8 replies to this topic

[prabhubct]

     How did you identify if Mycobacterium tuberculosis is growing or not before 3 weeks. How to differentiate contamination from mycobacterial colony? Thanks.

Edited by prabhubct, 13 September 2012 - 10:06 AM.

[bob1]

You can't easily tell - you could do cell counts or a C14 release assay.

PCR 16s rRNA gene.

[metionina]

If you use a Löwenstein–Jensen medium it's easy: only mycobacteria can grown.
In other kind of media, you can differentiate mycobacterial colonies from other bacteria colonies (that normally you can see already after few days) thanks to the characteristic colony aspect: granular colony, irregular contours (you can see many pictures in the web).
But if you are not sure, you can using the Ziehl–Neelsen stain.

[bob1]

Ah, I was assuming that the OP was meaning distinguish from other mycobacterial contaminants.

[prabhubct]

bob1, on 13 September 2012 - 01:02 PM, said:

You can't easily tell - you could do cell counts or a C14 release assay.

PCR 16s rRNA gene.

Thank you for your reply. How can I detect by cell count or C14 release assay or PCR 16s rRNA gene? how I could differentiate between required colony and satellite colony?

Edited by prabhubct, 18 September 2012 - 08:50 AM.

[prabhubct]

metionina, on 13 September 2012 - 01:03 PM, said:

If you use a Löwenstein–Jensen medium it's easy: only mycobacteria can grown.
In other kind of media, you can differentiate mycobacterial colonies from other bacteria colonies (that normally you can see already after few days) thanks to the characteristic colony aspect: granular colony, irregular contours (you can see many pictures in the web).
But if you are not sure, you can using the Ziehl–Neelsen stain.

      Thank You for your reply.
Mycobacterua grows very slowly. How I can detect if colony is growing or not at very initial stage before 3 weeks? Will PCR be good option? tough surface with tip at specified location and then PCR, it? but plated initial culture  will give positive PCR then.

[metionina]

prabhubct, on 18 September 2012 - 08:50 AM, said:

Mycobacterua grows very slowly. How I can detect if colony is growing or not at very initial stage before 3 weeks? Will PCR be good option? tough surface with tip at specified location and then PCR, it? but plated initial culture  will give positive PCR then.

yes, mycobacteria grow very very slow! The only thing you can do is wait.
I don't think a PCR like you said would work. Wait for some colonies and after that try a PCR or a Ziehl–Neelsen stain.

[bob1]

The Carbon14 release asssay works by having access to a gas chromatograph-mass spec- you put the C14 into the specific medium, and look for release of the C14 as CO2 in the atmosphere above the culture.  I think this would work best as a liquid culture rather than as a  plate with colonies.


PCR will tell you if you have the right species, but you should also do the standard tests for M. tuberculosis- morphology and ziehl-neelson stain (specific for mycobacteria only, not a species).

[Phil Geis]

Mycobacteria are not the only bacteria that grow on Lowenstein Jensen - another is Pseudomonas aeruginosa and other pseudomonads probaly do as well.