cincibuddy99
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viable bacteria count. Turbidimeter or Spectrophotometer ?

Postby cincibuddy99 » Mon Oct 02, 2017 6:26 pm

Hello,
My project is about e.coli and bacteria resistance. I want to create 5 generations of e.coli applying an antibiotic. For each generation I want to be able to count the number of bacteria. I don't want to measure areas of inhibition, I want to be able to actually count the number of e.coli in each generation.
I thought I could do this by measuring OD600 with a turbidimeter but my professor says that's not a good method. I don't want to use the plating method to count (petri dishes) because it takes too long and it's not accuate.
Which would be the best method to count e.coli bacteria? Is there any equipment (Turbidimeter or Spectrophotometer) I could purchase that will not be too costly?

For an antibiotic resistance experiment involving several generations. Do I count all cells or just the viable cells?

Thanks,
Sebastian

Moderator note: I combined your two posts since they seem directly related to the same project. Please keep your related posts in the same thread so the experts helping you can keep track! Thanks!

SciB
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Re: viable bacteria count. Turbidimeter or Spectrophotometer ?

Postby SciB » Thu Oct 05, 2017 5:53 pm

Hi Sebastian,

The method for counting bacteria used by most microbiologists is making serial dilutions, plating the dilutions on agar and counting colonies. Using a turbidimeter to measure bacterial growth by optical density at 600 nm is a valid way to compare cultures but it will not tell you how many bacteria per mL you have in a culture--only plating will do that.

If done correctly and carefully, making serial dilutions and counting colonies on agar plates is a highly accurate way to determine the number of E coli per mL in a culture. However, there are several sources of error in the method that you have to be aware of. If the agar is too wet or too dry, or spreading of the drop of culture is done for too short or too long a time, these can cause the number of colonies to appear larger or smaller than they actually are. Pipetting errors in making the serial dilutions are a common source of inaccuracy in bacterial counts.

I would suggest watching some of the Youtube videos that show how to do serial dilutions and spread bacteria on agar:

https://www.youtube.com/watch?v=Ppe_bgnPFHU

If you need further help with the methods, please let us know.

Sybee

cincibuddy99
Posts: 5
Joined: Fri Sep 09, 2016 11:18 am
Occupation: Student

Re: viable bacteria count. Turbidimeter or Spectrophotometer ?

Postby cincibuddy99 » Mon Oct 09, 2017 8:37 pm

Thank you Sybee! That was very helpful.

cincibuddy99
Posts: 5
Joined: Fri Sep 09, 2016 11:18 am
Occupation: Student

Re: viable bacteria count. Turbidimeter or Spectrophotometer ?

Postby cincibuddy99 » Tue Oct 10, 2017 8:10 pm

Hello,

I just have 2 questions if you have enough time to answer:

1. I don't want to do the typical experiment with the circular filter papers with antibiotic and measuring te areas of inhibition. I would like to plate the e-coli in a petri dish, then use a spreader to spread it evenly and then pour the antibiotic on top. Then after 24 hour I would like to count the remaining surviving bacteria using the standard plate counting (trying different dilutions). After that I would like to collect the surviving bacteria to use them for the next cycle(generation). Is this possible?

2. I like microbiology. If #1 is not possible, are there any other good ideas I can use for my experiment? I'm a High School freshman.

Sebastian

SciB
Former Expert
Posts: 1352
Joined: Fri Feb 01, 2013 7:00 am
Occupation: Retired molecular biologist, university researcher and teacher

Re: viable bacteria count. Turbidimeter or Spectrophotometer ?

Postby SciB » Sun Oct 22, 2017 8:20 pm

Sorry I missed your last question which was buried way down in the list.

I think what you re aiming at in this project is to determine how many bacteria have become resistant to an antibiotic--is that correct? Accurate counting will be necessary to calculate the percent mutation to antibiotic resistance. It will be interesting to compare different antibiotics to see which ones have the highest resistance rates.

You could also tie in the affects of a mutagen on the rate of resistance. I don't know which substance to use as a mutagen but it would be most interesting if there was a compound that the bacteria would be commonly exposed to that would increase the likelihood of their acquiring antibiotic resistance--maybe some other drug. You could do a search online of scientific publications and see if any research has been done in that area and what they found.

Hope this helps. Post again and i will try to respond right away!

Good luck,

Sybee


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