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I did this experiment last month using E.coli isolated from raw milk and have following data via uv-spec:

time    OD 66o nm
0  -    0;
2  -    -0.091;
4  -    -0.009;
6  -    0.08;
8  -    0.215;
10 -    0.47;
12 -    0.735;
14 -    0.93;
16 -    1.29;
18 -    1.29;
20 -    1.29;
22 -    0.96;
24 -    0.88;

The control and the growth media were Luria broth with the addition of casein and lactose 2% w/v. Data triplicated; average used.

I would like to know the reasons about this phenomenon. My inferences were:

  1. Exhaustion of the casein and lactose by bacteria during lag phase.
  2. Instrumental and procedural errors.

I was planning an enzyme activity assay, but stopped, thinking that the readings would be a reflection of procedural errors.

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thanks @Armatus for the corrections. Well, I'm a newbie here. –  Tripti Sharma Mar 12 '13 at 15:45
1  
Could you explain which aspect of the data you are concerned about? Apart from the odd values at the beginning of the series the A660 values show a pretty good log phase followed by a stationary phase. The fall in A660 in the later time points could be due to a bit of lysis but it could also be due to clumping. Is there any reason to think that E. coli will use casein as a nutrient in these conditions? –  Alan Boyd Mar 12 '13 at 16:46
    
I'm only concerned about the aspect where it shows the negative values. And I think that it must utilize casein (to a certain extent) because I isolated it from raw milk. –  Tripti Sharma Mar 12 '13 at 17:13
1  
The negative absorbance may be due to changes in the optical properties of the solution because of small changes in LB concentration. i don't think caesin protein alone would change absorbance of the baseline. –  shigeta Mar 12 '13 at 20:54
    
What is the concentration of casein in the medium? Does it contribute significantly to A660? What exactly was used for the inoculation of the culture? I don't think that the negative values are meaningful in terms of the growth of the cells - they will be caused by something changing in the medium during the initial part of the incubation. Even if the cells are able to use casein as a carbon source they will first use any glucose in the LB (there is some, from the yeast extract) then the lactose, and that would be enough to generate the growth curve that you have produced. –  Alan Boyd Mar 13 '13 at 11:06
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