doing phage titrering - (Oct/09/2004 )

hi guys!

i am doing phage titrering but have difficulty in chosen the right values. please give me some ideas. thanks in advance!


here is what we did:
dilute phages 5X with dilution buffer. add 1ul of the diluted phage to 200ul of the XL1-Blue overnight culture. Add 2ml melted top agar. Quickly pour onto a plate. the day after count pfu/ml.

pfu/ml: (number of plaque*dilution factor*10^3ul/ml )/ul of diluted phage plated.

my problem is: what is the dilution factor? is that factor 5 or should i also take 200ul of the XL1-Blue cells and 2ml melted top agar in consideration? (practically it seemed like the phages is diluted more than 5X.)

what is the "ul of diluted phage plated"? is that the 1ul diluted phage i added to the other volumes or is that the total volume (1ul phage+200ul XL1-Blue cells+2ml top agar)?


thanks!

usually one is interested in the phage count per µl in one's phage stock solution. so you can ignore the dillution in bacteria an medium.

your formular should be correct so far. when using 1µl phage solution 1:5 dilluted:

phages/µl = pfu * 5

naturally phages/ml will be the above number *1000.

and to your last question: yes, "µl of diluted phage plated" is in your case 1, just as you said in your experimental setup:

quote " ... add 1ul of the diluted phage..."

mike