Biot 120-111MB Spring 2018 2. A0.2 mL sample of detached cells was removed from
ID: 260853 • Letter: B
Question
Biot 120-111MB Spring 2018 2. A0.2 mL sample of detached cells was removed from a T-75 flask containing 10 mL of media and was used for counting. 30 uL of the counting sample was diluted with 270 ul of HBSS. A 50ul aliquot of the diluted cells was removed and mixed with 300ul of trypan blue. After 2 min the hemocytometer was loaded and a total of 200 cells were counted over 10 squares. 5 of the counted cells were blue Calculate the % viability and the live cell concentration. Is this count reliable? 21 You need to seed 1 x T-75 flask and your co-worker has asked that you seed 3 x T225 flasks for him as well. All the flasks are to be seeded at 6 x 105 cells/ml. Do you have enough cells to seed all the flasks? Indicate the volumes of cells and media required for all the flasks that can be seeded.Explanation / Answer
Viable (live) cells concentration C = N*D*103 where
N=Number of viable cells counted in 10 subgrids = 200 - 5 = 195
(Live cell do not take the dye)
D=Dilution factor = 10
103 = Hemocytometer correction factor
Hence, C= 195*10*103 = 195*104 cells/ml
% viability (V) = N / T = number of live cells counted per 10 subgrids / total number of cells counted
= 195 / 200 = 97.5 %
Hemocytometer is a manual method of counting cells and hence human errors can happen in mixing, handling, dilution, calculations and procdure. However, modern day automations has largely taken care of these human errors.
According to Protocol for Maintenance of Cell Culture, T25 flask can have 5 to 22.5 ml medium and T225 flask can have 45 to 67.5 ml medium for average cell yield of 1*105 cells /cm2 from a 100% confluent culture.
The viable cells available will not be sufficient enough to seed all the flasks.
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