Hi,

on the basis of what you are saying, I think that the problem could be the accurancy of your spectrophotometric measurements; I mean, when the OD value exceeds 1 (one), then you have to dilute your sample. If you measure 0.968 and 0.843 by diulting 2 times it means that your cell colture have an OD of about 2.1 after 3 hours. However, If you measure 1.159 and 1.340 by diulting 2 times after 8 hours it doesn't mean that your cell colture have an OD of about 2.1: the reason why by diluting the same sample 10 (ten) times you obtain an OD of 0.385 and 0.390 is that your real OD value after 8 hours is about 3.9, that I think that could be a good value in LB medium. 

Anyway, your data are suggesting you that the growth rate of wt and mutant is approximately the same!

I hope to be helpful!

Nicola

Hi,

on the basis of what you are saying, I think that the problem could be the accurancy of your spectrophotometric measurements; I mean, when the OD value exceeds 1 (one), then you have to dilute your sample. If you measure 0.968 and 0.843 by diulting 2 times it means that your cell colture have an OD of about 2.1 after 3 hours. However, If you measure 1.159 and 1.340 by diulting 2 times after 8 hours it doesn't mean that your cell colture have an OD of about 2.1: the reason why by diluting the same sample 10 (ten) times you obtain an OD of 0.385 and 0.390 is that your real OD value after 8 hours is about 3.9, that I think that could be a good value in LB medium. 

Anyway, your data are suggesting you that the growth rate of wt and mutant is approximately the same!

I hope to be helpful!

Nicola

Dear Mr. Nicolas, 

Thank you very much for your instructions. I will try to dilute more to get the appropriate numbers. If I have another problems on this experiment. I would like to receive your advice again. 

Hi there,

Depending on the spectrophotometer, it is advised for bacterial growth monitoring to read absorbance not exceeding 0.6 in order to get reliable and quantitative readings. So during growth you have to dilute your culture adequately to respect this and then use the dilution factor to finally get true numbers.

I conquer with Dominiquer Liger on your quiz

Yes, you have to dilute continuously your samples to read absorbance not exceeding 1, but it well be better if it in the range 0.5-1 OD.

Dear Mr. Dominique, Mr. Eric, Mr. Asem Shehabi. 

First of all, thank you very much for your advice. Actually, I tried to dilute the sample to get the OD600 less than 1. However, there are some opinions that dilute to the OD600 from 0.1-0.5 is more accurately, some suggested the OD600 should be from 0.5-1. I tried all ( for example: one with 3 fold dilution and one with 5 fold dilution and then calculate the real numbers), and it is nearly the same with variation ranges around 10%. 

Nevertheless, I also faced the problem with how to collect the sample correctly for accurate measurement. For example, I tried to collect the 2 samples after 16 hours of E.coli inoculation and measure, each time it gives the different values with high variations ( For example. sample 1: OD600= 0.471 (8 folds dilution) ; sample 2: OD600= 0.505 (8 fold dilution) . 

0.471 is not that much different from 0.505... What you can do is to calculate the average from a serie of measurements to limit errors.

When withdrawing aliquots from a culture you have to make sure of its homogeneity so don't hesitate to shake the flask or tube right before withdrawing. The same has to apply when diluting and when measuring OD...

About OD linearity window, it depends on the spec you use. Personnally I always prefere reading OD under 0.5 and therefore apply dilution as soon as OD reaches this limit.