This study outlines a method for quantifying antibiotic-resistant bacteria in mouse tissues following chronic intestinal infection. It details the procedures for tissue collection, homogenization, and bacterial plating to estimate viable bacterial loads.
Begin with a euthanized mouse infected with an antibiotic-resistant bacterial strain that models chronic intestinal infection.
Expose the abdominal cavity to access the cecum and spleen.
Harvest the cecal and splenic tissues, which represent intestinal and systemic sites of bacterial colonization.
Place each tissue into a separate tube containing a buffer and a steel bead.
Homogenize the tissue by rapidly agitating the tubes, causing the steel beads to collide with the tissue, break it down, and release bacteria into the suspension.
Perform serial dilutions of the homogenates in the buffer to reduce bacterial concentration.
Plate aliquots from each dilution onto nutrient agar supplemented with an antibiotic to selectively recover antibiotic-resistant bacteria.
Incubate the plates to allow viable bacteria to grow into colonies.
Count the colonies to estimate the number of viable bacteria per gram of tissue and compare bacterial loads in the cecum and spleen.
First, set out 2 milliliter safe-lock round-bottom microtubes, add 1 milliliter of sterile PBS and an autoclaved stainless steel bead to each tube. Pre-weigh the tubes prior to tissue collection.
After euthanizing mice, resect their cecal and splenic tissues, making sure to collect tissue from individual animals into separate tubes. Weigh each tube to determine the tissue weights.
Use a mixer mill apparatus to homogenize the tissues at 30 hertz for 15 minutes. Then, transfer 900 microliters of PBS into each well of a 96-well 2 milliliter mega block. Pipette 100 microliters of tissue homogenates into the first well, and mix well.
Perform serial dilutions by adding 100 microliters to subsequent wells, until a 10 to the negative sixth dilution is obtained. Plate 10 microliters of each dilution in triplicates onto LB agar that contains Streptomycin. Then, count the average colony forming units and determine the colony forming units per gram of tissue as outlined in the text protocol.
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