I have performed anti-mycobacterial assay, but the colonies in the plates are not seperated. Therefore I could not count the colonies.
1) I have used LB agar plate, should I use MH agar plate?
2) Is is necessary to pass the culture from a 26-30 gauge needle for a proper suspension culture?
With regards
Thanks
Is it necessary to count the colonies? Will it be okay if you find out the anti-mycobacterial activity instead? Because to find out the activity, you just need to M.I.C. assay. I am not working on Mycobacteria, but still.
Hi
The anti-mycobacterial method assay is different anti bacteria. In my work to use the method in attache file
Dear Praksh
For colony counting you must have given a brief bath sonication treatment to break the clumps or you may pass through syringe. Colonies generally appears merged. So you have to identify the ridges bulding above makes one colony. Then you may be able to count.
Or elase you may dilute your suspension by serial 1:10 dilution and calculate the isolated colonies in plate and calculate accordindly by multiplying with dilution factor.
Which Mycobacterium you are using ?
Middlebrook 7H10 is recommended media.
Hope this helps!
I guess you are working on M.smegmatis as you used LB plates, to reduce the clumping of mycobacteria you have different choices
1- sonication.
2- passing the culture through small needle
3- adding Tween 80 to LB broth you used.
4- M.smegmatis is fast-growing mycobacteria so do not leave it more than 3-5 days in the incubator
5- dilute your bacteria
Hopefully this might help
Hi
You can use Middelbrook 7H10 plates to count your colonies, the critical point is to standardize your inoculum.
We use the sensititre (TREK Diagnostic) protocole for MIC determination for slow and rapidly growing mycobacteria. The test is done on MH (or 7H9) liquid media, but the counting is done on 7H10 plates.
Briefly, colonies are resuspended in dry glass beads, separated by vortexing for few secondes
the culture is prepared to 0.5 McFarlan (you can also add a turbidity step to be sure)
Dilute to 5. 10e3, inoculate (100 ul) in each well,
For counting we dilute this suspension 1/10 and plate 100 ul.
Good luck
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