Hello Christopher.

Your medium turned into orange yellow, meaning too many

Streptococcus equi bacteria in the co-culture with immune cells.

Does your sterile control mean immune cell without the bacteria? You need this and medium alone without immune cells and the bacteria to see what went wrong.

Found a similar work mouse BMC + bacteria co-culture. Have a look at the link. http://iai.asm.org/content/78/2/854.full.pdf+html

Regards.

Thank you for the answer.

I grow the bacteria in the medium without BMMCs (sterile, just medium). And there are not many bacteria, I started with an OD of 0.05 and they grew to 0.15.

The link is from my lab and I am working exactly on this project. but the paper does not include growth experiments in serum containing medium.

Hi Christopher.

That really embarrasses me! However, I have suggestions.

You have two groups: medium alone and co-culture. You should to set up another group: BMMCs alone to monitor any other bacteria contaminants from cell recovery procedure. It often happens since they are primary cells collected from the body.

The possible interpretations would be;

1) The number of BMMCc in your culture. If the BMMCs were cultured at high density in the tube, the nutrients in the medium will be depleted that soon, and the metabolic wastes accumulated within several hours, leading to acidic condition of the medium. This acidity , the lack of nutrients, and the accumulation of waste accelerated arrest of bacteria growth.

2) Other contamination from the collected BMMCs, making the above-phenomena rapidly. This is why you should have BMMCs alone tubes.

3) Transferrin or other components in heat-inactivated FBS would not any harm to your bacteria sub-species. This cannot be a reason.

Best wishes.

It does not have to, there is no way to know that therefore I stated nowhere. I apologize for causing inconvenience.

To make it more clear:

I want to look at the growth in the co-cultivation medium (without any BMMCs involved). So I just have two groups:

Medium (Sterile control)

Medium + bacteria

BMMCs or any other cells are not used in this setup. So there cannot be any contamination from BMMCs. I guarantee the medium only contains bacteria. So the explanation must be either in the medium itself or in the way how the medium is conditioned by the bacteria (e.g. something is secreted that makes the medium more acidic).

Edit: Sorry, above it is "BMMCs" instead of "bacteria".