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measuring cfu/ml using spectrophotometer

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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measuring cfu/ml using spectrophotometer

Postby dreamy » Tue Nov 03, 2009 8:12 am

Hi all,
For my work, I have to perform a new test procedure (EN 1500) to test my company's products. Stated in the test, I have to prepare 1.5-5.0 x 10^8 (or 10^7 for fungi) cfu/ml test suspension according to the standard which stated that I must use 620 nm wavelength. So can anybody advise me how to do it? As for a fungi (A. niger), is there any other way to prepare the suspension or using haemacytometer is easier?
I have limited experience using spectrophotometer and usually measure a suspension at 470 nm so I hope someone can help me. :(
Thanks in advance for your help! I really appreciate it.......
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Postby JackBean » Tue Nov 03, 2009 8:27 am

I think, that the wavelength doesn't matter for how difficult the measurement is, does it? :)

Well, you can prepare the suspension by dilution and measuring OD (in your case OD620), but first you need to do some calibration, so you can assign OD620 to the particle concentration ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby canalon » Tue Nov 03, 2009 5:16 pm

Wavelength are not usually essential, as long as you have a good blank. Since you use the spec just to measure the amount of light that get dispersed, not some kind of change in the absorption of the medium. Usually, it is just a question of habits what wavelength is used in each lab.
The best way to do your work is to prepare a suspension of your culture, count the cells in it and measure the OD at your favourite wavelength of a few dilutions (in the original growth medium) of the suspension. This will allow you to calibrate what wavelength you want to have to get the desired concentration, and to see that you are measuring in the linear range of your machine.

Have fun
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Re: measuring cfu/ml using spectrophotometer

Postby dreamy » Wed Nov 04, 2009 6:46 am

What do you mean about having a good blank? And how to prepare dilutions? As in 10-1, 10-2.......10-8 ? I've found a lab guide http://a-s.clayton.edu/furlong/BIOL3250/lab/objectives_instructions/Labs/Bacterial%20Enumeration08.pdf on how to measure the spectrophotometry but I'm confused by the dilutions used. Can you please explain it more clearly? Thanks....
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