For the next hemocytometer counts calculations The original

For the next hemocytometer counts calculations

·      The original culture concentration in CFU/ml

·      The concentration of the sample loaded in the hemocytometer

·      The hemocytometer factor used to calculate each count

1) 50 ul were taken from a bacterial culture flask and dilution using 950uL of PBS, the hemocytometer was loaded with 10ul and counted in the microscope. For counting the cells the 0.00625 mm^3 volume squares were used.

·      Squares 1-80

·      Squares 2-75

·      Squares 3-50

·      Squares 4-60

2) The laboratory technician has given you a tube with a bacterial culture diluted one hundred times, after you load the hemocytometer you count the bacterial cells using the 0.1 mm^3 volume squares

·      Squares 1-65

·      Squares 2-35

·      Squares 3-91

·      Squares 4-150

You are asked to make a series of dilutions to plate 100 and 1000 colonies using the original culture. Please include the volume of solute and solvent in each dilution and how much volume would you use from you last dilution to plate.

Solution

Answer

1)- Square 4-60 should be used

2)Squares 1-65 should be used where Dilution factor =100

Dilution Factor, or how much the original sample is diluted:
here Initially 50uL in 950ul = Final Volume / Sample volume =1000/ 10 = 100

Then two serial dilutions of 1/100.

Total Dilution Factor = 10 * 100 *100 = 10^5

CFU/mL = cfu/ml = (no. of colonies x dilution factor) / volume of culture plate

= (100 * 10^5)/0.1
=10^8

10 ul volume should be used for each dilution.

So Total colony forming units = 1.37*10^8 CFU/mL