Polymicrobial infections in mice. Bacterial cultures were grown overnight and then subcultured the following day into fresh media appropriate for each bacterium.Three milliliters of each culture was pelleted and washed three times with phosphate-buffered saline (PBS) to remove residual medium and toxins. Bacterial suspensions were diluted in PBS to give a final concentration of 2×106 to 3×107 CFU/ml (as determined by rial dilutions and plating of the PBS suspensions of bacteria). The polymicrobial infections for the young and aged diabetic mice were parated into eight experimental groups, as shown in Table2. One hundred-microliter samples of each bacterial suspension (2×105 to 3×106 CFU), singly or in combination, were injected subcutaneously into the inner thighs of the mice, as previously described (7). Each experimental group (Table2) was comprid of 24 mice. At 1, 8, and 22 days postinjection, eight mice from each experimental group were euthanized by cervical dislocation following the induction of deep anesthesia with 100% CO2 gas. Six mice were ud for the determination of the number of CFU in the injection area; the remaining two mice were ud to asss the pathology of the injection site. Blood from all mice was removed from the caudal vena cava and/or directly from the heart and placed i
n blood vials containing EDTA. The samples were nt to a reference labo-ratory (Ani Lytics, Inc., Gaithersburg, MD) for determining complete blood cell counts.
From six of the mice, the tissue surrounding the area at the site of injection was excid and the spleens were removed. Each sample was homogenized using a tissue homogenizer and resuspended in a final volume of 1 ml of PBS. Appropriate dilutions were plated on a lective medium specific for growth of each of the three organisms ud. E. coli-containing samples were plated on eosinmethylene blue agar (Becton Dickinson, Cockeysville, MD) and incubated aerobically at 37°C overnight. B. fragilis-containing samples were plated on Bacteroides bile esculin agar (Becton Dickinson, Cockeysville, MD) and incubated anaerobically at 37°C for approximately 2 days. C. perfringens-containing samples were plated on either trypto-sulfate cyclorine agar ba (EM Science, Gibbstown, NJ) or Shahidi Ferguson Perfringens agar ba (Becton Dickinson, Sparks,MD) and incubated anaerobically at 44°C for 8 to 12 h.
The remaining two mice were necropsied, and the area of injection and spleens were exa
mined for pathological changes in the infection. Five-micron-thick ctions of the abscess area were stained with hematoxylin and eosin and mounted on microscope slides.
Experimental protocols involving mice were examined and approved by the Virginia Tech Institutional Animal Care and U Committee.
