Thayer-Martin agar is another selective medium composed of chocolate agar supplemented with

several antibiotics that suppress the growth of nonpathogenic Neisseria and other normal and abnormal

flora. This medium is normally used to isolate Gonococci.

When submitting samples for culture, the physician must alert the laboratory to likely pathogens

whenever possible, especially when unusual organisms are suspected. This allows inclusion of

selective media that might not be used routinely.

Bacterial Identification

The most widely used identification scheme involves determining the morphologic and metabolic

properties of the unknown bacterium and comparing these with properties of known microorganisms.

Alternate identification schemes using nucleic acid–based methods are used also. It is essential to start

identification tests with pure bacterial isolates grown from a single colony.

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Single enzyme test for bacterial identification

Different bacteria produce varying spectra of enzymes; e.g., some enzymes are necessary for the

bacterium’s individual metabolism, and some facilitate the bacterium’s ability to compete with other

bacteria or establish an infection.

Tests that measure single bacterial enzymes are simple, rapid, and generally easy to interpret. They can

be performed on organisms already grown in culture and often provide presumptive identification.

1) Catalase test: The enzyme catalase catalyzes the degradation of hydrogen peroxide to water

and molecular oxygen (H2O2 → H2O + O2). Catalase positive organisms rapidly produce

bubbles when exposed to a solution containing hydrogen peroxide. The catalase test is key in

differentiating between many gram-positive organisms; e.g., Staphylococci are catalase positive,

whereas Streptococci and Enterococci are catalase negative. The production of catalase is an

important virulence factor because H2O2 is antimicrobial, and its degradation decreases the

ability of neutrophils to kill invading bacteria.

2) Oxidase test: The enzyme cytochrome c oxidase is part of electron transport and nitrate

metabolism in some bacteria. The enzyme can accept electrons from artificial substrates (such as a

phenylenediamine derivative), producing a dark, and oxidized product. This test assists in

differentiating between groups of gram-negative bacteria. Pseudomonas aeruginosa; e.g., is

oxidase positive.

3) Urease: The enzyme urease hydrolyzes urea to ammonia and carbon dioxide (NH2CONH2 +

H2O → 2NH3 + CO2). The ammonia produced can be detected with pH indicators that change

color in response to the increased alkalinity. The test helps to identify certain species of

Enterobacteriaceae, Corynebacterium urealyticum, and Helicobacter pylori.

4) Coagulase test: Coagulase is an enzyme that causes a clot to form when bacteria are

incubated with plasma. The test is used to differentiate Staphylococcus aureus (coagulase

positive) from coagulase-negative Staphylococci.

Automated systems for bacterial identification

Microbiology laboratories are increasingly using automated methods to identify bacterial pathogens;

as in the Vitek System, small plastic reagent cards containing micro liter quantities of various

biochemical test media in 30 wells provide a biochemical profile that allows for organism

identification. An inoculum derived from cultured samples is automatically transferred into the card,

and a photometer intermittently measures color changes in the card that result from the metabolic

activity of the organism.

The data are analyzed, stored, and printed in a computerized database. There are many commercial

variants of these automated systems and several can be used for simultaneous identification and

antimicrobial susceptibility determination.

Tests based on the presence of metabolic pathways (API; analytical Profile Index)

These tests measure the presence of metabolic pathways in a bacterial isolate, rather than a single

enzyme. Commonly used assays include those for oxidation and fermentation of different

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carbohydrates, the ability to degrade amino acids, and use of specific substrates. A widely used manual

system for rapid identification of members of the family Enterobacteriaceae and other gram-negative

bacteria makes use of twenty micro tubes containing substrates for various biochemical pathways. The

test substrates in the micro tubes are inoculated with the bacterial isolate to be identified, and, after 5

hours incubation, the metabolic profile of the organism is constructed from color changes in the micro

tubes. These color changes indicate the presence or absence of the bacteria’s ability to metabolize a

particular substrate. The results are compared with a data bank containing test results from known

bacteria. The probability of a match between the test organism and known pathogens is then

calculated.

Immunological bacterial identification

In the diagnosis of infectious diseases, immunologic methods take advantage of the specificity of

antigen–antibody binding, as known antigens and antibodies are used as diagnostic tools in

identifying microorganisms.

Serologic detection of a patient’s immune response to infection, or antigenic or nucleic acid evidence

of a pathogen in a patient’s body fluids, is frequently useful. Immunologic methods are useful when

the infecting microorganism is difficult or impossible to isolate or when a previous infection needs

to be documented.

I- Detection of microbial antigen with known antiserum

These methods of identification are often rapid and show favorable sensitivity and specificity; unlike

microbial culturing techniques, these immunologic methods do not permit further characterization of

the microorganism, such as determining its antibiotic sensitivity or characteristic metabolic patterns:

1. Quellung reaction: Some bacteria having capsules can be identified directly in clinical specimens

by a reaction that occurs when the organisms are treated with serum containing specific antibodies.

The Quellung reaction makes the capsule more refractile and thus more visible, but the capsule does

not actually swell. This method can be used for all serotypes of S. pneumoniae, H. influenzae type b,

and Neisseria meningitidis groups A and C.

2. Slide agglutination test: Some microorganisms, such as Salmonella and Shigella species, can be

identified by agglutination (clumping) of a suspension of bacterial cells on a microscopic slide.

Agglutination occurs when a specific antibody directed against the microbial antigen is added to the

suspension, causing cross-linking of the bacteria.

II-Identification of serum antibodies

Detection in a patient’s serum of antibodies that are directed against microbial antigens provides

evidence for a current or past infection with a specific pathogen; and it characterized by:

1) Antibody may not be detectable early in an infection

2) The presence of antibodies in a patient’s serum cannot differentiate between a present and a prior

infection

3) A significant rise in antibody titer over a 10 to14-day period does distinguish between a present or

prior infection

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Techniques such as complement fixation and agglutination can be used to quantitate antimicrobial

antibodies.

1. Complement fixation: It is the older method but still useful method for detecting serum

antibody directed against a specific pathogen employs the ability of antibody to bind complement.

A patient’s serum is first incubated with antigen specific for the suspected infectious agent,

followed by the addition of complement. If the patient’s serum does contain immunoglobulin

(IgG or IgM) that target the specific antigen (indicating past or current infection), then the added

complement will be sequestered in an antigen–antibody–complement complex (“complement

fixation”).

Then the sensitized (antibody-coated) indicator sheep RBCs are added to the solution. If

complement has been fixed (because the patient’s serum contained antibodies against the added

antigen), then little complement will be available to bind to the antibody–RBC complexes, and the

cells will not lyse.

If complement has not been depleted by initial antigen–antibody complexes (because the patient’s

serum does not contain antibodies to the specific antigen), the complement will bind to the antibody–

RBC complexes, causing the cells to lyse. As hemolyzed RBCs release hemoglobin, the reaction can

be monitored with a spectrophotometer.

2. Direct agglutination: Direct bacterial agglutination testing is sometimes ordered when a suspected

pathogen is difficult or dangerous to culture in the laboratory. This test measures the ability of a

patient's serum antibody to directly agglutinate specific killed (yet intact) microorganisms. This test is

used to evaluate patients suspected of being infected by Brucella abortus or Francisella tularensis.

3. Direct hemagglutination: Antibodies directed against RBCs can arise during the course of various

infections; such antibodies are typically found during infectious mononucleosis caused by EpsteinBarr virus. When uncoated (native) animal or human RBCs are used in agglutination reactions with

serum from a patient infected with such an organism, antibodies to RBC antigens can be detected. The

patient’s antibodies cause the RBCs to clump. This test is, therefore, a direct hemagglutination

reaction. In the case of some diseases, including pneumonia caused by Mycoplasma pneumoniae, IgM

auto antibodies may develop that agglutinate human RBCs at 4o C but not at 37o C ((termed the “cold

agglutinins” test))

Other tests used to identify serum antigens or antibodies

1. Latex agglutination test:

Latex and other particles can be readily coated with either antibody (for antigen detection) or antigen

(for antibody detection). Addition of antigen to antibody-coated latex beads causes agglutination that

can be visually observed; such methods are used to rapidly test CSF for antigens associated with

common forms of bacterial or fungal meningitis. When antigen is coated onto the latex bead,

antibody from a patient’s serum can be detected. Latex agglutination tests are widely used for the

identification of β-hemolytic Streptococci group A.

2. Enzyme-linked immunosorbent assay:

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Elisa is a diagnostic technique in which antibody specific for an antigen of interest is bound to the

walls of a plastic micro titer well. Patient serum is then incubated in the wells, and any antigen in the

serum is bound by the antibody on the well walls. The wells are then washed, and a second antibody is

added. This one is also specific for the antigen but recognizes epitopes different from those bound by

the first antibody. After incubation, the wells are again washed, removing any unattached antibody.

Attached to the second antibody is an enzyme, which, when presented with its substrate, produces a

colored product, the intensity of the color produced being proportional to the amount of bound antigen.

Elisa can also be used to detect or quantitate antibody in a patient’s serum; the wells are coated with

antigen specific for the antibody in question. The patient’s serum is allowed to react with the bound

antigen, the wells are washed, and a secondary antibody (that recognizes the initial antibody)

conjugated to a color product–producing enzyme is added to the well. After a final washing, substrate

for the bound enzyme is added to the well, and the intensity of the colored product can be measured.

3. Fluorescent-antibody tests: Organisms in clinical samples can be detected directly by specific

antibodies coupled to a fluorescent compound such as fluorescein. In the direct Immunofluorescence

antibody technique, a sample of concentrated body fluid (like CSF or serum), tissue scraping (like

skin), or cells in tissue culture is incubated with a fluorescein-labeled antibody directed against a

specific pathogen.

The labeled antibody bound to the microorganism absorbs ultraviolet light and emits visible

fluorescence that can be detected using a fluorescence microscope. A variation of the technique, the

indirect Immunofluorescence antibody technique, involves the use of two antibodies. The first is

unlabeled antibody (the target antibody), which binds a specific microbial antigen in a sample; and

this clinical sample is subsequently stained with a fluorescent antibody that recognizes the target

antibody. Because a number of labeled antibodies can bind to each target antibody, the fluorescence

from the stained microorganism is intensified.