162
- Mice are sacrificed at different time points post treatment.
- Lungs and spleens from mice are removed rapidly by a sterile
autopsy after sacrifice. - The organs are transferred into 2 mL tubes each containing
1 mL sterile distilled water and 2 mm diameter glass beads. - Lungs and spleens of mice are homogenized using a reciprocal
shaker (Thermo Hybaid Ltd) for 40 s at 6.5 speed. - CFU counts and broth counts from each lung and spleen are
performed using serial dilutions of the homogenates (see
Subheading 3.2) and expressed as log CFU/organ for CFU
counting or Log viable cells/organ for broth counting (see
Subheading 3.6). - After 11 weeks of treatment, mouse organs are most likely to
become CFU count free. Therefore, at the late stage of treat-
ment, the entire organ homogenates (the total volume of each
organ homogenate is approximately 1.5 mL including the
organ and 1 mL of water) from 8 to10 mice are aliquoted
equally into three tubes. - Tube 1. CFU counting by addition of the homogenate to
2 mL of sterile distilled water following by plating out the
entire organ homogenate suspension on six selective 7H11
agar plates (see Note 12). - Tube 2. Culturing in 5 mL of selective Kirchner liquid medium
for 4 weeks with subsequent sub-culturing of the entire culture
onto Löwenstein-Jensen slopes for a further 4 weeks (see Note
13 ). - Tube 3. Resuscitation of persistent bacteria (see Subheading
3.6). - Culture negative organs are defined as no colonies grown on
7H11 agar plates and no growth in selective Kirchner liquid
medium following inoculation on Löwenstein-Jensen slopes. - After 8 weeks of hydrocortisone treatment, CFU counts from
lungs and spleens are performed to determine disease relapse.
The CFU counts will be performed by dilution of the organ
homogenizes in a tenfold serial and plating 3 × 100 μL of all
dilutions including the entire tissue homogenate on selective
7H11 agar plates (see Note 14).
Broth counting is performed as serial tenfold dilutions [ 7 , 8 ].- Add 4.5 mL of the culture filtrates (see Subheading 3.1, step
5 ) in 7 mL Bijou tubes. - Add 0.5 mL of in vitro cultures or tissue homogenates in
4.5 mL culture filtrates in triplicate and mix well.
3.5 Assessment
of Infection
and Treatment
Efficacy
3.6 Resuscitation
of M. tuberculosis
Persisters In Vitro
and in Mice
Yanmin Hu and Anthony Coates