I am trying to grow up some Mycobacterium thermoresistibile for some experiments, and have got the freeze dried stocks but don't know how long to plate them for or how long to subsequently grow them up in liquid media for to make glycerol stock. Has anyone got any experience working with the organisms or any ideas? I intend on growing them in Middlebrook 7H9 broth and Middlebook 7H10 agar.
Replication Dynamics of Mycobacterium tuberculosis in Chronically Infected Mice†
Ernesto J. Muñoz-Elías,‡ Juliano Timm,‡ Tania Botha,§ Wai-Tsing Chan,¶ James E. Gomez, and John D. McKinney*
ABSTRACT
The dynamics of host-pathogen interactions have important implications for the design of new antimicrobial agents to treat chronic infections such as tuberculosis (TB), which is notoriously refractory to conventional drug therapy. In the mouse model of TB, an acute phase of exponential bacterial growth in the lungs is followed by a chronic phase characterized by relatively stable numbers of bacteria. This equilibrium could be static, with little ongoing replication, or dynamic, with continuous bacterial multiplication balanced by bacterial killing. A static model predicts a close correspondence between “viable counts” (live bacteria) and “total counts” (live plus dead bacteria) in the lungs over time. A dynamic model predicts the divergence of total counts and viable counts over time due to the accumulation of dead bacteria. Here, viable counts are defined as bacterial CFU enumerated by plating lung homogenates; total counts are defined as bacterial chromosome equivalents (CEQ) enumerated by using quantitative real-time PCR. We show that the viable and total bacterial counts in the lungs of chronically infected mice do not diverge over time. Rapid degradation of dead bacteria is unlikely to account for the stability of bacterial CEQ numbers in the lungs over time, because treatment of mice with isoniazid for 8 weeks led to a marked reduction in the number of CFU without reducing the number of CEQ. These observations support the hypothesis that the stable number of bacterial CFU in the lungs during chronic infection represents a static equilibrium between host and pathogen.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC538940/
I work with non-pathogenic mycobacterial species such as M. amegmatis, M. bovis BCG and others. They are generally slower growing than other common lab bacterial such as E. coli. Some mycobacteria require additional supplements in the media. Are you using ADC or OADC in your growth media?
I am using OADC in my media, and have discovered that it sticks to the side of falcons unless 0.05% Tween-80 is supplemented in the media.
Hi,
M. thermoresistibile is a moderatly rapidly growing Mycobcterium (slowly growing M tuberculosis can take more than 21 days). From freezed stock stock it will take 5-7 on solid 7H10 or 7H11, and 3 to 5 days in liquid media (7H9+OADC).
To avoid clumps formation, use Tween and orbital shaking of your culture, You can also stop the culture when reaching 1 Mc Farland standars (availaible commercially or you can prepare it in house). Plate some of your aliquots on Blood agar and incubae at 37 to check fot the purity.
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