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Measuring OD for a bacteria culture - Directly or diluted? (Feb/09/2009 )

Dear all,

First off, I read in a paper which mentioned "culture from the log phase (OD600=0.8)...". I'm not sure if that reading was a diluted reading or one measured directly.

I cultured my bug for about 28 hours to obtain the growth profile. I wanted to measure the dry cell weight but since I'm waiting for the freezing to complete, I took the OD reading directly. The mid log phase is about 14 hours with an OD600nm of about 1.9...

Did I do anything wrong? Do I have to dilute the culture before taking a reading?

When we say we "standardize" our inoculum for a batch culture, it's standardized ad libitum right? I mean, I can set the inoculum size as I wish and maintain it for every flask? Does 2 mL of culture at OD600nm= 2.0 sound like a standardized inoculum for my batch culture?

I know it's a loaded one. Thanks very much.


the reading is direct, undiluted, or it would have been mentioned in the text.

what do you blank against? it should be against un-inoculated medium.

also keep in mind that there may be some differences in the readings with different spectrophotometers (for various reasons).

also keep in mind pathlength. most cuvettes have a 10mm path.


If I would have to guess, OD =0.8 is the reading of the culture at full concentration.

OD readings pass 1.0 aren't accurate anymore (it has passed the linear phase of the spectrophotometer), and you need to dilute the culture to calculate readings of that density.


You need to look at the dynamic range your spectrophotometer is capable of reading accurately. If you are above the dynamic range limit, the readings cannot be trusted, and you must dilute your sample.


Ok. Thanks a lot.

For the record, I'm using a Hitachi U-1900



I would dilute already from 0,6-0,7 on 1:10 because I saw in my experiments that already from this density there is a mistake in the measurement when you do not dilute...


Should I use a blank (medium) before measure the OD?!


Ivanov_br on Aug 27 2009, 03:30 PM said:

Should I use a blank (medium) before measure the OD?!

yes you have to

-little mouse-