ALPF Medical Research

Characteristics to Identify Prokaryotes

Phenotypic characteristics such as cell morphology, colony morphology, biochemical traits, and the presence of specific proteins can all be used in the process of identifying microorganisms. Most of these methods do not require sophisticated equipment and can easily be done anywhere in the world. Methods used to identify prokaryotes are summarized in table 10.3.

Microscopic Morphology

An important initial step in identifying a microorganism is to determine its size, shape, and staining characteristics. Microscopic examination gives information very quickly and is sometimes enough to make a presumptive identification.

Size and Shape

The size and shape of a microorganism can readily be determined by microscopically examining a wet mount. Based only on the size and shape, one can readily decide whether the organism in question is a prokaryote, fungus, or protozoan. In a clinical lab, this can sometimes provide all the information needed

10.2 Using Phenotypic Characteristics to Identify Prokaryotes 249

Table 10.3 Methods Used to Identify Prokaryotes

Method

Comments

Phenotypic Characteristics

Microscopic morphology

Metabolic differences Serology

Fatty acid analysis Genotypic Characteristics

Nucleic acid hybridization

Amplifying specific DNA sequences using PCR

Sequencing rRNA genes

Most of these methods do not require sophisticated equipment and can easily be done anywhere in the world.

Size, shape, and staining characteristics such as Gram stain can give suggestive information as to the identity of the organism. Further testing, however, is needed to confirm the identification.

Culture characteristics can give suggestive information. A battery of biochemical tests can be used to confirm the identification.

Proteins and polysaccharides that make up a prokaryote are sometimes characteristic enough to be considered identifying markers.These can be detected using specific antibodies.

Cellular fatty acid composition can be used as an identifying marker and is analyzed by gas chromatography. These methods are increasingly being used to identify microorganisms.

Probes can be used to identify prokaryotes grown in culture. In some cases, the method is sensitive enough to detect the organism directly in a specimen.

Even an organism that occurs in very low numbers in a mixed culture can be identified.

This method requires amplifying, cloning, and then sequencing rRNA genes, but it can be used to identify unculturable organisms.

for diagnosis of certain infections. For example, a wet mount of vaginal secretions is routinely used to diagnose infections caused by yeast or by the protozoan Trichomonas (figure 10.2). A wet mount of stool is examined for the eggs of parasites when certain roundworms are suspected. The size, shape, and special features of the eggs are often sufficient to allow identification of the intestinal parasite. ■ wet mount, p. 47 ■ vaginal infections, p. 639

Gram Stain

The Gram stain is a differential stain that distinguishes between Gram-positive and Gram-negative bacteria (see figure 3.14). This relatively rapid test narrows the possible identities of an organism by excluding numerous others and provides suggestive information that can be helpful in the identification process.

In a clinical lab, the Gram stain of a specimen by itself is generally not sensitive or specific enough to diagnose the cause of most infections, but it is still an extremely useful tool. The clinician can see the Gram reaction, the shape and arrangement of the bacteria, and whether the organisms appear to be growing as a pure culture or with other bacteria and/or cells of the host. However, most medically important bacteria do not have distinctive shapes or staining characteristics and usually cannot be identified by Gram stain alone. For example, Streptococcus pyogenes, which causes strep throat, cannot be distinguished microscopically from the other streptococci that are part of the normal flora of the throat. A Gram stain of a stool specimen cannot distinguish Salmonella species from E. coli. These organisms generally must be isolated in pure culture and tested for their biochemical attributes to provide precise identification.

In certain cases, the Gram stain gives enough information to start appropriate antimicrobial therapy while awaiting more accurate identification. For example, if a urine sample from an otherwise healthy woman reveals more than one Gram-negative rod per oil immersion field, the clinician will suspect a urinary tract infection caused by E. coli, the most common cause of such infections. Likewise, a Gram stain of sputum showing numerous white blood cells and Gram-positive encapsulated diplococci is highly suggestive of Streptococcus pneumoniae, an organism that causes pneumonia (figure 10.3a). In certain other cases, the result of a Gram stain is enough for accurate diagnosis. For example, the presence of Gram-negative diplococci clustered in white blood cells in a sample of a urethral secretion from a male is considered diagnostic for gonorrhea, the sexually transmitted disease caused by Neisseria gonorrhoeae (figure 10.3b). This diagnosis can be made because N.gonorrhoeae is the only Gramnegative diplococcus found inhabiting the normally sterile urethra of a male. ■ pneumonia, p. 576 ■ gonorrhea, p. 644

Special Stains

Certain microorganisms have unique characteristics that can be detected with special staining procedures. For example, Fihbasidiella (Cryptococcus) neoformans is one of the few types of yeast that produce a capsule. Thus, a capsule stain of cerebrospinal fluid that shows the presence of encapsulated yeast is diagnostic for cryptococcal meningitis (see figure 3.16). Members of the genus Mycobacterium are some of the few microorganisms that are acid-fast (see figure 3.15). If a patient has symptoms of tuberculosis, then an acid-fast stain will be done on a sample of their sputum to determine whether Mycobacterium tuberculosis can be detected. ■ capsule stain p. 47 ■ acid-fast stain, p. 47

Chapter 10 Identification and Classification of Prokaryotes

Candida albicans

Epithelial cell

Chapter 10 Identification and Classification of Prokaryotes

Candida albicans

Epithelial cell

Roundworm egg

Figure 10.2 Wet Mounts of Clinical Specimens (a)Vaginal secretions containing yeast (Candida albicans, 410x); (b) Trichomonas vaginalis attached to squamous epithelium, as viewed through a Nomarski microscope (400x); and (c) roundworm (Ascaris) eggs in a stool (400x).

Roundworm egg

Figure 10.2 Wet Mounts of Clinical Specimens (a)Vaginal secretions containing yeast (Candida albicans, 410x); (b) Trichomonas vaginalis attached to squamous epithelium, as viewed through a Nomarski microscope (400x); and (c) roundworm (Ascaris) eggs in a stool (400x).