specimens for culture is to recognize that they contain living organisms whose viability is influenced by transport conditions. These organisms may be killed by changes in temperature, drying of the specimen, exposure to oxygen, lack of vital nutrients, or changes in specimen pH. Transport conditions that support the viability of any clinically significant organisms present in the specimen should be established. It should also be noted that the longer the transport takes, the less likely it is that viability will be maintained. Rapid transport of specimens is important for maximal accuracy at the analytical stage.
If the correct test is selected, the proper specimen is collected and transported, but the specimen is labeled with the wrong name, the test findings might be harmful to two different patients. The patient from whom the specimen came might not receive the proper therapy, while a second patient whose name was mistakenly used to label the specimen might receive a potentially harmful therapy.
DIRECT EXAMINATION
Macroscopic
Once a specimen is received in the clinical laboratory, the first step in the determination of the cause of an infection is to examine it. Frequently, infected urine, joint, or cerebrospinal fluid specimens will be “cloudy” because of the presence of microorganisms and white blood cells, suggesting that an infectious process is occurring. Occasionally, the organism can be seen by simply looking for it in a clinical specimen or by looking for it on the patient. Certain worms or parts of worms can be seen in the feces of patients with ascariasis or tapeworm infections. Careful examination of an individual’s scalp or pubic area may reveal lice, while examination of the anal region may result in the detection of pinworms. Ticks can act as vectors for several infectious agents, including Rocky Mountain spotted fever, Lyme disease, and ehrlichiosis. When they are found engorged on the skin, physicians may remove and submit these ticks to the laboratory to determine their identity. This is done because certain ticks (deer ticks) act as a vector for certain infectious agents (Borrelia burgdorferi, the organism that causes Lyme disease). Knowing the vector may help the physician determine the patient’s diagnosis.
Microscopic
Because most infectious agents are visible only when viewed with the aid of a light microscope, microscopic examination is central to the laboratory diagnosis of infectious diseases. Microscopic examination does not have the overall sensitivity and specificity of culture or the newer molecular diagnostic techniques. However, microscopic examination is very rapid, is usually relatively inexpensive (especially when compared with molecular techniques), is available around the clock in at least some formats in most institutions, and in many clinical settings, but by no means all, is highly accurate when done by highly skilled laboratorians. The organisms can be detected either unstained or by using a wide variety of stains, some of which are described below. Microbes have characteristic shapes that are important in their identification. Morphology can be very simple, with most clinically important bacteria generally appearing as either bacilli (Fig. 1a) or cocci (Fig. 1b). The bacilli can be very long or so short that they can be confused with cocci (coccobacilli); they can be fat or thin, have pointed ends, or be curved. The arrangement of cocci can be very helpful in determining their identity. These organisms can be arranged in clusters (staphylococci), pairs or diplococci (S. pneumoniae), or chains (various streptococcal and enterococcal species).
Fungi are typically divided into two groups based on morphology. One is a yeast (Fig. 2), which is a unicellular organism, and the other is a mold, which is a multicellular organism with complex ribbon-like structures called hyphae (Fig. 3). Organisms that are referred to as parasites may be unicellular—the protozoans (Fig. 4)—or highly complex—the nematodes and cestodes (Fig. 5). Parasites are typically identified on the basis of morphology alone.
Because of their small size, viruses cannot be visualized by light microscopy. Alternative approaches described below are needed to detect these microbes in clinical specimens.
Wet mounts
The wet mount technique is extremely simple to perform. As the name implies, the clinical specimen is usually mixed with a small volume of saline, covered with a glass coverslip, and examined microscopically. It is most commonly utilized to examine discharges from the female genital tract for the presence of yeasts or the parasite Trichomonas vaginalis. Wet mounts are also used to make the diagnosis of oral thrush, which is caused by the yeast Candida albicans. Using a special microscopic technique—dark-field microscopy—scrapings from genital ulcers and certain skin lesions can be examined for the spirochete Treponema pallidum, the organism that causes syphilis. This technique is not particularly sensitive but is highly specific in the hands of an experienced microscopist. It is typically done in STI clinics where large numbers of specimens are available, enabling the microscopist to maintain his or her skill in detecting this organism.
The wet mount can be modified by replacing a drop of saline with a drop of a 10% KOH solution to a clinical specimen. This technique is used to detect fungi primarily in sputum or related respiratory tract specimens, skin scrapings, and tissues. The purpose of the KOH solution is to “clear” the background by “dissolving” tissue and bacteria, making it easier to visualize the fungi.
Another modification of the wet mount is to mix a drop of 5% Lugol’s iodine solution with feces. This stains any protozoans or eggs of various worms that may be present in the stool, making them easier to see and identify.
Gram stain
The most frequently utilized stain in the microbiology laboratory
