2. Detecting Viruses: Plaque Assay Explain what the pour-plate technique is and

Question

2. Detecting Viruses: Plaque Assay Explain what the pour-plate technique is and why it is used for plaque assays? a. You inoculated a plate with 0.1 mL of a 10-4 dilution. The plate now contains 10-5of original phage sample. Suppose you subsequently counted 47 plaques on the plate. What would the original phage density be? Show work. b. You performed a plaque assay using the stock of T4 bacteriophage. Your results show an average of 380 plaques when you assay 0.1ml of a dilution that was prepared by mixing 1 part of the original virus solution that contained 999,999 parts of buffer. What is the titer of your original stock of bacteriophage? Show work c.

Explanation / Answer

(a)Pour plate method is usually the method of choice for counting the number of colony-forming bacteria or plaque forming units present in a liquid specimen. The pour plate allows for uniform distribution/spreading of the phage and bacteria. Hence allows for counting of plaques.

(b)

Original PFU/ml = ?

Dilution factor = 10^-4

Number of plaques= 47

Volume of sample =0.1 mL to the plate

cfu/ml = (no. of plaques)/( dilution factor x volume of culture plate)

cfu/ml = (47)/ (10^-4 x 0.1)

pfu/ml = 47 x 10^5 pfu/mL

(c)

Original PFU/ml = ?

Dilution factor = 1 in 999,999 parts = 1/10^6

Number of plaques= 380

Volume of sample =0.1 mL to the plate

cfu/ml = (no. of plaques)/( dilution factor x volume of culture plate)

cfu/ml = (380)/ (10^-6 x 0.1)

pfu/ml = 38 x 10^8 pfu/mL

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