counting cells-dilution - (Jan/29/2012 )
The other day I had to count cells after resuspending my cells in 4mL. I took 10uL of cells mixed with 10uL of trypan blue and loaded onto a hemocytometer...I had soo many cells I couldn't count so I diluted my cells 15 fold (3.3uL of cells + 46.7uL of media +50uL of trypan blue)..I counted around 381 cells in the grids all up so just wondering when its time to count the total amount of cells in that whole 4mL do I use the following formula.
(381*15)*2*10^4=1.143*10^8 multiplied by 4 =4.6*10^8
seeing as i diluted 15 fold i assume i multiply the total number of cells by 15 and then because theres a 1:1 ratio of trypan blue I multiple by 2 and then 10^4 becuase one set of 16 corner squares is equivalent to the number of cells x 104 / ml and then i multipled by 4 because thats how much i resuspended in...have i got this right? I think I may have got the typan blue dilution factor wrong because when I previously counted cells I never had to dilute my cells as they were countable...so i just used 2 as the dilution factor..have i got this new dilution factor right?..hope im making sense!
You would have to consider Trypan Blue in final dilution factor. ......<3.3 in 96.7 ul diluent i.e 3.3:100 = 1:30>
30 is your overall dilution factor.
Total cells (in 4 ml) = Average count in four grids x dilution factor x 104 x 4
Thank you so much for that reply!! ...much appreciated
I just realised that my way and your way produce the same result
cell # * 30 * 10^4 * 4 is the same as:
cell # * 15 * 2 * 10^4 * 4
You also need to take into account the number of squares(divisions) of the main grid you counted. For a standard haemocytometer, the whole grid is divided into 9 large squares (sometimes bounded by 3 lines, depending on the model), which are then subdivided into 16 or more smaller grids. Each of the 9 large squares, is equivalent in volume to 10-4 mm2, counting all 9 and not dividing by 9 will give you an erroneous density. If you are only counting one of the 9 squares you are doing it wrong! Your error will be somewhere in the 20-50% range!