Several methods for diagnosing bacterial or fungal infections require isolation of the suspected pathogen in pure culture from a properly obtained clinical specimen. This is accomplished by streaking the specimen onto media that supports the growth of the suspected pathogen and incubating under appropriate atmospheric conditions (aerobic or anaerobic) to obtain well isolated colonies.
Media can be “selective,” containing compounds that only allow certain bacteria to grow (e.g., antibiotics, salts, or dyes), and/or “differential,” containing other compounds that allow one type of bacteria to be distinguished from another based on a biochemical reaction (e.g., detecting hemolysis on blood agar plates or pigment formation). Table 1 contains a list of various bacteriologic agars commonly used in the diagnostic laboratory and the function of these agars. Once pure, well-isolated colonies are further characterized (e.g., analyzing biochemical and enzymatic activities) and antibiotic susceptibility testing is performed.
Table1. Commonly Used Bacteriologic Agars and Their Function
Several advances in specimen collection devices and trans port media and containers have significantly improved pathogen recovery rates. Liquid-based transport medium has become the mainstay of specimen collection. High recovery of a homogenous distribution of organisms in liquid transport medium facilitates the use of automated plating instruments, allowing for many more plates to be inoculated from the same clinical specimen. This shifts the practice of microbiology toward a more accurate and efficient automated sample processing pipeline using plates and assays that can be incubated, monitored, and read automatically. Automation of specimen processing and incubation decreases institutional costs, technician errors, and time-to-specimen identification, all of which improve patient care and safety.
Specimen Collection for Culture
Common specimen cultures and a general description of how they are carried out are described below. Table 2 lists these culture methods, the syndromes that indicate their use, and common pathogens that are isolated from these cultures.
Table2. Common Culture Methods, the Syndromes That Indicate Their Use, and Common Pathogens That Are Isolated from These Cultures
Blood Cultures
Blood cultures are performed when sepsis, endocarditis, osteomyelitis, meningitis, or pneumonia is suspected. Blood (typically 10 mL from venipuncture) is added to two bottles of rich growth medium containing an indicator for carbon dioxide (CO2) production. One bottle is incubated aerobically and one anaerobically. Production of CO2 indicates organism growth. Gram stain, subculture, and antibiotic sensitivity tests are then performed.
Throat Cultures
Throat culture involves using a sterile swab to rub the surface of one or both tonsils, the tonsillar pillars, and/or the posterior pharyngeal wall to collect the pathogen specimen. The swab is then used to apply the collected specimen onto a sector of a blood agar plate, which is then streaked across the plate using a loop. Because there is an abundant oral microflora, it is important to streak for well-isolated colonies to separate the pathogen from the normal oral flora.
Sputum Cultures
Sputum cultures are performed to determine infectious etiologies of pneumonia or to test for active pulmonary tuberculosis. It is critical to collect a good sputum sample with minimal saliva or nasopharyngeal secretions from the upper airway. A reliable specimen has >25 leukocytes and active tuberculosis is suspected, an acid-fast stain will be done to look for the organism in the sputum, though this microscopic method is not very sensitive.
Cerebrospinal Fluid Cultures
Cerebrospinal fluid (CSF) cultures are performed when a neurologic infection (e.g., meningitis) is suspected. CSF specimens from tissue-centric cases, including encephalitis, brain abscess, and subdural empyema, may show negative cultures. Acute meningitis is a medical emergency. Specimens should be immediately taken for gram stain, which can guide immediate empirical treatment. Suspected meningitis caused by acid-fast bacteria such as M. tuberculosis should be followed up with an acid-fast stain and culture of CSF in addition to nucleic acid amplification methods.
Stool Cultures Most cases of acute diarrhea are self-limiting and don’t require empiric antimicrobial therapy or stool culture. However, testing is performed for patients with severe, persistent, or bloody diarrhea, who are immunocompromised, or outbreak-or healthcare associated diarrhea. When bacterial culture is recommended, feces should be collected during the acute phase of symptoms. Specimens should be processed by the clinical lab within 2 hours of collection to maximize pathogen detection. Selection of primary plating media used for routine culture typically includes (1) MacConkey agar; (2) a selective/differential medium (e.g., eosin-methylene blue agar [EMB]) to maximize recovery of Salmonella and Shigella; (3) a medium to recover Campylobacter (e.g., Campy-CVA); and (4) a medium to recover E. coli O157, like MacConkey-sorbitol medium. Antigen detection assays to test for Shiga toxin I and II or C. difficile toxin A/B should also be performed.
MacConkey and EMB agars are both selective and differential. They are selective because they allow gram-negative rods to grow but inhibit many gram-positive organisms. Their differential properties are based on the fact that Salmonella and Shigella do not ferment lactose, whereas many other enteric gram-negative rods do. On EMB agar, colonies of E. coli, a lactose fermenter, appear dark and have a green sheen. In contrast, colonies of nonlactose fermenters, such as Salmonella and Shigella, appear colorless.
Urine Cultures
Urine cultures are performed primarily when pyelonephritis or cystitis is suspected. A midstream specimen, voided after washing the external orifice, is used for urine cultures. In special situations, suprapubic aspiration or catheterization may be required to obtain a specimen. Because urine is a good culture medium, any organisms present in the specimen can multiply, leading to erroneous results regarding type and number of organisms present at the time of collection. Thus, it is essential that the cultures be done within 1 hour after collection or stored at 4°C for not more than 18 hours. It is commonly accepted that a bacterial count of at least 100,000/mL must be found to conclude that significant bacteriuria is present (in asymptomatic persons). There is evidence that a bacterial count as low as 1000/mL is significant in symptomatic patients. For this determination to be made, quantitative or semiquantitative cultures are performed. There are several techniques: (1) a calibrated loop that holds 0.001 mL of urine can be used to streak the culture; (2) serial tenfold dilutions can be made and samples from the dilutions streaked; and (3) a screening procedure suitable for the physician’s office involves an agar-covered “paddle” that is dipped into the urine—after the paddle is incubated, the density of the colonies is compared with standard charts to obtain an estimate of the concentration of bacteria.
Genital Tract Cultures
Genital tract cultures can be performed on specimens from individuals with an abnormal discharge or on specimens from asymptomatic contacts of a person with a sexually transmit ted disease. Specimens are obtained by swabbing the urethral canal (for men), the cervix (for women), or the anal canal (for men and women). Because of the nonsterile nature of these sites, laboratory diagnosis now typically employs nucleic acid techniques, but can also include microscopy, and/or serology depending on the suspected agent (see below). Culture is still important to determine antimicrobial susceptibility in cases of treatment failure.
Wound & Abscess Cultures
Wound and abscess infections are often polymicrobial. The bacteria most frequently isolated differ according to anatomic site and predisposing factors. Anaerobes are frequently involved in these types of infection, so it is important to place the specimen in anaerobic collection tubes and transport it promptly to the laboratory. Because many of these infections are due to multiple organisms, including mixtures of anaerobes and non-anaerobes, the specimen should be cultured on several different media under different atmospheric conditions.
Sometimes an organism is not recovered by culturing, either because it is nonculturable on bacteriologic media or it is intermittently present or only present in limited numbers, and other techniques must be used. Table3 describes some approaches to making a diagnosis when the cultures are negative, which include immunologic and molecular methods discussed below.
