2. Add 10 µl of spore solution to each side of the hemacytometer. 3. Count number of spores in zones A, B, C, D and E on both sides of the hemacytometer, record them, and calculate the average of the two sides. a. If a spore falls on the left or bottom line DO NOT count it. b. If a spore falls on the right or top line DO count it
A B
C D
4. To determine the number of spores per mL
a. (A + B + C + D + E) x 2000 5. Dilute the spore solution to desired concentration (spores/mL) a. Use C1V1= C2V2 formula i. Ex. If spore solution is found to be 67,000 spores/mL and you want 40mL of 50,000 spores/mL ii. 67,000 x = 40 * 50,000 a. x = 2,000,000 = 29.85 ~=30mL b. 67,000 iii. So, you will add 30mL spore solution to 10mL sterile dH20 (40-30=10) to give a final concentration of 50,000 spores/mL in 40mL 6. You can use the Excel worksheet: C:\Scab data\Protocols\Current\Hemacytometer Worksheet.xls to print out a data entry form or to calculate spore concentrations.
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