If you observe significant cell lysis in E. coli culture first of all test your inoculating material for a phage contamination. Also something could be wrong with your culture medium. Normally there should not be cell lysis up to stationary phase

Glycerol sometimes works, but in my hands the effect is not dramatic. You can try...

If you observe significant cell lysis in E. coli culture first of all test your inoculating material for a phage contamination. Also something could be wrong with your culture medium. Normally there should not be cell lysis up to stationary phase

You should definitively check for a contamination as Marina already said. Normally cell lysis does not occur in the extent you are mentioning. I kept cultures active for several months in the fridge without excessive lysis. If there is no contamination I would suggest, if possible, to reduce the amount of nutrients to make cellular growth a bit slower. Worked fine for me, but you have to add extra nutrients after approx. 24-36 hours of growth to keep your cells highly active.

Of course, I agree with the comment about possible phage contamination!!.

Agree with Marina.

Not entire sure if I understand the question clearly. if the lysis happened during stationary phase, it was normal. You may reduce the inoculated cell number so the stationary phase could come later.

In addition, is that because your control cells produce some intrinsic proteases at the early phase even during exponential-phase? In this case, you may need to

1) change cell line,

2) refresh medium more frequently

3) restrict this enzyme synthesis by removing essential elements for the synthesis or inhibit the activity by adding inhibitors, which doesn't affect your target enzyme synthesis and activity.

Also, I wonder if you have added any reagents for the activity test to the medium during culturing of cells? May those reagents be toxic to cells?

First of all, repeat everything again with a fresh batch of cells coming from your cell stock, like Nilabh Suggested. Still having problems?

Make a control without adding your reagent. Check for lysis using a method that will detect molecules that should be inside cells and not in the medium :). If your control gives different results, then your reagent (or its solvent) may be causing some stress. If the results are not significantly different, then the problem lies elsewhere.

Check for phage contamination as suggested above (good one by the way, Marina).

Re-check your pH, temperature (to high will increase lysis), salinity of the medium. Cells have to feel comfortable, so avoid stress conditions.

Do not decrease the amount of nutrients, as stationary phase will come sooner and not later.

Does your medium also contain antibiotics? or ROS-generating compounds such as heavy metals? These will cause lysis even during exponential phase.

Take care!

It means the enzyme is extracellularly secreting in the broth. Well, it may be unusual to notice the lysis of E.coli in the early phase of growth. Sometimes it could be due to medium components used would cause the production of unknown organic acids during exponential growth phase causes autolysis. May be mother culture got contaminated with the E.coli specific phage or look for the pH of the enzyme assay buffer or the Assay methodology. If the enzyme under investigation is assayed (spectrophotometrically) by using synthetic substrates one has to look for standardized assay method (particularly for ionic strength of the buffer, pH, temperature and duration of assay).

If phage contamination isn't the problem (which I really hope for you)

you should TEST YOUR HYPOTHESIS that spontaneous lysis of the bacterial cells during the growth is the reason for your positive "negative controls".

You could lyse different ammount of E.coli and check if you "signal/activity" is related to the ammount of lysed cells, which should be the case, if cell lysis is the reason.

Using a different E.coli strain might also be an option, because the E.coli you are using might be secreting an enzymatic activity and another strains may be not.

Check "empty broth" as negative control to check for the point Shaik mentioned.

Best Kai

I strongly agreed with Marina. Please check for contamination with other lytic enzymes.

Just make sure that the conditions your working on is sterile and repeat the experiments and see what happens, making sure that everything you are using is sterile of any kind of contaminants.

In agreement with the other suggestion, probably you could add a little glycerol (1% or so), provided that does not affect your colorimetric assays...

Is this result different from previous tests or is this new to the lab? If it is a repeat - has the growth conditions changed? gas sparge rate? rotation rate of platform? different flask? also I would check all of the raw materials - has a vendor, lot or preparation changed? All of these can lyse cells. If this is a new test and everything checks out as sterile, then I would suggest adding antifoam to approximately 10 ppm.

Thanks to all for your contributions. Curiously, we have already solved our problem just by diluting 1/10 the medium we used in our enzymatic assays with a non-selective medium. Apparently the original medium components were the cause of the autolysis observed during the exponential growth phase. since this was a commertially prepared medium, we are not sure of the total composition of this medium.

The culture medium should be checked for the pH. Alkaline culture medium will facilitate the lysis of the bacterial cells. So, if the culture broth is alkline in pH, use acid to neutralise the medium.