Here is a good reference.

Breaking down the wall: Fractionation of mycobacteria

Mandana Rezwana, Marie-Antoinette Lanéelleb, Peter Sandera, c, Corresponding author contact information, E-mail the corresponding author, Mamadou Dafféb

J Microbiol Methods. 2007 Jan;68(1):32-9. Epub 2006 Jul 12.

Hope it is useful.

Sandeep

You could try heat inactivation or UV irradiation - you have to grow the "inactivated" bacteria for 4-6 wks on agar before you can safely remove it. The issue with sonication is the possibility of generating an aerosol if you are using a probe - it will of course have to be done in a hood. Consult your safety officer.

thanks a lot for the answers , just wonder Ian Teo if the heat inactivation ( 80 C' for 20 min) is enough to kill mycobacteria coz there is a lot of articles debating the efficiency of heat inactivation for killing mycobacteria. I am planning to plate them on agar plates for 4-6 weeks before moving the bacteria outside P3 lab.and if some one could help me with optimum Sonication time and power to start isolating good yields of M.tuberculosis DNA for Whole genome sequencing .

Our dept use 50 min at 80C. If you are going to extract DNA for sequencing, can you not prepare the DNA in the P3 rather than taking the material out? Sonication may not be good idea if you are doing sequencing, as it is can shred your DNA.

if you do not open the tubes, you can take them out of from the BL3 lab and directly sonicate and then heat.

could somebody help me with an optimum sonication protocol for mycobacterium tuberculosis ?

Sonication of virulent Mtb in a P3 lab seems contradictory to the standard operating procedures of minimizing aerosolization.

The kill methods we use vary dependent upon culture volume and downstream experiments. Variations that we use are as follows:

1. 80C for 20mins for culture volumes up to 0.5mL

2. Incubation with 5% formalin for 1hour

3. Addition of phenol or 1:1 phenol:choloroform for 15mins

4. Addition of 1:128 vesphene for 10mins

5. Bead beating (2-3 45sec cycles at 6,000 rpm) of culture followed by 99C incubation of supernatant with SDS loading buffer for 20 mins

See link for a publication on heat killing.

It should be noted that any kill method for virulent Mtb should be verified independently (ex. CFU plating) in your lab on your equipment before making it a standard practice.

Article The efficacy of the heat killing of Mycobacterium tuberculosis

BCG (risk group 2) can be handled in BSL2 level  but not M tuberculosis (Risk Group 3)

Just Be carrefull when you want to transfer M tb from BSL3 to low level.

Sonication is not recommanded, it produces a lot of aérosols and its depend on your aparatus , power and probe. You can heat your suspension first and sonicate after for more cells lysis.

In our BSL3 facility, we perform a heating first ( 100C for 25 minutes) and after we sonicate for 5 minutes. and then we proceeed to BSL2 level. In each experiment, we include a strain of H37Ra as inactivation control, that is incubated in liquid MGTI 960 tube for at least 6 weeks. We never had a positive culture on these control tubes.

Some poeple who recommended UV irradiation, this procedure is not efficient.

Hi Nader,

 we heat kill M. tuberculosis at 90oC for 45 minutes. This has been validated to show no live organisms. Then it is safe to to take out of BSL3 Lab. The organisms then can be used for DNA and sequencing.

Thanks for all the tips! This was extremely helpful