Riccardo Muzzi

Greetings.

I am treating an industrial wastewater containing dimethylacetamide, with Electro Fenton processes and I am measuring the concentration of formaldehyde generated, to investigate it as a source of toxicity.

I am using the DNPH-GCMS derivatization method, and obtain coherent data.

Upon trying to replicate the toxic effect with formaldehyde solution, I get stronger toxicity response with the "handmade" solution of identical concentration.

Is it possible that I am observing some interference from other components in the wastewater that increases the reading vs the calibration curve?

The wastewater contains Iron, chlorine and sulfate as the main other components.

I am observing some presence of acetic acid, but if it comes from acetaldehyde the DNPH should react with it too and shoudl exit the GC column at a later time and I would be able to detect it, but none is observed.

Edit: ~~I realised the formaldehyde solution I used for the calibration is stabilized with 10% methanol. I read conflicting reports on the effect of methanol on the derivatization. Could this be the source of the deviation? ~~

Edit: I tested extraction with 1ml sample, 1ml sample/250µl methanol and 1ml sample/1ml methanol to a final volume of 10 ml.

The peak area in the ones additioned with methanol are ~5% and 20% larger than the sample without.

Hello all,

I find myself in need to perform serial dilution of E.coli cells for a luminometric ATP assay.

Normally for serial dilution I would use PBS, but from what I understood the concentration of salts (Na and K) is too high, which interferes with luciferase activity.

Diluting in DI water should cause too much stress, right? Passing from LB growth culture to nothing could cause shock.

Is there a different buffer that keeps the cells alive and well while not providing a too high concentration of monovalent metal ions?

EDIT:

I came up with a possible solution. I could centrifuge 5 ml of the culture broth with 5 ml of 0.1 Peptone water (to retrieve the cells, remove salts and not to shock the E.coli too much) at 5000 rpm in Falcon tubes, discard the supernatant and resuspend the pellet in Peptone water, which I would use for subsequent serial dilution. The time between dilution and test would be ~ 1 hour, so the bacteria would not suffer too much from the change of nutrients/osmotic pressure?

Hello all,

I wish to perform a luciferase luminescent measurement of ATP in E. coli.

I have acquired a kit that requires less than 1 µM of KCl (and other monovalent metal salts). Also divalent metals must be in the ~3 mM range, and EDTA inhibits the reaction.

Normally, I would perform serial dilutions of the cells in PBS, but this raises the concentration of salts too much.

I thought about using Tris-HCl 0.01 M, but the only protocols i found are for cell lysis. I want to lyse the cells but not immediately.

Can i use Tris 0.01 M at 7.4 pH for the serial dilution of my cells without them suffering stress?

Greetings,

I am currently trying to determine the toxicity of the effluents of an Electro-Fenton reactor with macrotoxicity method and colony count.

The concentrations of H2O2 I calculated (maximum 6 mg/l) is very far from the MIC reported for H2O2 against E. coli (between 469 and ~2500 mg/l).

Other components are weak organic acids and 0.2 mM of Iron.

Despite the low concentrations, I am observing a marked reduction in the colonies. Is there a chance that the prolonged interaction, even as such low concentration, between bacteria and H2O2 before plating is preventing them to create colonies?

I need to perform toxicity testing on the byproducts of an electrochemical treatment for wastewater.

To test the effect on E. coli, I prepared serial dilutions of the overnight bacterial culture and had them interact (for 5 hours) with the same concentration treated wastewater in 50-50 diluted broth-treated wastewater. From the mixture, I took 0.1 ml and plated in LB-agar for a bacterial count.

My reasoning being that if the concentration of toxic compounds in the treated wastewater is somewhat effective (acting on, but not completely inhibiting the growth or killing off all the bacteria), the bacterial count would be lower than a negative control with diluted broth-sterile water.

Would this reasoning be sound or am i overlooking some aspect?

Greetings,

i am currently performing an experiment with wastewater to verify the presence of coliforms, with TTC medium and filter paper.

My problem is that after treatment, there are no coliforms found, but lots of blue tinged colonies, whose number is not influenced by the dilution (~ 100 for all three dilutions i used, 10^-3,10^-4 and 10^-5). How could this be?

These blue colonies are only on the filter paper, so i think the medium is not contaminated. I tested the water i used for dilutions with the same filter papers, but there were no colonies on them, so the water is not contaminated.