Hello,
I have a question related to an in vivo infection assay I'm doing. My experiment involves infecting mice with bacteria via intraperitoneal injection. At a given time point, the mouse is sacrificed and a peritoneal lavage is carried out to collect macrophages in the peritoneum. I then process this peritoneal lavage fluid to look at the amount of free bacteria and bacteria present within cells. I then process these two samples and I do serial dilutions of the bacteria to determine the CFU/ml.
I'm having issue with the serial dilutions. My dilutions are serial 1 in 10 dilutions however, my plate counts do not reflect this in some cases. For example at a serial dilution of 10^-3 I may count around 130 bacterial colonies. However at the next diltuion (10^-4) I count around 100 colonies. I have tried vortexing each dilution before proceeding to make the next and I vortex my dilutions before plating them on circle pates to hopefully break apart clumps of bacteria. Has anyone else come across a similar problem? Are there any good techniques that can aid in producing cleaner results with bacterial serial dilutions?
you can think on this technique
https://www.researchgate.net/post/How_to_perform_cell_counting_bacteria_using_hemocytometer
https://logosbio.com/automated-cell-counters/microbial/bacteria/quantom-tx
Hi Subothan,
I do not have a direct answer, except that I have seen this effect as well. One of the solutions is to change tips after each transfer which I am going to test next. At best I have seen 8-9 fold reduction after dilution when the dilution factor has been 10. Everybody seems to know of the phenomenom, but nobody has given me a clear explanation of it. So, looking forward for some peer-wisdom on the matter.
I have tested:
- Diluent media (broth or PBS)
- Dilution scale (at 250 ul, 500ul and 1000ul)
- Dilution factor (1/5, 1/10)
- Using vortexing vs mixing with pipette.
No obvious improvements, sometimes a bit better, more often not...
Abhijeet's suggestion is a fair one. However I would suggest carefully reading critiques of the staining based viability determinations such as this one:
Article Critical aspects of using bacterial cell viability assays wi...
We have never had problems getting accurate counts when doing serial dilutions. How are you distinguishing between free and engulfed bacteria? Are you spinning your lavage down and then plating the supernatant vs. the cells? Do you have the same issue when plating the supernatants and the cells? Also are you lysing your cells before plating them? If not, you may have cells that are clumped together. When trying to enumerate intracellular bacteria, we used to lyse the cells (PBS.1%SDS.0.1%Triton-X) before doing serial dilutions. Like Juha suggested, make sure you plate immediately after vortexing and go from the highest dilution to the lowest dilution. And change pipette tips and L-spreaders between each dilution.
Juha Kotimaa One of my volunteers has also tried altering the dilution scale, we got better results but it was still a problem. And yes I do change tips between dilutions. Thanks for your insight, I will definitely provide any updates on technique, if I see improvements in results.
Julie Joseph Yes, I spin my lavage, separate the supernatant from the cells and lyse the cell pellet with Triton as well. And yes the issue is present in both supernatant and lysate. Thanks for the input! I will be repeating this experiment soon and hopefully will get cleaner results!
Please read the following article:
Article Measuring Bacterial Load and Immune Responses in Mice Infect...