I have fed the 2-3 weeks old swiss albino mice with E. coli (by gavage) and am trying to get a count of these bacteria that has caused infection in the colon region. After oral gavage the mice were sacrificed at day 7. I tried homogenization of the tissue with beads but after plating the homogenate, there was no bacteria at all. Is it possible that the bacteria did not infect at all? or the technique of homogenization was not correct? In that case:
How can I homogenize the tissue so that only the mice cells are lysed but not the bacterial cells; so that I can get a plate count of these bacteria on LB agar plates (CFU/mL) ?
(1) Is sonication a right method for such kind of lysis, then what amplitude can be used for the same?
(2) Or can the cells be burst open to release the bacteria with specific concentrations of Triton-X-100 or SDS?
(3) Or the method for bursting the cells with beads could be the right way for the experiment?
(4) Can any other method be used for the lysis without affecting the bacteria within?
Hi
If you do not want to destroy bacteria cells only bursting the cells with beads could be the right way for the experiment. 1 and 2 can cause lysis of bacteria,
Jarek
Remember that any procedure used will cause some kind of bacterial cell lysis, the important thing is the comparison with your negative control (murine tissue without experimental infection), the important thing in your case to my particular judgment is the viable bacterial colonies count Get, you can according to the availability of resources with which you count, compare between techniques and make a decision. I think sonication is a very aggressive procedure. Regards!!!!!!
José
Hi Sathi,
Generally a tissue homogenizer is used to homogenize the tissues. Thereafter you can serially dilute the homogenate and plate it preferably, using Miles and Misra method (https://en.wikipedia.org/wiki/Miles_and_Misra_method). E. coli used in labs is usually non-pathogenic i.e., it is unable to colonize and thus cause infection. So it could have been cleared by normal digestive processes. Increasing the inoculum (challenge dose, cfu/mL) might help.
AK
Thank you Jaroslaw and Jośe. I will definitely avoid the procedures including sonication and detergents.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Histological Techniques