In: Biology
Show all calculations in a neat and organized manner. Be sure that I understand your logic. For your experiment you must treat 1 X 109 cells with 5 nM cycloheximide (MW=281.35). Cycloheximide is both toxic and expensive. You have counted the cells with a hemocytometer and found the following numbers when counting 5 “4x4” sections (each 4x4 is 0.004 µl) of a 1:10 dilution of you chlamy stock: 90, 103, 123, 99, 107.
1) What is the concentration of the initial Chlamy culture in cell/ml? _______________________
2) How many mls of cells of the culture must you use for the experiment? _____________________
3) Describe how you will treat the cells with cyclohexamide. Clearly show all calculations.
1) volume of each (44) section= 0.004 uL
volume of 5 (44) section= 50.004
= 0.02 uL
1 uL= 10^-3 mL
0.02 uL= 0.02 10^-3
= 2 10^-5 mL
average number of cells counted= 90+103+123+99+107=104.4
number of cells in 2 10^-5 mL = 104.4
number of cells in 1 mL= 104.4/( 2 10^-5)
= 5.22 10^6 cells.
number of cells in 1 mL of the original culture= number of cells per mL in the diluted culture dilution factor
dilution=1/10
dilution factor=1/dilution=10
number of cells in 1 mL of the original culture= 5.22 10^610
= 5.22 10^7 cells
2) number of cells required=10^9
5.22 10^7 cells is present in 1 mL
1 cell is present in 1/(5.22 10^7)=1.9210^-8 mL
10^9 cells is present in 1.9210^-8 10^9= 19.2 mL
volume of cells required= 19.2 mL
3) 1 mL= 10^-3 L
19.2 mL= 19.2 10^-3=1.92 10^-2 L
molarity= number of moles/volume of the solution in liters
1 nM=10^-9 M
5 nM=510^-9 M
510^-9 = number of moles/ 1.92 10^-2
number of moles= 510^-91.92 10^-2
= 9.610^-11
number of moles= mass in gram/gram molecular mass
9.610^-11 = mass in gram/281.35
mass in gram= 9.610^-11281.35
= 2.70110^-8 g
so mass required is very less to be measured.
so let`s make stock solution 10 mL of 5 mM ( 5mM= 510^-3 M)
molarity= number of moles/volume of the solution in liters
510^-3 = number of moles/ 1010^-3 ( 10 mL= 1010^-3 L)
number of moles= 510^-310 10^-3
= 510^-5
number of moles= mass in gram/gram molecular mass
mass in gram= number of moles gram molecular mass
=510^-5281.35
=0.0140675 g.
take 0.0140675 g of cyclohexamide and dissolve in 10 mL water.
then
M1= 5 mM
M2=5 nM=510^-6 mM ( 1 nM= 10^-6 mM)
V1=?
V2= 19.2 mL
M1V1=M2V2
V1=M2V2/M1
= 510^-619.2/ 5
=1.9210^-5 mL
1 mL= 10^3 uL
1.9210^-510^3= 0.0192 uL
take 0.0192 uL of the stock solution and add to the cell culture.