How did they measure total cells? (genetics of bacteria)

[outxbreak]

1. About how many cells were plated to count His+ revertant colonies?
How was total cell number determined?

I know that the mutation freq was out of 10^7 survivors. So they had to measure the total cell number and come up with 10^7 survivors? But I'm not sure how they got this exact number of 10^7 survivors?

Then to measure His+ revertants I would think they need to plate the same concentration of cells as they did to measure the total cell count?

 

[epenguin]

Answer the second part of the question first, and that should also lead to the answer to the first.

Imagine yourself doing the experiment - what do you actually do in order to get results?

 

[outxbreak]

Hmm well replica plate from the survival plate (his+ min + glu) onto another plate (-his +min +glu)

But I'm really confused as to how every time point had a survival plate with 10^7 survivors? I guess they started with something like 1*10¹⁴ cells and mutagenized them with UV... so is the y-axis saying if the survival plate has 10^14 survivors and the mutant plate has 5 mutants then to find the number of actual mutants that you plot set up:

5 mutants/ 10^14 survivors= x mutants/10^7 survivors
x=5*10^-7 mutants per 10^7 survivors..?

 

[epenguin]

Hmm well replica plate from the survival plate (his+ min + glu) onto another plate (-his +min +glu)

But I'm really confused as to how every time point had a survival plate with 10^7 survivors? I guess they started with something like 1*10¹⁴ cells and mutagenized them with UV... so is the y-axis saying if the survival plate has 10^14 survivors and the mutant plate has 5 mutants then to find the number of actual mutants that you plot set up:

5 mutants/ 10^14 survivors= x mutants/10^7 survivors
x=5*10^-7 mutants per 10^7 survivors..?

Your questions come with very little context; fortunately these are very common types of experiment for mutagenesis etc. so we can reasonably guess.

You ask about time points. There are no time points in what you have told us. There is a sketch without experimental points of a graph of slightly processed or calculated numbers showing the frequency in the population of revertants from auxotrophy (i.e. his⁺) as function of uv dose. This is how such data would normally be presented except there would be experimental points.

Survivors and revertants are measured by the number of colonies you get on a plate, which you can count. You don't, and you don't need to, measure every cell in this way - it would take a long time to count 10¹⁴ colonies. You typically aim to have of the order of 100 colonies per plate.

So you have to arrange the experiment to have reasonably countable numbers of colonies for the various conditions. You have the advantage of knowing some expected numbers. (When you don't know beforehand what the numbers are, how are you going to do that?)

Are doing this stuff with no laboratory practice? It sounds like either that or you are not connecting these things up with the lab work.

I think replica platings are not needed and do you no good in this case when you are looking for small frequencies. You do everything by dilutions!

 

[outxbreak]

She gave us no other context. :/

In order to measure total survival don't you need to have a survival plate (min+his) and then replica plate these survivors onto the mutant plate (min-his). This ensures that the mutants came from those survivors.

I'm still not sure the total cells to start with.
I would guess 1*10¹⁴ cells in a tube.
Mutagenize the cells in this tube with UV dose.
Then plate onto survival plate after a 100,000 fold dilution. If you get 100 cells it means you had 1*10⁷ survivors/10⁹ cells.. I don't know any other way to get 10^7 survivors.

 

[epenguin]

She gave us no other context. :/

In order to measure total survival don't you need to have a survival plate (min+his) and then replica plate these survivors onto the mutant plate (min-his). This ensures that the mutants came from those survivors.

where else can they come from but survivors?
To know the number of survivors, yes you have to plate on min + his.
According to the sketch about 10^-5 of his⁺ revertants not requiring histidine to form colonies are being easily measured.
I think it is obvious that whatever the volumes etc., to see a convenient number of these, you need to dilute them 10^-5 less than you do to measure total survivors.
If you measured by replica plating you would have to look at 10⁵ p!ates to see one colony - there is no point in it

I'm still not sure the total cells to start with.
I would guess 1*10¹⁴ cells in a tube.
Mutagenize the cells in this tube with UV dose.
Then plate onto survival plate after a 100,000 fold dilution. If you get 100 cells it means you had 1*10⁷ survivors/10⁹ cells.. I don't know any other way to get 10^7 survivors.

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