Additional reagents and equipment for euthanasia (unit 1.8)

1. Prepare L. monocytogenes (see Basic Protocol, steps 1 to 2), but adjust concentration to 106 bacteria/ml.

2. Inject mice as described (see Basic Protocol, steps 3 and 4).

Although inbred mice have predictable responses to infected L. monocytogenes, there is still significant animal-to-animal variation in the clearance of bacteria. Therefore, it is best to use at least three, but preferably five, mice per group for experiments comparing the degree of specific immunity.

In this case, the 0.2-ml inoculum will contain 2 x 105 bacteria. This dose of L. monocytogenes represents ~10 x the LD50 for L. monocytogenes strain 10403s in BALB/c mice, and thus is only appropriate for mice that have some degree of immunity.

If desired, naive mice can be used to examine specific and innate immunity to L. monocy-togenes by injecting mice i.v. with a sublethal dose of bacteria (see Basic Protocol).

3. Place mice in a BL-2 animal facility for 24 to 48 hr.

Following a large dose of L. monocytogenes, mice should be monitored at least daily for illness (ruffling of fur, lethargy), and moribund mice should be euthanized.

4. Place stainless steel wire mesh screens into two separate 50-mm sterile Petri dishes, each containing 5 ml of 0.05% Triton X-100.

5. At 24 to 48 hr after L. monocytogenes infection, sacrifice mice by cervical dislocation (unit 1.8), remove the spleen and liver, and place each organ on a separate sterile wire mesh screen.

If desired, assessment of the competence of innate and specific immunity in naive mice should be performed 48 to 72 hr and 7 to 10 days after infection, respectively.

6. Use the rubber plunger of a 5-ml syringe to press the spleen and liver through the wire mesh screens, and mix the Triton X-100 and tissues.

The organs will disperse into the Triton X-100 and should be mixed to form a homogenous solution.

7. Perform three serial 1/10 dilutions of the organ homogenates. Prepare three sterile 12 x 75-mm polystyrene tubes containing 0.9 ml of 0.05% Triton X-100. Add 0.1 ml homogenate to the first tube, mix, and then transfer 0.1 ml to the next tube. Repeat dilution to the third tube.

8. Place 10 |l of each dilution in the middle of a fresh but dry BHI agar plate and disperse the cultures with a sterile glass spreader. Place the plates in a 37°C incubator and incubate 24 hr.

9. Count colonies on each plate. Factoring in the dilution and volume plated, calculate the total number of bacteria per organ (see Anticipated Results).

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