Question

In: Biology

Show all calculations in a neat and organized manner. Be sure that I understand your logic....

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.

Solutions

Expert Solution

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.


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