Hi all
I am currently working with Mycobacterium tuberculosis and need to disrupt Mtb clumps/generate single cell suspension.
Stock is defrosted, centrifuged, the supernatant removed and the pellet resuspended in PBS. Glass beads (2, 3 or 5mm) are added and then container is shaken and vortexed. Solution is allowed to settle for short time (to allow glass beads to sink to bottom) and the supernatant is removed.
I have yet to perform this as I am currently investigating the best possible way to generate single cell suspension. So, is this the best way? Should the glass beads be added in solution or once supernatant removed (1st time) should glass beads be added without PBS - then container vortexed? Does the solution need to settle to allow glass beads to sink to the bottom? (I am also looking at filtration and sonication, but this question pertains specifically to the use of glass beads).
Any help is appreciated.
Hi Micheael,
The way we use glass beads for producing single cell suspension is to add resuspend your bactrial pellet in 0.2ml PBS. Add in your glass beads 2.0mm ( about 50). Stir into the inoculum well. Vortex for 30 seconds.
Leave for 30 minutes for aersols to settle.
Depending on your starting inoculum , add either 0.8ml of water to make 1ml or 9.8ml to make 10ml.
Then vortex briefly again before using.
There are either sonicators that will give you single cells without killing the bacteria or the other alternative is a sonicating water bath which works just as well.
hope that helps
Jasvir
Please follow this link;
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418728/
Hi,
Its dépends what are you doing with single cell suspension:
For microbiology work, inoculum preparation...etc , We add glass beads before adding buffer or water, vortex for 30 - 60 s, resupend.
For FACS, macrophage or other cell infection, we use sonicating water bath followed by syringing (at least 20 time by paasing through insulin syringe). it gave a good single cell suspension that we confirmed several time with confocal microscopy.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology