Antibiotic Resistance Protocols (Methods in Molecular Biology)

160

1. Mice: BALB/c mice, female and aged 6–8 weeks (Harlan UK
   Ltd.).


2. Rifampicin, isoniazid and pyrazinamide are obtained from
   Sigma-Aldrich.


3. Hydrocortisone acetate is obtained from Sigma-Aldrich.

3 Method

As transmission of M. tuberculosis is via aerosols, workers must be

protected from laboratory acquired infections caused by M. tuber-

culosis. Working with M. tuberculosis must be in Biosafety cabinet

(BC) within the Biosafety level 3 laboratories.

Culture supernatant containing resuscitation promoting factors

(RPF) or 7H9 medium is used as described previously [ 5 , 7 , 8 ].

1. M. tuberculosis H37Rv is grown in serials of 10 mL 7H9 broth
   media in 30 mL screw-capped universal tubes without distur-
   bance (see Subheading 3.2) for 15–20 days until an optical
   density of 1–1.5 (optical density reader, Biochrom WPA
   CO8000) is reached (see Note 4).


2. The bacteria are removed by centrifugation at 3000 × g for
   15 min.


3. The culture supernatants are collected and sterilized by filtra-
   tion with 0.2 μm filter (Sartorius) twice.


4. The sterilized culture filtrates are made selective by addition of
   Selectatab (Mast Diagnostica GmbH) (see Subheading 2.1,
   item 11).


5. The selective culture filtrates are used immediately for broth
   counting of the most probable number (MPN) of the bacilli
   (see Subheading 3.6).


6. Serials of 10 mL 7H9 broth media in 30 mL screw-capped
   universal tubes are inoculated with 1 mL of 10-day M. tuberculosis
   culture (see Note 5).


7. The cultures are incubated in an upright position without
   disturbing for up to 100 days.


8. The numbers of viable M. tuberculosis in the cultures are deter-
   mined by surface plate counts on 7H11 agar.


9. Prior to inoculation, the agar plates are incubated upside down
   at 37 °C for 24 h in order to check sterility and to ensure the
   surface is sufficiently dry.


10. Cultures are vortexing in 30 mL screw-capped bottles with
    1 mm glass beads (VWR UK) for 2–5 min (see Note 6).

2.2 Cornell
mouse Model

3.1 Preparation
of Culture Filtrates
Containing RPF

3.2 In Vitro Hypoxia
Model of M.
tuberculosis Growth

Yanmin Hu and Anthony Coates