Question

1. Vibrio bacteria are abundant in ocean and aquatic environments. Suppose you wish to isolate Vibrio- specific bacteriophages from seawater. You enrich your sample by spiking the seawater with growth medium and Vibrio fischer. You then isolate your phage and plate it on lawns of bacterial growth to enumerate how many PFUs/mL are in your sample. a. You set up a series of 10-fold phage dilutions out to a TDF of 10', and you plate each of those dilutions mixed with V. fischeri. Your lab partner also wants to check for phage using the related Vibrio pocabgemolyticus as a host. You do so, but you only bother to plate out to your 10 dilution, assuming you will have significantly fewer plaques on these plates. Why is this a fairly safe assumption? (1 pt.) b. You obtain the following results from your plaque assays: Host Species V. fischeri V. parabgemolyticus Countable Number 45 200 TDF of Countable Plate 10 10 Assuming you plated 0.1 ml of your phage dilution, calculate the phage density (PFU/mL) for each of the two host strains. (2 pts.) c. Which of these values more accurately reflects the actual number of phages in your sample? (1 pt.) d. Now suppose you skipped the enrichment step and isolated phages directly from a sample of seawater. If you plated your sample using V. fischeri as a host, would the number of plaques accurately reflect the total number of phages that you were able to isolate? Explain your rationale. (1 pt.)

Answer 1

Answer 1.a)

1. Basically, to state the specificity in terms of bacteriophage, V.parahaemolyticus is stated to have a more specific isolating potential. KVP40 strain is the bacteriophage stated to show more specificity to the above mentioned Vibrio bacteria. It is used to check and calculate the infection titer in order to check the number of viable bacteriophage able to form colonies or more specifically plaque forming units (PFU).
2. Total dilution factor is the sum of individual dilution factors and it accounts to 10^-6 in a ten fold dilution ( 1ml in 9ml ) which can be stated as (10^-1) + (10^-2) + (10^-3) which may account to 10^-6 as the TDF. So, the final Individual dilution factor is 10^-3 which is 1/1000 and hence shows fewer plaques.

Answer 1.b)

1. PFU/ml = Number of observed plaques(colonies) / (Dilution factor) × (Volume of diluted phage)
2. This accounts to (45) / (10^-4) (0.1) = 4500000 PFU/ml (4.5×10⁶ PFU/ml) for V.fischeri.
3. This accounts to (200) / (0.0001) (0.1) = 20000000 PFU/ml (2×10⁷ PFU/ml)