For the next hemocytometer counts calculations · The original culture concentrat
ID: 223451 • Letter: F
Question
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.
* I asked this questions already, however, the only answer I received was which squares to use. All the squares need to be incorporated by the following questions followed by the calculations.
Explanation / Answer
1) When counting cells using the 0.00625mm3 volume squares, then the volume of such square in ml = 0.00625/1000 = 0.00000625ml
Average count per square = (Square count + Square 2 count + Square 3 count + Square 4 count)/4 = (80+75+50+60)/4 = 265/4 = 66.25 = ~66 cells/square or 66cells/0.00000625ml
To determine the number of cells per ml = (66*1ml)/(0.00000625ml) = 1.056 x 107 cells/ml
Dilution factor used before loading cells on hemocytometer = Total volume/volume of cells = (950+50)/50 = 1000/50 = 20
Therefore original culture concentration =1.056x107 x 20 = 2.112 x 108 CFU/ml
Concentration of sample loaded in the hemocytometer = 1.056x107 CFU/ml
Hemocytometer factor used to calculate each count = 1/0.00000625 = 16x104
Answer:
2) When counting cells using the 0.1mm3 volume squares, then the volume of such square in ml = 0.1/1000 = 0.0001ml
Average count per square = (Square count + Square 2 count + Square 3 count + Square 4 count)/4 = (65+35+91+150)/4 = 341/4 = 85.25 = ~85 cells/square or 85cells/0.0001ml
To determine the number of cells per ml = (85*1ml)/(0.0001ml) = 8.5 x 105 cells/ml
Dilution factor used before loading cells on hemocytometer = 100 as mentioned
Therefore original culture concentration =8.5x105 x 100 = 8.5 x 107 CFU/ml
Concentration of sample loaded in the hemocytometer = 8.5x105 CFU/ml
Hemocytometer factor used to calculate each count = 1/0.0001 = 104