Please help me solve this problem A 1 ml phage suspension ly

Please help me solve this problem

A 1 ml phage suspension (lysate) was serially diluted in saline by the following protocol and the indicated samples plated using a standard plaque assay: a. What is the total dilution of the phage suspension in tube 4? (Think carefully) b. Calculate the phage titer of the original suspension. Assume each plaque is caused by a single phage, (show your work). 4. You have been given a broth culture of E. coli at 5 times 10\" CFU/ml. For your experiment you need 4 cultures that are 1 times 10, 1 times 10-, 1 times 10-, 1 x 10\', and 1 x 10- CFU/ml. Devise a dilution scheme to provide all of the above concentrations of bacteria.

Solution

Given Original undiluted Phage Lysate sample = 1 ml

In tube 1, 100 microlitre lysate was added to 900 microlitre of saline (Total volume formed = 1000 microlitre). Thus, this dilution is 100 microlitre of phage lysate in 1000 microlitre of total solution fromed, i.e., 100/1000 = 1:10 dilution.

thus Tube 1 is 1:10 diluted.

Similarly, 100 microlitre from tube 1 added to 900 microlitre of saline in tube 2 to makes it more 1:10 diluted, this forms the 1:100 diluted solution of the original undiluted sample in tube 2 (1:10*1:10), Thus tube 2 is 1:100 diluted

Similarly, Tube 3 is 1:1000 diluted solution of Original undiluted phage lysate sample.

Since NO SALINE was added in tube 4, the dilution remains same as tube 3, i.e., 1:1000

Answer (a) = Tube 4 dilution is 1:1000 (1:10^-3) (Dilution = 1x10^-3).

Numbers of plaques obtained in 3 samplings = 22, 17, 29

Average number of plaques = (22 + 17 + 29)/3 = 68/3 = 22.6 = round off to 23.

Assuming that each plaque is caused by a single phage, we calculate Plaque forming units (Pfu) to determine the concentration in Original undiluted sample.

It is calculated as follows:

Pfu/ml = (Average No. of plaques obtained)/(Dilution of phage solution added * Volume of phage solution added)

= 23 / (10^-3 * 0.1 ml )

= 23 / (10^-3 * 10^-1 )

= 23 / (10^-4 )

= 23x10⁴ Pfu per ml

Answer (b): The phage concentration of original undiluted suspension is 23x10⁴ Pfu/ml