Growth on Media

Suitable criteria for bacterial classification include many of the properties that were described in the preceding chapter. One criterion is growth on different types of bacteriologic media. The general cultivation of most bacteria requires media rich in metabolic nutrients. These media generally include agar, a carbon source, and an acid hydrolysate or enzymatically degraded source of biologic material (eg, casein). Addition ally, these types of media may be supplemented by vitamins and even intact red blood cells in the case of blood agar media. Because of their undefined composition, these types of media are referred to as complex media.

Clinical samples from normally nonsterile sites (eg, the throat or the colon) contain multiple species of organisms, including potential pathogens and resident microbial flora. Media can be nonselective or selective; the latter are used to distinguish among the various bacteria in a clinical sample containing many different organisms.

A. Nonselective Media

Blood agar and chocolate agar are examples of complex, nonselective media, which support the growth of many different bacteria. Different bacterial species growing on these types of agar often give rise to colonies with distinctive morphologies—for example, large or small, yellow versus white, serrated or smooth. Colonial growth patterns on different types of media can be useful in the identification of a particular bacterial species.

 B. Selective Media

Because of the diversity of microorganisms that typically reside at some sampling sites (eg, the skin, respiratory tract, intestines, vagina), selective media are used to eliminate (or reduce) the large numbers of irrelevant bacteria in these specimens. The basis for selective media is the incorporation of an inhibitory agent that specifically selects against the growth of irrelevant bacteria. Examples of such agents are:

• Sodium azide selects for Gram-positive bacteria over Gram-negative bacteria.

• Bile salts (sodium deoxycholate) select for Gram-negative enteric bacteria and inhibit Gram-negative mucosal and most Gram-positive bacteria.

• Colistin and nalidixic acid inhibit the growth of many Gram-negative bacteria.

Examples of selective media are MacConkey agar (containing bile) that selects for the Gram-negative rods and CNA blood agar (containing colistin and nalidixic acid) that selects for Gram-positive cocci.

 C. Differential Media

Upon culture, some bacteria produce characteristic pigments, and others can be differentiated based on their complement of extracellular enzymes; the activity of these enzymes often can be detected as zones of clearing surrounding colonies grown in the presence of insoluble substrates (eg, hemolysis around colonies on agar medium containing intact animal red blood cells).

Many of the members of the Enterobacteriaceae can be differentiated by their ability to metabolize lactose. For example, pathogenic salmonellae and shigellae that do not ferment lactose on a MacConkey plate form white colonies, while lactose fermenting members of the Enterobacteriaceae (eg, E. coli) form red or pink colonies. There are many types of differential media, far too many to describe in a chapter focused on taxonomy.

Microscopy

Historically, the Gram-stain, together with visualization by light microscopy, has been among the most informative methods for classifying the eubacteria. This staining technique broadly divides bacteria based on their fundamental differences in the structure of their cell walls (see Chapter 2). This typically represents the first step in identifying individual microbial specimens (eg, are they Gram-negative or Gram-positive) grown in culture or even directly from patient specimens (eg, urine or cerebral spinal fluids).

Biochemical Tests

Tests such as the oxidase test, which uses an artificial electron acceptor, can be used to distinguish organisms by detecting the presence or absence of a respiratory enzyme, cytochrome C, the lack of which differentiates the Enterobacteriaceae from other Gram-negative rods. Similarly, catalase activity can be used, for example, to differentiate between the Gram-positive cocci; the species staphylococci are catalase positive, whereas the species streptococci are catalase negative. If the organism is demonstrated to be catalase positive (Staphylococcus spp.), the species can be subdivided by a coagulase test into Staphylococcus aureus (coagulase positive) or Staphylococcus epidermidis (coagulase negative) as demonstrated in Figure 1.

Fig1. Algorithm for differentiating the Gram-positive cocci.

Ultimately, there are many examples of biochemical tests that can ascertain the presence of characteristic metabolic functions and be used to group bacteria into a specific taxon. A list of common biochemical tests is given in Table 1.

Table1. Common Microbial Biochemical Tests Used to Differentiate Among Bacteria

 Immunologic Tests—Serotypes, Serogroups, and Serovars

 The designation “sero” simply indicates the use of anti bodies (polyclonal or monoclonal) that react with specific bacterial cell surface structures such as lipopolysaccharide (LPS), flagella, or capsular antigens. The terms “serotype,” “serogroups,” and “serovars” are, for all practical purposes, identical—they all use the specificity of these antibodies to subdivide strains of a specific bacterial species. In essence, these reagents are “forensic” agents, establishing a fingerprint that links the source of an organism to a disease caused in an individual. In certain circumstances (eg, an epidemic), it is important to distinguish among strains of a given species or to identify a specific strain. This is called subtyping and is accomplished by examining bacterial isolates for characteristics that allow discrimination below the species level. Classically, subtyping has been accomplished by biotyping, serotyping, antimicrobial susceptibility testing, and bacteriophage typing. For example, more than 130 serogroups of Vibrio cholerae have been identified based on antigenic differences in the O-polysaccharide of their LPS; however, only the O1 and O139 serogroups are associated with epidemic and pandemic cholera. Within these serogroups, only strains that produce a toxin-coregulated pili and cholera toxin are virulent and cause the disease cholera. By contrast, nontoxigenic V. cholerae strains, which are not associated with epidemic cholera, are generally isolated from environmental specimens, from food, or from patients with sporadic diarrhea.

Clonality with respect to isolates of microorganisms from a common source outbreak (point source spread) is an important concept in the epidemiology of infectious diseases. Etiologic agents associated with these outbreaks are generally clonal; in other words, they are the progeny of a single cell and, for all practical purposes, are genetically identical. Thus, subtyping plays an important role in discriminating among these microorganisms. Recent advances in biotechnology have dramatically improved our ability to subtype microorganisms. Hybridoma technology has resulted in the development of monoclonal antibodies against cell surface antigens, which have been used to create highly standardized antibody-based subtyping systems that describe bacterial serotypes. This is an important tool for defining the epidemiologic spread of a bacterial infection.

Some organisms cannot be identified as unique serotypes. For example, some bacteria (eg, Neisseria gonorrhoeae) or viruses (eg, human immunodeficiency virus [HIV] and hepatitis C) are transmitted as an inoculum composed of quasispecies (meaning that there is extensive antigenic variation among the bacteria present in the inoculum). In these cases, groups of hybridomas that recognize variants of the original organisms are used to categorize serovariants or serovars.

Genetic Diversity

 The value of a taxonomic criterion depends on the biologic group being compared. Traits shared by all or none of the members of a group cannot be used to distinguish its members, but they may define a group (eg, all staphylococci produce the enzyme catalase). Developments in DNA sequencing now make it possible to investigate the relatedness of genes or genomes by comparing sequences among different bacteria. It should be noted that genetic instability can cause some traits to be highly variable within a biologic group or even within a specific taxonomic group. For example, antibiotic resistance genes or genes encoding toxins may be carried on plasmids or bacteriophages , extrachromosomal  genetic elements that may be transferred among unrelated bacteria or that may be lost from a subset of bacterial strains identical in all other respects. Many organisms are difficult to cultivate, and in these instances, techniques that reveal relatedness by measurement of DNA sequence analysis may be of value.

Related topics

Serodiagnosis Of Streptococcus,Enterococcus, and Similar Organisms

Approach To Identification of Streptococcus, Enterococcus, and Similar Organisms

Taxonomy—The Vocabulary of Medical Microbiology

Mycobacterium Leprae

Treatment of Mycobacterium tuberculosis

Pathogenesis and Spectrum of Disease of Streptococcus Pneumonia and Viridians Streptococci

Pathogenesis and Spectrum of Disease of Beta- Hemolytic Streptococci

Morphology and Identification of Mycobacterium Tuberculosis

Neisseria gonorrhea

Yersinia enterocolitica and Yersinia pseudotuberculosis

Yersinia pestis and Plague

Pseudomonas