotor with an opposing tube if necessary. 3. Centrifuge for 6 minutes at 4000 rpm. 4. Remove the test tube being careful not to disturb the sediment. 5. Measure the total volume in the tube and the volume of supernatant and from the difference - volume of sediment. 6. Calculate the percentage of sedimentation by the following formula: % sediment = sediment volume/total volume х 100 7. Determine the rate of sample dilution according to Table 1, depending on the determined % sediment. B. With known indicative sample cell concentration Usually, in beer, wine and bioethanol production it is known in what range cell concentration varies at different stages of production. In this case, it is not necessary to determine % spun solids, but the sample is directly diluted with distilled water. Table 1 shows how many times the initial sample is diluted according to cell concentration in the sample. The sample dilution for determination of dead cell count is 10 times lower than the sample dilution for determination of total cell count. Table 1. Dilution of the cell suspension depending on the initial cell concentration in order to reach the optimal measuring interval for total and dead cell count determination. Spun solids Total number of cells in the initial sample,?cells/ml Dilution of the initial sample, how many times for a total number of cells?(transparent eppendorf) for dead cells?(colored eppendorf) ? 80% 1500 - 2500 х 106 1000 100 50-80% 500 - 1500 х 106 500 50 5-50% 100 – 500 х 106 100 10 ? 5% ? 100 х 106 20 2 If the concentration of the cells in the undiluted sample is below 1x105 cells/ml, the concentration should be increased by centrifugation (> 10000xg, 15min) with subsequent resuspension of the pellets with PBS.