Introduction In recent years, substantive basic science and clinical research have been conducted to evaluate the mechanisms of wound healing, the efficacy of various modalities for treatment of wounds, and the best methods for diagnosing wound infection. A great deal of this effort has been directed toward evaluating the most accurate and reproducible methods for diagnosing chronic wound infection. From the surface, this seems like a fairly simple clinical question. However, it quickly becomes apparent that this question is quite involved. For example, chronic wounds often harbor bacteria at levels many times that which constitute infection in an acute surgical wound (i.e., >= 105 microorganisms/gm of tissue); yet, many of these chronic wounds go on to closure despite levels of microorganisms as great as 108/gm of tissue.1–3 Chronic wounds are thought to be able to tolerate these high numbers of microorganisms because of the type and content of the microorganisms present in the wound bed. Chronic wounds often contain three or more microorganisms. These organisms include both gram-positive and -negative bacteria as well as both aerobic and anaerobic bacterial species. In fact, the cohabitation of these organisms and the resultant competition for nutrients and space is thought to decrease their virulence. As a result, higher numbers of microorganisms can be tolerated because of their decreased ability to harm tissue.1,4 Because of these intrinsic differences in the way acute and chronic wounds respond to varying levels of microorganism numbers,4 a greater emphasis is currently being placed on holistic assessment with clinical signs and symptoms playing key roles in diagnosis of chronic wound infection. The classical signs of infection (erythema, edema, heat, purulent exudate, and pain) have been modified to include serous exudate with concurrent inflammation, delayed healing, discoloration of granulation tissue, friable granulation tissue, pocketing at the base of the wound, foul odor, and wound breakdown as these signs appear to be more predictive of chronic wound infection. Increasing pain and wound breakdown have been shown to be particularly good predictors of infection in the chronic wound.5 However, there are several intrinsic limitations to diagnosing a wound infection and establishing a treatment paradigm via clinical signs and symptoms alone. Of particular concern is the constantly evolving number of microorganisms with antibiotic resistance. While the evaluation of clinical signs and symptoms may prove to be a very cost-effective and expedient method for diagnosing chronic wound infection, the use of this method alone does not inform the wound care clinician of the most appropriate chemotherapeutic approach to treatment. Use of clinical signs and symptoms alone leaves the provider to select a therapeutic agent based on little specific information about the particular pathogen(s). As a result, broad-spectrum chemotherapeutic agents may be initiated that only serve to facilitate the development of antibiotic resistance. The use of clinical signs and symptoms to predict the need for wound culturing has been suggested in the literature.5,6 However, this points to another controversy in the practice of diagnosing wound infection—which methods are best? The method used for collection of wound specimens can influence the data obtained from microbiological culturing. Currently, collection of a biopsy specimen is the gold standard for determining the presence and identity of microorganisms within the wound bed tissue. However, there are limitations as to which healthcare providers can collect biopsies, availability of laboratories offering microbiological culture testing on biopsies, the expenses involved with performance of these tests, and the potential for further tissue damage and delay of healing when biopsies are taken.