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Mouse Lymphoma Assay Calculation of mutation frequency When using the published formulae for MF in the microwell version of the MLA, the sum of the large and small colony mutation frequency does not equal the total mutation frequency. Specifically, the published formulae give incorrect results for the small colony mutation frequency. Why is this? This phenomena occurs because any given well can only yield one of three possible outcomes:
Corrected calculations As per accepted practice, Plating Efficiency (PE) is: PE = [-loge (EW ÷ TW)] ÷ n Where EW = the number of empty wells, TW = the total number of readable wells, and n= the the mutation frequency. The mutation frequency (MF) is calculated in terms of mutant cells per 106 viable plated cells. It is based on the plating efficiency of the mutation plates and the viability plates. MF = PEmutation ÷ PEviability × 106 The small colony mutation frequency (SCMF) is calculated in terms of small colony forming mutant cells per 106 viable plated cells and require an adjustment to the PEmutation formula. This is because a well containing a large colony may obscure the presence of a small colony within, and wells containing large colonies must be excluded from the SCMF calculations thusly: SCMF = {[-loge (EW ÷ OW)] ÷ n} ÷ PEviability × 106 Where EW = the number of empty wells, OW = the total number wells not containing a large colony and n= the total number of cells plated per well. The large colony mutation frequency (LCMF) is calculated as the total mutation frequency (MF) as: LCMF = MF – SCMF Alternately, or for the purpose of verification, it can also be calculated as: LCMF = {[-loge (EW ÷ TW)] ÷ n} ÷ PEviability × 106 Where EW = the number of empty wells, TW = the total number of wells and n= the total number of cells plated per well. When using the above calculations, the sum of the large and small colony mutation frequency always equals the total mutation frequency.
See also: An introduction to genetic toxicology |
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