Course: Medical Microbiology, PAMB 650/720 2009/2010

Lecture 33: Culture and identification of infectious agents

Faculty: Dr. Alvin Fox, Phone: 733 3288, E-mail: alvin.fox@uscmed.sc.edu,

Office: C-19, Building 2

Suggested reading: Murray, 6th Edition, Chapter 2, 14-17, 19

KEY WORDS
Identification after culture
Isolation (culture) 1. Biochemical (physiological) tests
Agar plate/colonies 2. Genetic tests (genomics):
Liquid media PCR
Sequencing
Restriction digestion
DNA-DNA homology (probes)
3. Immunological
4. Fatty acid profiling
5. Protein profiling (proteomics)

Identification & taxonomy Non culture based detection

Family Polymerase chain reaction (PCR)
Genus Antigen detection
Species Stain (e.g. Gram strain)
Type Serology (antibody levels)
Isolate

Taxonomy

The basis of bacterial identification is rooted in taxonomy. Taxonomy is concerned with

cataloging bacterial species and nowadays generally uses molecular biology (genetic)
approaches. It is now recognized that many of the classical (physiology-based) schemes for
differentiation of bacteria provides little insight into their genetic relationships and in some
instances are scientifically incorrect. New information has resulted in renaming of certain
bacterial species and in some instances has required totally re-organizing relationships within
and between many bacterial families. Genetic methods provide much more precise identification
of bacteria but are more difficult to perform than physiology-based methods.

Family: a group of related genera.

Genus: a group of related species.
Species: a group of related strains.
Type: sets of strain within a species (e.g. biotypes [example, phage recognition], serotypes).
Strain: one line or a single isolate of a particular species.

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 The most commonly used term is the species name (e.g. Streptococcus pyogenes or
Streptococcus pyogenes abbreviation S. pyogenes). There is always two parts to the species
name one defining the genus in this case "Streptococcus" and the other the species (in this case
"pyogenes"). The genus name is always capitalized but the species name is not. Both species
and genus are underlined or in italics. Nowadays underlining is rarely used.

The Diagnostic Laboratory

The diagnostic laboratory uses taxonomic principles to identify bacterial species from
patients. When patients are suspected of having a bacterial infection, it is usual to isolate visible
colonies of the organism in pure culture (on agar plates) and then speciate the organism.
Physiological methods for speciation of bacteria (based on morphological and metabolic
characteristics) are simple to perform, reliable and easy to learn and are the backbone of the
hospital clinical microbiology laboratory. More advanced reference laboratories, or laboratories
associated with larger medical schools or hospitals additionally use genetic testing.

Isolation by culture and identification of bacteria from patients aids treatment. Since
infectious diseases (caused by different bacteria) have a variety of clinical courses and
consequences. Susceptibility testing of isolates (i.e. establishing the minimal inhibitory
concentration [MIC]) can help in selection of antibiotics for therapy. Recognizing that certain
species (or strains) are being isolated atypically may suggest that an outbreak has occurred e.g.
from contaminated hospital supplies or poor aseptic technique on the part of certain hospital
personnel.

Steps in diagnostic isolation and identification of bacteria

Step 1: Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are streaked on culture
plates and isolated colonies of bacteria (which are visible to the naked eye) appear after
incubation for one - several days. It is not uncommon for cultures to contain more than one
bacterial species (mixed cultures). If they are not separated from one another, subsequent tests
can’t be readily interpreted. Each colony consists of millions of bacterial cells. Observation of
these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly
important as a first step in bacterial identification. Whether the organism requires oxygen for
growth is another important differentiating characteristic.

Step 2: Colonies are Gram stained and individual bacterial cells observed under the microscope.

Step 3: The bacteria are speciated using these isolated colonies. This often requires an additional
24 hr of growth.

Step 4: Antibiotic susceptibility testing is performed (optional).

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THE GRAM STAIN, a colony is dried on a slide and treated as follows:

Step 1: Staining with crystal violet.

Step 2: Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue.

Step 3: Extraction with alcohol or other solvent. Gram negative bacteria are generally
decolorized but Gram positive are not.

Step 4: Counterstaining with safranin. Gram positive bacteria are already stained with crystal
violet and remain purple or blue. Gram negative bacteria are stained pink.

Under the microscope the appearance of bacteria are observed including: Are they Gram
positive or negative? What is the morphology (rod, coccus, spiral, pleomorphic [variable form]
etc)? Do cells occur singly or in chains, pairs etc? How large are the cells? There are other less
commonly employed stains available (e.g. for spores and capsules).

Another similar colony from the primary isolation plate is then examined for biochemical
properties (e.g. will it ferment a sugar such as lactose). In some instances the bacteria are
identified (e.g. by aggregation) with commercially available antibodies recognizing defined
surface antigens. As noted above genetic tests are now widely used.

Genetic characterization of bacteria

The coding DNA of a representative strain of most major human pathogens has been
sequenced, and this is referred to as genomics. This huge data-base of sequences is highly useful
in helping design diagnostic tests. There can be substantial variability in sequences among
individual strains. Thus several representative genomes are often sequenced. Genetic tests are
designed to detect individual genes that are conserved in sequence among strains of a species;
multiple strains can be easily compared. Thus for practical reasons diagnostic tests are at the
gene not genomic level. Metagenomics refers to genomic characterization of a population (e.g.
the human flora, or microbiome).

1. Sequencing of 16S ribosomal RNA molecules (16S rRNA) has become the "gold standard" in
bacterial taxonomy for differentiating bacterial species or populations. The molecule is
approximately sixteen hundred nucleotides in length.

2. Once the sequence is known, specific genes (e.g. 16S rRNA) are detected by amplification
using the polymerase chain reaction, PCR. The amplified product is then detected most simply
by fluorescence (“real time” PCR) or by gel electrophoresis (MW, molecular weight).

3. Restriction enzymes recognize and cut at specific DNA sequences; differentiate species by
producing different size DNA fragments.

4. DNA-DNA homology (how well two strands of DNA from different bacteria bind [hybridize]

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is employed to compare the genetic relatedness of bacterial strains/species. If the DNA from two
bacterial strains display a high degree of homology (i.e. they bind well) the strains are
considered to be members of the same species. DNA-DNA homology is rarely commonly used
nowadays. However, probe-based diagnostic assays work on the same principle.

5. The guanine (G) - cytosine (C) content (% GC) is now only of historical value.

Fatty acid profiling: a. The chain length (and other characteristics) of structural (long chain) fatty
acid monomers present in the membranes of bacteria are determined. b. Characterization of
secreted metabolic products (e.g. volatile alcohols and short chain fatty acids) is also employed.

Proteomics and protein profiling: Proteomics is characterization of the entire set of proteins
expressed (from the genome) under specific conditions. This is technically complex and time
consuming and not used in routine bacterial diagnostics. However low MW proteins (often
2,000-10,000 range) is extracted from a few colonies; the protein profile (MW versus
abundance) obtained in a mass spectrometer rapidly and simply identifies bacterial species.

Rapid diagnosis without prior culture: Certain human pathogens (including the causative agents
of tuberculosis, Lyme disease and syphilis) either can’t be isolated in the laboratory or grow
extremely poorly. Successful isolation can be slow and in some instances currently impossible.
Direct detection of bacteria without culture is possible in some cases; examples are given below:

Bacterial DNA sequences can be amplified directly from human body fluids using PCR.
For example, great success has been achieved in rapid diagnosis of tuberculosis. PCR is also
commonly used as an alternative or supplement to culture for many other bacterial species.

Rapid diagnosis (as an example of antigen detection) is used in many doctor's offices for
the group A streptococcus. The patient's throat is swabbed and streptococcal antigen extracted
directly from the swab (without prior bacteriological culture). The bacterial antigen can be
simply detected by aggregation (agglutination) of antibody coated latex beads. A more current
example of the test is described in powerpoint/lecture 45).

Direct microscopic observation of certain clinical samples for the presence of bacteria
can be helpful (e.g. detection of M. tuberculosis in sputum). However, sensitivity is poor and
many false negatives occur.

Serologic identification of an antibody response (in patient's serum) to the infecting agent
can only be successful several weeks after an infection has occurred. This is commonly used in
the diagnosis of Lyme disease.

Epidemics: Defining that a particular strain is being isolated from multiple sources (e.g.
tomatoes or spinach) suggests the occurrence of an outbreak. However this is not the usual
function of the local hospital clinical laboratory but the state or federal laboratory system.