Semiquantitative Bacteriologic Culture of Tissue

Principle:

The degree or extent of bacterial wound contamination is directly related to the risk of wound sepsis. Because of this relationship, physicians use the results of a quantitative culture (the number of colony- forming units [CFUs] per gram of the eschar biopsy) in their management of severely burned patients.

Method:

1. Cut a piece of tissue, measuring several cubic millimeters, aseptically onto a small, reweighed, sterile container.
2. Determine the weight of the tissue by subtracting the weight of the aluminum foil from the total weight.
3. Place the specimen and 2 mL of sterile nutrient broth in a sterile tissue grinder; macerate the specimens.
4. Inoculate 0.1 mL of sample to a blood agar plate, in duplicate, and an anaerobic blood agar plate (if indicated), in duplicate. In addition, inoculate 0.01 mL of sample using a calibrated loop to a blood agar plate, in duplicate. Spread the inoculum on the plates with a sterile glass spreading rod or a loop.
5. Incubate plates in 5% to 10% carbon dioxide overnight, and count the colonies of bacteria on the plate that contain 30 to 300 CFUs. If more than 300 colonies are obtained on both plated dilutions, the factor 300 is used as N for calculations and the result is considered greater than the value.
6. Calculate the number of CFUs per gram of tissue with following formula: 

 Number of CFUs counted x Reciprocal of volume of homogenate inoculated (10^-1 or 10^-2) x 2 (volume of diluent used for tissue homogenization) / weight of tissue.