To begin, unclamp the body of the sonicator converter unit and clean the probe with a 70% alcohol swab. Then, switch the sonicator on, and adjust the power setting to 4, by turning the power control knob.
Next, to generate a fine suspension of the Mycobacterium tuberculosis H37Ra in PBS, immerse the probe's tip into the PBS-containing mycobacterial powder. Sonicate the mixture on ice for 30 seconds, then, pause for 30 seconds, and repeat for a total of 5 minutes to fully disperse the powder into an even suspension without heating the liquid. Next, add 2.5 milliliters of Freund's incomplete adjuvant to the mixture, and repeat the sonication process on ice until the emulsion forms a toothpaste-like consistency.
For the subcutaneous injection, load 200 to 300 microliters of the mycobacterial emulsion in a 1-milliliter syringe. Expel the air from the syringe, and continue filling the syringe, with intermittent inverting and tapping, until filled.
Next, place the subcutaneous injections on either the dorsal surface of the hips, or on the ventral surface of the legs proximal to the region of the inguinal lymph nodes of a 6 to 10-week-old, anesthetized, C57Black/6J mouse. Carefully insert the needle, taking care not to penetrate the muscle, and inject 50 microliters of the emulsion into the subcutaneous space. Do not remove the needle immediately to allow the thick emulsion to be fully injected.
