non-lytic phage titer on X-gal plates -urgent help needed - (Jan/16/2007 )

Hello
I'm having a problem with establishing the phage titer in a phage-display system using X-gal plates.
The cells infected by the recombinant phages should turn out blue. Once I have the blue colonies I count them, and then what? How do you calculate the phage titer? I was looking for basic microbiology for that, but the university only has books dated from '77 (imagine...), and online I only can find calculations for transformation efficiency. I don't think the equation I use is correct -they don't make sense. The 1/10000 titer is always smaller than the 1/100000, for example. They should give the same results, right?

Is there a good source for this kind of basics? My microbiology books are left in Europe, and the last time I was working with that stuff was 6 years ago...)

Thank you for the help.

I think I know what you're asking but I'm not sure.

If you're asking how to figure out plaque forming units per ml (pfu/ml). Then here's how I do it for bacteriophages.

Take 50ul of you stock phage solution and dilute it into 4.95ml of solution. This is a 1:100 or a -2.

From your -2 take 50ul and dilute into another 4.95ml tube. This is another 1:100 or a -4.

From your -4 take 50ul and dilute into another 4.95ml tube. This is another 1:100 or a -6.

From the -6 take 200ul and dilute into a 1.8ml tube. This is a 1:10 or a -7.

Now take three petri plates. Label one "-8", "-7", and "-5".

You will take 100ul from your -7, -6, and -4 tube. The 100uls from the -7 goes into the "-8" plate. The 100uls from the -6 goes into the "-7" plate, and 100uls from -4 goes into the "-5" plate.

Add you host and agar to the plates they swirl and let solidify. Incubate then count when appropriate.

The plate you count should have between 30 and 300 plaques. If there are more of less you need to look at a different plate.

If on the "-7" plate you count 150 plaques then you have a viral titer of 1.5x10^9. The "-8" should have somewhere around 15 plaques on it because the "-8" plate is one logarithm greater than the "-7" plate. The "-5" is countable you would have 15,000 plaques on it.

Since I don't know what titer you have I just chose a 10^9 titer as an example since that is what I typically deal with. If your titer is lower then you may only need to use 1.8ml tubes and just do a few 1:10 dilutions.

Just remember:

50ul always goes into a 4.95ml tube to give you a 1:100 or -2. 4.95 always counts for 2 log basically.

200ul always goes into 1.8ml tube to give a 1:100 or -1. 1.8 always counts for 1 log.

When you take 100uls from the dilution tube you need to keep in mind that 100ul is 1/10 of 1ml so that is why you number your plate one number higher than the tube.

When counting the plates if you have more than 10 plaques the exponent is increased by one. If you have more than 100 plaques the exponenet is increased by two.