INTRODUCTION

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From a microbiological perspective, the primary function of normal, intact skin is to control microbial populations that live on the skin surface and to prevent underlying tissue from becoming colonized and invaded by potential pathogens. Exposure of subcutaneous tissue following a loss of skin integrity (i.e., a wound) provides a moist, warm, and nutritious environment that is conducive to microbial colonization and proliferation. However, the abundance and diversity of microorganisms in any wound will be influenced by factors such as wound type, depth, location, and quality, the level of tissue perfusion, and the antimicrobial efficacy of the host immune response. Whereas the microflora associated with clean, surgical wounds would be expected to be minimal, the presence of foreign material and devitalized tissue in a traumatic wound is likely to facilitate microbial proliferation unless early prophylactic antibiotic treatment and surgical debridement is implemented (201).

Since wound colonization is most frequently polymicrobial (25, 27, 44, 166, 226), involving numerous microorganisms that are potentially pathogenic, any wound is at some risk of becoming infected. In the event of infection, a wound fails to heal, the patient suffers increased trauma, treatment costs rise, and general wound management practices become more resource demanding. An analysis of postsurgical wound infections following head and neck surgery demonstrated an increase in the average hospitalization period from 14 days when wounds healed without complication to 24 days when the wounds became infected (118). In a similar analysis of 108 postsurgical wounds, Zoutman et al. (249) concluded that 10.2 days per case was directly attributable to wound infection and that the associated hospital cost was $3,937 per infected patient.

Thus, concern among health care practitioners regarding the risk of wound infection is justifiable not only in terms of increased trauma to the patient but also in view of its burden on financial resources and the increasing requirement for cost-effective management within the health care system. From a clinical perspective, fears associated with wound infection have paralleled the increasing use of occlusive dressings since the 1960s. The primary function of dressings such as polyurethane films, polyurethane foams, and hydrocolloids is to maintain a moist and optimal environment for wound healing. Although they have been reported to encourage microbial proliferation in wounds (95, 128), the infection rate is lower under occlusive dressings than under conventional dry dressings (24, 113) and wound healing is not impaired (95).

Although microorganisms are responsible for wound infection, widespread controversy still exists regarding the exact mechanisms by which they cause infection and also their significance in nonhealing wounds that do not exhibit clinical signs of infection. One school of thought is that the density of microorganisms is the critical factor in determining whether a wound is likely to heal (100, 102, 151, 196, 202). However, a second school of thought argues that the presence of specific pathogens is of primary importance in delayed healing (59, 130, 149, 181, 216, 217), while yet others have reported microorganisms to be of minimal importance in delayed healing (4, 70, 80, 95, 98, 214, 237).

There is also debate about whether a wound should be sampled for culture, the value of wound sampling in determining the cause of infection and subsequent treatment, and the sampling technique required to provide the most meaningful data. Regarding the role of the microbiology laboratory, consideration must be given to the relevance of culturing polymicrobial specimens, the value of identifying one or more microorganisms, and which microorganisms (if any) should be assayed for antibiotic susceptibility. By questioning and justifying the need to sample and perform microbiological analyses on any problematic wound, long-term savings in cost, labor, and time to both the wound management team and the microbiology laboratory could be considerable. In this respect, the value of the Gram stain as a quick and inexpensive additional or alternative test is also worthy of consideration.

Although appropriate systemic antibiotics are considered essential for the treatment of nonhealing, clinically infected wounds, there is debate about the relevance and use of systemic and topical antibiotics and of topical antiseptics in the treatment of nonhealing, noninfected wounds. Other, nonmicrobiological approaches to controlling potentially pathogenic microbial populations in wounds must also be considered part of a multidisciplinary wound management effort.

In view of the fears, uncertainties, and controversies regarding the role of microorganisms in wounds, this review aims to capture current opinion, evaluate the role of the microbiologist and the microbiology laboratory in wound management, and clarify the relevance of treatment and treatment options in controlling microbial colonization and infection in wounds.

Wounds can be broadly categorized as having either an acute or a chronic etiology. Acute wounds are caused by external damage to intact skin and include surgical wounds, bites, burns, minor cuts and abrasions, and more severe traumatic wounds such as lacerations and those caused by crush or gunshot injuries (60). Irrespective of the nature of the cutaneous injury, acute wounds are expected to heal within a predictable time frame, although the treatment required to facilitate healing will vary according to the type, site, and depth of a wound. The primary closure of a clean, surgical wound would be expected to require minimal intervention to enable healing to progress naturally and quickly. However, in a more severe traumatic injury such as a burn wound or gunshot wound, the presence of devitalized tissue and contamination with viable (e.g., bacterial) and nonviable foreign material is likely to require surgical debridement and antimicrobial therapy to enable healing to progress through a natural series of processes, including inflammation and granulation, to final reepithelialization and remodeling.

In marked contrast, chronic wounds are most frequently caused by endogenous mechanisms associated with a predisposing condition that ultimately compromises the integrity of dermal and epidermal tissue (60). Pathophysiological abnormalities that may predispose to the formation of chronic wounds such as leg ulcers, foot ulcers, and pressure sores include compromised tissue perfusion as a consequence of impaired arterial supply (peripheral vascular disease) or impaired venous drainage (venous hypertension) and metabolic diseases such as diabetes mellitus. Advancing age, obesity, smoking, poor nutrition, and immunosuppression associated with disease (e.g., AIDS) or drugs (e.g., chemotherapy or radiation therapy) may also exacerbate chronic ulceration. Pressure or decubitus ulcers have a different etiology from other chronic wounds in that they are caused by sustained external skin pressure, most commonly on the buttocks, sacrum, and heels. However, the underlying pathology often contributes to chronicity, and in this situation, pressure sores, like all chronic wound types, heal slowly and in an unpredictable manner.

WOUND MICROBIOLOGY

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Microbial Colonization

Exposed subcutaneous tissue provides a favourable substratum for a wide variety of microorganisms to contaminate and colonize, and if the involved tissue is devitalized (e.g., ischemic, hypoxic, or necrotic) and the host immune response is compromised, the conditions become optimal for microbial growth. Wound contaminants are likely to originate from three main sources: (i) the environment (exogenous microorganisms in the air or those introduced by traumatic injury), (ii) the surrounding skin (involving members of the normal skin microflora such as Staphylococcus epidermidis, micrococci, skin diphtheroids, and propionibacteria), and (iii) endogenous sources involving mucous membranes (primarily the gastrointestinal, oropharyngeal, and genitourinary mucosae) (65). The normal microfloras of the gut, the oral cavity, and the vagina are both diverse and abundant, and these sources (particularly the oral and gastrointestinal mucosae) supply the vast majority of microorganisms that colonize wounds. Detailed microbiological analyses of wounds demonstrate close correlations between the species found in the normal flora of the gut or oral cavity and microorganisms present in wounds in close proximity to those sites (33-35, 43, 46). Whereas a minor, healing wound may allow sufficient time for a only relatively small number of skin contaminants to take residence, the continued exposure of devitalized tissue associated with a slowly healing chronic wound is likely to facilitate the colonization and establishment of a wide variety of endogenous microorganisms. Dental plaque, the gingival crevice, and the contents of the colon contain approximately 10¹¹ to 10¹² microorganisms/g of tissue, of which, up to 90% of the oral microflora (16) and up to 99.9% of the colonic microflora (105) are anaerobes. In view of this situation, it is reasonable to predict that wounds with a sufficiently hypoxic and reduced environment are susceptible to colonization by a wide variety of endogenous anaerobic bacteria. However, to date, widespread opinion among wound care practitioners is that aerobic or facultative pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and beta-hemolytic streptococci are the primary causes of delayed healing and infection in both acute and chronic wounds. Such opinion has been formed on the basis of referenced comments and studies performed largely during the last two decades that have investigated the role of microorganisms in wound healing (58, 59, 81, 94, 146, 182, 216, 217, 238; D. J. Leaper, Editorial, J. Wound Care 7:373, 1998). A common oversight in these and other studies and opinions is that the culture and isolation of anaerobic bacteria was minimal or omitted, whereas when wounds are investigated by appropriate microbiological techniques anaerobes are found to form a significant proportion of the microbial population in both acute and chronic wounds (25, 27, 28, 33-38, 41-45, 64, 80, 98, 143, 166, 185, 213, 226). On the basis of the studies reviewed in Table 1, which involved detailed microbiological analyses of clinically noninfected (i.e., colonized) wounds of varied etiology, anaerobes constituted, on average, 38% of the total number of microbial isolates per study. It should be emphasized that the studies reported did not investigate specifically the effect of microorganisms on wound healing.

Recognition of the fact that anaerobes are too often overlooked, although many are potentially highly virulent, has led experts in the field to define members of this group of bacteria as being "the secret pathogens" (74) and "invisible villains" (18). Nichols and Smith (175) reported that endogenous anaerobic bacteria were the likely cause of postoperative infections when wound specimens failed to yield bacterial growth on routine culture.

The failure to recognise the prevalence of anaerobic bacteria in wounds may be due to several reasons. (i) Anaerobes are not regarded as being detrimental to normal wound healing (70, 80, 150, 217). (ii) Compared with aerobic and facultative microorganisms, the culture, isolation, and identification of anaerobic bacteria is more time-consuming, labor-intensive, and expensive and is often deemed to be too demanding for many diagnostic microbiology laboratories. The relevance of culturing specimens for anaerobic bacteria is discussed in "Microbiological analysis of wounds" below. (iii) Since anaerobes are often perceived to die rapidly in air, the method of specimen collection and transportation to the laboratory is assumed to be critical for maintaining viability and for effective culture. However, many of the frequent wound colonizers, including Bacteroides, Prevotella, Porphyromonas, and Peptostreptococcus spp., will survive for several days in the presence of air (17, 26, 99, 142). Consequently, the methods for sampling and transportation are probably less critical than the microbiological methods used to ensure effective isolation of anaerobic bacteria. However, this does not imply that specimen collection and transport should be performed without the utmost care and meticulous procedures.

Both acute and chronic wounds are susceptible to contamination and colonization by a wide variety of aerobic and anaerobic microorganisms, as indicated in Table 2.

Surgical wounds will heal rapidly if blood perfusion is maximized, thus delivering oxygen, nutrients, and cells of the immune system to the site of injury and providing minimal opportunity for microorganisms to colonize and proliferate (110). This situation is exemplified by wounds in the anus, which, despite being susceptible to gross microbial contamination, are very well perfused and rarely become infected (110). The probability of wound healing is extremely high if the tissue oxygen tension (pO₂) is >40 mm Hg, but healing is unlikely to occur at levels of <20 mm Hg (110). In well-perfused periwound tissue, reported oxygen tensions of 60 to 90 mm Hg compare with levels of near zero in central dead wound space (176).

In contrast, chronic, nonhealing wounds are frequently hypoxic (218) as a consequence of poor blood perfusion (ischemia), and host and microbial cell metabolism contributes further to a lowering of the local pO₂. Oxygen tensions of between 5 and 20 mm Hg have been recorded in nonhealing wounds (218), and values of less than 30 mm Hg have been recorded in infected and traumatized tissue (164); this correlates with a reported pO₂ requirement of approximately 30 mm Hg for active cell division (111). Thus, cell death and tissue necrosis caused by tissue hypoxia or anoxia are likely to create ideal growth conditions for members of the wound microflora, including fastidious anaerobes that will proliferate as residual oxygen is consumed by facultative bacteria. Such interaction between microorganisms was recognized as long ago as 1915 by Alexander Fleming in his studies on gunshot wounds during the First World War (63). As well as being essential for cell growth and wound healing, oxygen is a critical component of the respiratory burst activity in polymorphonuclear leukocytes (PMNs), resulting in the intracellular production of highly potent antimicrobial metabolites. A significant reduction in the killing capacity of PMNs at a pO₂ of <30 mm Hg has been reported (107), and in this respect, poorly perfused wound tissue is considered to be far more susceptible to infection than are wounds involving well-perfused tissue (176).

Although many endogenous anaerobes survive prolonged periods of exposure to air (17, 26, 99, 142, 230) and tolerate oxygen tensions up to 60 mm Hg (8% oxygen) (184), the redox (oxidation-reduction) potential (E_h) of tissue is also important for their survival (11). Generally, a low E_h (measured in millivolts) favors the growth of anaerobic bacteria, as demonstrated in the colon, where values can be as low as 250 mV, compared with approximately +150 mV in normal tissue and up to +250 mV in circulating blood. Although some anaerobes have been reported to survive in an aerated broth culture medium that was maintained at low E_h (73), other investigations have demonstrated that several intestinal pathogens (Clostridium perfringens, Bacteroides fragilis, and Peptostreptococcus magnus) were inhibited in the presence of oxygen at an E_h of 50 mV (73). Also, the tolerance of anaerobes to E_h is influenced by pH, as demonstrated by C. perfringens, which has a growth-limiting E_h of +30 mV at pH 7.8 but can survive at +250 mV at pH 6.0 (73).

Although opinion is conflicting regarding the importance of the redox potential in supporting the growth of anaerobic bacteria, a wound environment that has a low oxygen tension (hypoxia or anoxia) and a low redox potential will facilitate the development of polymicrobial aerobic-anaerobic populations.

Infection occurs when virulence factors expressed by one or more microorganisms in a wound outcompete the host natural immune system and subsequent invasion and dissemination of microorganisms in viable tissue provokes a series of local and systemic host responses. Characteristic local responses are a purulent discharge or painful spreading erythema indicative of cellulitis around a wound (186). The progression of a wound to an infected state is likely to involve a multitude of microbial and host factors, including the type, site, size, and depth of the wound, the extent of nonviable exogenous contamination, the level of blood perfusion to the wound, the general health and immune status of the host, the microbial load, and the combined level of virulence expressed by the types of microorganisms involved. Most acute and chronic wound infections involve mixed populations of both aerobic and anaerobic microorganisms, and this is demonstrated in Table 3, which collates some of the published literature regarding the microbiology of a variety of infected wound types.

The overall average percent frequencies of anaerobic bacteria in noninfected and infected wounds, based on data presented in Tables 1 and 3, are 38 and 48%, respectively. These numbers compare very closely with those observed by Bowler and Davies (28) specifically in noninfected and infected leg ulcers (36 and 49%, respectively); a correlation between the incidence of anaerobic bacteria and wound infection is thus evident.

Surgical wound infections. The risk of infection is generally based on the susceptibility of a surgical wound to microbial contamination (196). Clean surgery carries a 1 to 5% risk of postoperative wound infection, and in dirty procedures that are significantly more susceptible to endogenous contamination, a 27% risk of infection has been estimated (174). The Guideline for Prevention of Surgical Site Infection, 1999 issued by the Centers for Disease Control and Prevention classified surgical wound infections as being either incisional (involving skin, subcutaneous tissue, or deeper fascia and muscle tissue) or organ/space, involving any internal organs or anatomical spaces (151). Examples of the latter include surgery associated with the large intestine and the head and neck, where extensive endogenous wound contamination, and hence a higher probability of wound infection, is likely.

Acute soft tissue infections. Acute soft tissue infections include cutaneous abscesses, traumatic wounds, and necrotizing infection. Microbiological investigations have shown that S. aureus is the single causative bacterium in approximately 25 to 30% of cutaneous abscesses (41, 158), and the same organism has also been recognized as being the most frequent isolate in superficial infections seen in hospital Accident and Emergency Departments (180). However, other studies have demonstrated that approximately 30 to 50% of cutaneous abscesses (41, 42, 226), 50% of traumatic injuries of varied etiology (40, 45), and 47% of necrotizing soft tissue infections (69) have a polymicrobial aerobic-anaerobic microflora.

Bite wound infections. The reported infection rate for human bite wounds ranges from 10 to 50% depending on the severity and location of the bite, and up to 20% of dog bites and 30 to 50% of cat bites become infected (92). Brook (33) reported that 74% of 39 human and animal bite wounds contained a polymicrobial aerobic-anaerobic microflora, with S. aureus, Peptostreptococcus spp., and Bacteroides spp. being the predominant isolates in both wound types.

Burn wound infections. Infection is a major complication in burn wounds, and it is estimated that up to 75% of deaths following burn injury are related to infection (200, 239). Although exposed burned tissue is susceptible to contamination by microorganisms from the gastrointestinal and upper respiratory tracts (239), many studies have reported the prevalence of aerobes such as P. aeruginosa, S. aureus, E. coli, Klebsiella spp., Enterococcus spp., and Candida spp. (13, 132, 154, 200, 239). In other studies involving more stringent microbiological techniques, anaerobic bacteria have been shown to represent between 11 and 31% of the total number of microbial isolates from burn wounds (46, 166, 197). While the aerobes isolated in the latter studies were similar to those reported previously, predominant anaerobic burn wound isolates were Peptostreptococcus spp., Bacteroides spp., and Propionibacterium acnes (46, 166). Mousa (166) also reported the presence of Bacteroides spp. in the wounds of 82% of patients who developed septic shock and concluded that such microorganisms may play a significant role in burn wound sepsis.

Diabetic foot ulcer infections. Plantar ulcers associated with diabetes mellitus are susceptible to infection due to the high incidence of mixed wound microflora (62) and the inability of the PMNs to deal with invading microorganisms effectively (8). However, with optimal treatment involving debridement of devitalized tissue, the use of appropriate dressings, and pressure relief, wound infection can be minimized. Boulton et al. (24) reported an infection rate of 2.5% in diabetic wounds treated with a moisture-retentive hydrocolloid dressing, compared with a 6% infection rate under a traditional gauze dressing. Laing (127) also observed a similar infection rate (2%) in diabetic foot ulcers treated with a hydrocolloid dressing, despite the number of species increasing during treatment.

Leg and decubitus (pressure) ulcer infections. The microflora of chronic venous leg ulcers is frequently polymicrobial, and anaerobes have been reported to constitute approximately 30% of the total number of isolates in noninfected wounds (28, 42, 98). Although S. aureus is the most prevalent potential pathogen in leg ulcers (28, 42, 98), Bowler and Davies (28) reported a significantly greater frequency of anaerobes (particularly Peptostreptococcus spp. and pigmenting and nonpigmenting gram-negative bacilli) in clinically infected leg ulcers than in noninfected leg ulcers (49 versus 36% of the total numbers of microbial isolates, respectively). The same investigators also suggested that aerobic-anaerobic synergistic interactions are likely to be more important than specific microorganisms in the pathogenesis of leg ulcer infection; this mechanism is not widely recognized in the management of surgical (207) and chronic wound infections.

Quantitative microbiology: significance of microbial numbers. The clinical significance of the microbial load in delaying wound healing was described in 1964 by Bendy et al. (19), who reported that healing in decubitus ulcers progressed only when the bacterial load was <10⁶ CFU/ml of wound fluid. In this study, quantification was determined by using superficial wound swab samples. Similar observations, placing emphasis on counts in tissue biopsy specimens, were reported in studies involving skin graft survival in experimental wounds inoculated with various types of bacteria (126), pressure ulcer healing (203), and delayed closure of surgical wounds (204, 205). Aligned with this early work and in recognition of the fact that quantitative culture of tissue biopsy specimens was demanding on the microbiology laboratory (102) and was of minimal value in facilitating prompt wound management (202), a rapid Gram stain technique was shown to reliably predict a microbial load of >10⁵ CFU/g of tissue if a single microorganism was seen on the slide preparation (102). Additionally, Levine et al. (139) consistently demonstrated a microbial load of 10⁶ organisms per quantitative swab sample taken from open burn wounds when bacterial cells were observed in a Gram-stained smear prepared from the same sample (139). The work of Robson and Heggers, in particular, has spanned more than three decades, and on the basis of their (and other) observations, one school of thought believes that acute or chronic wound infection exists when the microbial load is >10⁵ CFU/g of tissue. More recently, Breidenbach and Trager (31) demonstrated that a critical level of bacteria of 10⁴ CFU/g of tissue must be reached to cause infection in complex extremity wounds and that quantitative tissue cultures predict the likelihood of wound infection more effectively than swab cultures do. In contrast, Pruitt et al. (194) reported that quantitative cultures are incapable of differentiating between burn wound colonization and infection, and they described histological analysis as being the most effective and rapid method for determining invasive burn wound infection. Raahave et al. (196), using a velvet pad surface imprint technique, reported that the median infective dose of mixed aerobes and anaerobes in postsurgical wounds was 4.6 × 10⁵ CFU/cm², and Majewski et al. (150), using a surface swab method, demonstrated that skin grafting was more successful in patients with wound contamination of <5 × 10⁴ CFU/cm². A dermabrasion technique, considered to quantify tissue colonization while minimizing the degree of tissue invasion in burn wounds, has been shown to be more sensitive, both qualitatively and quantitatively, than a surface sample procedure (181). However, the technique requires specialized equipment, and the work of Pallua et al. (181) excluded investigation for anaerobic bacteria.

Qualitative microbiology: significance of specific microorganisms. The effect of specific types of microorganisms on wound healing has been widely published, and although the majority of wounds are polymicrobial, involving both aerobes and anaerobes, aerobic pathogens such as S. aureus, P. aeruginosa, and beta-hemolytic streptococci have been most frequently cited as the cause of delayed wound healing and infection (39, 58, 59, 81, 94, 146, 149, 216, 217, 238). As a specific example, S. aureus is considered to be the most problematic bacterium in traumatic, surgical, and burn wound infections (96, 123, 154, 175, 180), primarily based on the knowledge that its incidence is high in these, and other, types of wound (25, 27, 180). However, although polymicrobial wounds are frequently colonized with S. aureus, a correlation between the presence of this particular pathogen and wound infection is lacking (25, 28, 98).

View larger version (120K): [in this window] [in a new window]   FIG. 1.   Growth of P. loescheii in the presence of S. aureus.

In clinical practice, the presentation of a devitalized acute or chronic wound or a clinically infected wound is likely to prompt a practitioner to sample the wound for microbiological analysis. However, from a wound management perspective, there is little consensus regarding whether sampling is relevant, when and how a wound should be sampled, how a specimen should be transported to the laboratory, and what analyses should be requested. Confusion also exists in view of the fact that health care practitioners often consider a microbiological report to provide definitive information on whether a wound is infected (76, 173), and the provision of an antibiogram for a particular pathogen can often be misleading and prompt unnecessary treatment.

The aim of the following sections is to clarify current controversies in wound sampling and discuss the role of the health care practitioner and the microbiology laboratory in achieving clinically relevant outcomes.

Wound tissue sampling. The acquisition of deep tissue during biopsy following initial debridement and cleansing of superficial debris is recognized as being the most useful method for determining the microbial load and the presence of invasive pathogens (76, 173, 235). Tissue is obtained aseptically and is then weighed, homogenized, serially diluted, and cultured on selective and nonselective agar media under aerobic and anaerobic conditions to provide quantitative and qualitative information. Superficial, devitalized tissue removed by curettage, which is often used in the management of diabetic foot ulcers, may also be investigated for microbial content. Another technique involving dermabrasion has recently been described that enables the acquisition of deeper tissue without being as invasive as the biopsy method (181).

Following the acquisition of wound fluid or tissue for microbiological analysis, prompt delivery of the specimen to the laboratory is considered to be of utmost importance (32), particularly if anaerobic bacteria are being investigated. Aspirates of purulent fluid and tissue samples are considered to be preferable to swabs (32, 119) because they will maintain the conditions required to sustain microbial viability (i.e., a moist and reduced environment) if processed promptly. However, prereduced commercially available transport media offer advantages if specimen culture is delayed beyond 1 to 2 h after isolation. Since swab samples are susceptible to desiccation and oxygen exposure, a prereduced, nonnutritive transport medium is essential to maintain the viability of both aerobic and anaerobic microorganisms on cotton swabs.

Although anaerobic bacteria are commonly perceived to die in air, they have been shown to survive in mixed cultures over extended periods (24 h) (99, 142) and various anaerobes have been shown to survive in clinical specimens despite delayed processing (17). Also, pure cultures of various anaerobic bacteria (including sporing and nonsporing gram-positive and gram-negative bacteria) can survive in air for up to 72 h and can resume growth when reintroduced into an anaerobic environment (26). These observations correlate with those of Bowler and Davies (27), who isolated 157 anaerobes from 106 acute and chronic wounds, the majority of which had been swab sampled and the swabs had been transferred to the laboratory in a standard transport medium. Similarly, in a study that compared the efficacy of three commercially available transport media in facilitating microbial recovery from purulent wounds, a time delay between collection and plating did not affect the isolation of aerobic and anaerobic microorganisms in any of the systems (156). Thus, despite the obvious merits of specialized anaerobic transport media (116), transportation of a moist swab sample to the laboratory in a prereduced transport medium offers a cheap and effective method to enable the culture of both aerobic and anaerobic microorganisms (provided that the isolation and identification methods are adequate). For specimens that cannot be transferred to the laboratory within 1 to 2 h, storage at room temperature is considered to be appropriate for the maintenance of aerobic and anaerobic microorganisms; elevated temperatures may cause differential growth or death of some microorganisms, and lower temperatures will cause increased oxygen diffusion (225).

Gas-liquid chromatography for malodorous specimens. As previously discussed, an offensively malodorous wound specimen is indicative of the presence of anaerobic bacteria (29). Since these microorganisms can be characterized on the basis of the SCFAs produced as end products of metabolism, rapid determination of the presence of potentially pathogenic anaerobic bacteria can be performed by direct GLC analysis on the specimen (87). However, specimens that can benefit from this type of analysis are normally restricted to purulent drainage from enclosed abscesses.

Gram stain. Despite being used for over a century, Gram's stain is still the most important stain in microbiology (192) and is widely used as a rapid technique for guiding antibiotic therapy in life-threatening infections such as bacterial meningitis. However, the value of the Gram stain as a diagnostic tool is debatable. Although its use in the evaluation of tracheal aspirate samples has been reported as insufficiently reliable to guide antibiotic selection (172), the value of the Gram stain has been recognized in diagnosing the cause of peritonitis in continuous ambulatory peritoneal dialysis patients (21), in predicting skin catheter-related bacteremia (137), and in detecting bacterial vaginosis in pregnant women (231).