Plaque formation and Phage Concentration...please help.?

Question

Hi. Could anyone help me on either, but hopefully on both!, questions?!! All help is greatly appreciated as it for an assignment. Please...I do not want a copy/pasted version from Wikipedia!! and please..no silly/sarcastic answers!

Thanks in advance :-)

Q1) In microbiology, how do plaques form?

Q2) Why do results gained at the end of a experiment where, for example, E.coli was mixed with a bacteriophage at different dilutions give an idea of phage concentration present?

Seriously, any (useful, not stupid) help is greatly appreciated!!
10 points up for grabs aswell!!

Thanks again in advance

Ms Me
:-D

Answer 1

Plaques are areas of clearing on what microbiologists (and virologists) call bacterial lawns, caused by the infection of bacterial cells by a bacteriophage. Bacterial lawns are created by spreading a bacterial culture onto a petri dish that contains a nutrient solidified with agar.

Bacteriophage (phago= eat) are actually named so since it looks like they are 'eating' the bacteria from the plate!

One can estimate the concentration of bacteriophage in a sample by dispensing the same volume of bacteriophage sample into different dilutions of a known bacterial sample and then coounting the plaques. The number of infected bacteria in any dilution can be 'seen' by plating a constant volume of the bacteria-phage onto petri dishes and then counting the plaques formed.

In microbiology, the number of plaques must be in the range between 30-300 in order to get a good statistical validity. Also, usually three platings are done for each dilution in order to average the number of plaques per dilution (again, statistical value). Concentration of phage is generally reported in #/mL.

Example: add 1mL phage solution to 6 ten-fold dilutions of bacteria. Plate 0.1mL of each dilution + phage onto plates (do 3 plates per dilution).

Let's say that the first few dilutions have way too many plaques to count, and the last two dilutions only have a few plaques. The 10-4 dilution has between 30-300 plaques per plate. Count them and then average the three plates, ie 67 + 101 + 84 = 252, divided by three for an average of 84 per plate.

First you must multiply that number by 10 since you only plated 0.1mL bacteria-phage, and then you must multipy by the dilution concentration in order to get the #plaques/mL.

Therefore, 84 x 10 x 10exp5 = 840 x 10exp5
In scientific notation, this is 8.4 x 10exp7 PFUs/mL
PFU is plaque-forming units, ie. the number of phage in 1 mL of your phage solution.

Answer 2

Plaques are formed on a bacterial lawn when the phage introduced to the plate kills the bacterial cells in a specific area.

The results at the end of the experiment would give an idea of phage concentration because if there were lots and lots of plaques, you would know there had been lots and lots of phage introduced originally, meaning there was a high concentration of phage. If there were fewer plaques, you would know there were fewer phage, meaning a lower concentration.