Question

How to count colonies and determine the frequency of Ampicillin resistant transformants?

Answer 1

Ans- Best way to count is to divide the Petri plate into four quadrants, almost equal in size , using marker on the bottom surface of the plate.

*Counting should be done by marking the isolated and identical colonies only , because one colony is thought to have originated from a single parent cell, hence all members of the colony are clones of each other.

* If the spreading has been done properly and the colonies are distributed very well , that is each quadrant seems to have almost equal number of colonies , than you need not to count all the colonies across all the four quadrants, counting colonies of single quadrant and then multiplying it by four, will give almost the same result as in case of counting and totaling all the colonies across all the four quadrants.

* if the spreading is not proper, that is some quadrant have more number of colonies than the other, then in that case you have to count the colonies in each quadrant and then total them, to get number of transformed colonies.

* Total Colony count must be within 30-300 , count below 30 will be marked as Too low/Less to count (TLTC) , while count above 300 will be marked as Too Numerous to Count (TNTC).  

*Colony count below and above the range results in under or overestimation of the actual number of transformed cells, hence the colony count must be within 30-300.

* Diagram is just to depict the plate , how to divide into quadrants, how the colony distribution may appear. Depicted colonies may not be of same morphology or equally distributed on the plate. This is just to demonstrate you the process involved.

Hope this will help you .