Help measuring bacteria growth/death rates

[deleted-530123]

Hello, I hope everyone is doing well.

I am currently in the process of designing an experiment for an essay I have to write (I want to talk about the experiment), but I've run into a bit of a stump.

My idea was to get several species of gram positive vs gram negative bacteria and test how well they do in more acidic vs more alkaline environments. I was thinking of spreading bacteria uniformly over an agar plate and then applying a very thin coating of an acidic or alkaline substance (for example, diluted vinegar) over the plate.
The problem is, i'm not sure how I could measure how many bacteria that kills or how it affects their growth rates...

If anyone has any suggestions, they would be much appreciated. I have no problems changing some of my procedures or setup if it is required. Also, let me know if more details are needed in order to help me.

Thank you,
Mauricio

[deleted-287099]

Hello, Macullau.

Bacteria perform cellular respiration. You might add some sort of nutrient to the agar plate (sugar?) and wrap a balloon over the whole setup. You could then compare rates of inflation.

You may also purchase fairly cheaply (<$10) a bottle of a carbon dioxide indicator which would do the trick just as well.

Just some quick thoughts. Let me know what you think. Feel free to add to this thread if you need anything else or if you are dissatisfied with both of my solutions.

[deleted-530123]

Hello, thank you for the quick reply and suggestions.

I really like the balloon idea. However, i'm thinking that would not be very easy to set up, so i'm definitely considering it but would prefer finding another method if possible.

Also, i'm planning on growing the bacteria on Luria Broth. I want to mix the luria broth with whatever substance i'm testing (for example with vinegar) and then pouring that solution onto the plates. Would that work?

Thank you.

[deleted-287099]

Macullau,

The balloon setup should be fairly simple. I think you can tape/rubber band a balloon to the skinny end of a funnel, then place the funnel over the agar plate.

Alternatively, you might consider using a solution of bromothymol blue and water. When carbon dioxide is forced into water (you can do this by covering the agar plate with a funnel, connecting a straw/tubing to the funnel, then inserting the straw into the bromothymol blue solution), the molecules form a weak acid called carbonic acid. Bromothymol blue is a pH indicator, so you can use it to measure the acidity of the solution. Check this source out: http://cswnetwork.org/projects/pdf/219.pdf

There are also kits/equipment available for measuring carbon dioxide levels. These are especially useful for pet owners, so you might consider checking your local pet supply store.

I think mixing in the acids and bases into the lysogeny broth would work. Looks good.

[deleted-530123]

Hey, thanks again for helping me.

So then if I do use that carbonic acid approach, which ever turns more acidic is the one in which the most bacteria grew?

I think that could work well.

[deleted-287099]

Macullau,

Yes, it should be. More carbon dioxide means more carbonic acid which means a lower pH (and an overall more acidic substance).

We are here to help.

[deleted-530123]

Awesome.

So my plan is to have roughly 6 species of bacteria and test 6 different substances (3 acidic, 3 alkaline, 1 neutral). Do you think that method could get me data accurate enough to where I could clearly see the results of which bacteria grew more and which grew less?

Edit: So I would pretty much be using about 37 petri dishes.

[deleted-287099]

Thirty-seven? Not (7 pH levels)*(6 different bacteria)=42 petri dishes?

Yes, I believe this would work, though I am unsure why 37.

[deleted-530123]

My bad :/

When I thought control I was thinking 1, not 7. You're right, it's 42. I'm going to buy the materials and attempt to perform the experiment this weekend. I'll let you know how it goes or if I have any other questions.

Cheers