Situation: You love microbial physiology and want to see if LPS is inducing an i
ID: 253661 • Letter: S
Question
Situation: You love microbial physiology and want to see if LPS is inducing an immune response. You have three groups of mice (1) control mice, (2) mice injected with LPS, (3) mice infected with an E. coli strain that can't make LPS (E. coli). You have one control mouse, three LPS mice, and two E. coli mice. You want to determine if there is any immune stimulation from the LPS or if something else on the E. coli is causing the immune response Part I: Spleen cell isolation and Cell concentration calculations l. You resuspend the control group's spleen cells in a final volume of 5 ml. You take 10 ?! of your cell suspension and add it to 90 ul of trypan blue. In the microscope you count 10 squares and the average cell number per square is: 36 alive cells and 4 dead cells. a. What is the dilution factor? b. What is the concentration of the viable cells in cells/ml? c. What percentage of your cells are viable? d. How many total cells do you have? 2. You pool the three spleens from the LPS group, and you resuspend the cells in a final volume of 5 ml. You take 10 ?1 of cell suspension and add it to 90 l of trypan blue. You then take 10 ?1 of your first dilution and add it to another 90 ?1 of trypan blue. The average cell counts from the hemocytometer are: 19 alive cells and 3 dead cells per square a. What is your final dilution factor? b. Answer questions b - d above for the LPS spleen cell preparation. 3. You pool the two spleens from the E. coli group, and you resuspend the cells in a final volume of 7 ml. You then take 10 ?1 of you cell suspension and add it to 90 ?1 of trypan blue. You take 10 ul of your first dilution and add it to another 40 ul of trypan blue. The average cell counts from the hemocytometer are: 23 alive cells and 6 dead cells. What is the final dilution factor? Answer questions b - d above for the E. coli spleen cell preparation a. b.Explanation / Answer
1.a) dilution factor=10/ 10+90=10/100= 1/10 or dilution factor is 10
as 10?l was added to 90?l of trypan blue.
b) viable cell/ml= ( avarage no of cell/ per square) X dilution factor X 104
= 36 X 10 X104 = 360 X 104 =36 X 105 or 3.6 X 106
c) as we have 36 live and 4 dead cell,
we got out of 40 cell, 36 cell is viable
then out óf 100 cell = 100 X 36/ 40=90% cells are viable.
d) total cell/ml = 3.6 X 106 X 5= 18 X 106
2.a) 10 ?l in 90 ?l =1/10
further 10 ?l from this was added to 90 ?l
final dilution = 1/10 X 1/10 = 1/100
dilution factor =100
b) viable cell /ml= ( avarage no of cell/ per square) X dilution factor X 104
= 19 X 100 X 104 = 19 X106
c) percentage of viable cell:
19 cell in 22 cell
22 cell give 19 live cell
thus 100 cell will give 19 X 100/ 22=86.3 % cells are viable
d) total cell= 5 X 19 X106= 95 X106
3. a)
10 ?l in 90 ?l =1/10
further 10 ?l from this was added to 40?l
final dilution= 1/10 X 1/5= 1/50
dilution factor=50
b)viable cell /ml= ( avarage no of cell/ per square) X dilution factor X 104
= 23 X 50 X 104 = 1150 X 104 = 11.5 X106
c)
c) percentage of viable cell:
23 cell in 29 cell
29 cell give 23 live cell
thus 100 cell will give 23 X 100/ 29=79.3 % cells are viable
d) total cell= 7 X 11.5 X106= 80.5 X106