Biot 120- Spring 2018 \' Part IIl. Calculations (20 pts ea). Answer each questio
ID: 260852 • Letter: B
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Biot 120- Spring 2018 ' Part IIl. Calculations (20 pts ea). Answer each question completely - be sure to read and answer al parts of each question. Answer only in the space provided - answers written on the back of any page will NOT be considered. Be sure your writing is legible and easy to read. Partial credit will be given- so try to write something down. SHOW ALL WORK! A0.5 mL sample of detached cells was removed from a T-225 flask containing 20 mL of media and was used for counting. 25uL of the counting sample was diluted to 500ul with HBSS. A 50ul aliquot of the diluted cells was removed and mixed with 250uL of trypan blue. After 2 min the hemocytometer was loaded and a total of 419 cells were counted over 10 squares. 39 of the counted cells were blue 1. Calculate the % viability and the live cell concentration. Is this count reliable? You need to seed 3 x T-75 flask and your co-worker has asked that you seed 3 x T25 flask for her as well. All the flasks are to be seeded at 3 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
1) blue is a stain that only stains dead cells. Hence, dead cells will take up the blue colour. Live cells remain unstained.
Number of viable cells= Average count of live cells/square * Dilution Factor* 10000
Dilution factor= 50 ul sample + 250 ul trypan blue= 1+5= 6
Number of viable cells= 419/10*6*10000= 251.4 * 10000= 2.514 X 106 cells/ml
25 ul of sample was diluted in 500 ul HBSS. This is a 1: 20 dilution
Hence, Number of viable cells in original flask= Number of viable cells *20= 2.514 X 106 X 20
=5.048*107 cells/ml
% Viability= Live cell count/Total cell count *100= 419/419+39 *100= 91.48%
The Trypan blue staining is widely used as is considered the standard approach for calculating cell density. However, it has limitations. Trypan blue is toxic to cells after some time. Hence, counts should be taken within a specific time. In primary cells, it will bind to cellular proteins giving non-specific results. False positive and negative results may occur if the cells have intact membranes but are dead or cells are viable but have damaged membranes.
2) For T75 flask, culture volume range is 15-25 ml is added. For T25, range is 5-7.5 ml volume is added.
Consider that for one T75 ml flask, you add 20 ml and for each T25 flask, you add 5ml.
Hence, total volume required= 3* 20 + 3*5 ml= 75 ml
Prepare 100 ml so that there is enough medium
Viable count= 5.048* 107 cells /ml
In order to get 3 X 105 cells /ml,
Volume = 100 ml
C1V1= C2 V2
5.048 X 105 X A= 3 X 105 X 125 ml
A= 3 X 105 X 100/ 5.048 X 107
A= 0.6 ml
0.6 ml of stock cell + 99.4 ml of medium= 3 X 105 cells/ml
There are enough cells to seed the plates, as there is 19.5 ml of culture cells left.
Extra culture media is prepared to account for pipetting errors etc.
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