Diagnostic Microbiology In Action
  Microbiology of the Urinary and Genital Tracts
    Urine Culture Techniques
      Examination and Qualitative Culture of Voided Urine
      Quantitative Urine Culture
    Neisseria and Spirochetes
The spirochetes are slender, coiled organisms with a longitudinal axial filament that gives them motility. Seen in action, they are long, flexible, and always actively spinning or undulating. Their cell walls are extremely thin and not readily stainable. In unstained wet mounts they are too transparent to be seen by direct condenser light, but become quite visible by “dark-field” condenser illumination. A special condenser lens is used to block the passage of direct light through the mount and to permit only the most oblique rays to enter, at an angle that is nearly parallel to the slide. When viewed in such minimal light, the background of the mount is very dark, almost black, but any particles in suspension are brightly illuminated because they catch and reflect light upward through the objective lens. To stain spirochetes in fixed smears, stains containing metallic precipitates are used. Silver, for example, can be precipitated out of solution and will coat spirochetes on the slide, giving them a black color when viewed by ordinary light microscopy (see colorplate 7).

There are three major genera of spirochetes: Treponema, Borrelia, and Leptospira, each containing species associated with human disease. Many of these organisms are obligate parasites that grow only in human or animal hosts, and others are difficult to cultivate on artificial culture media. The leptospires are an exception, for they will grow in a special serum-enriched medium or in embryonated eggs. The laboratory diagnosis of spirochetal diseases is made by microscopic demonstration of the organisms in appropriate clinical specimens, when possible; in special cultures in the case of leptospirosis; or, most frequently, by serological methods for detecting antibodies in the patient’s serum.

Treponema: The most important member of this genus is Treponema pallidum, the agent of syphilis. The organism can be demonstrated by dark-field examination of material from the primary lesion of the disease, called a chancre. Diagnostic serological tests for syphilis are numerous. They are particularly valuable because syphilis can be a latent, silent infection, with few or no obvious symptoms in its early stages. It is a chronic, progressive disease, however, and if unrecognized and untreated it can have very serious consequences. Also, it is a sexually transmitted disease, highly communicable in its primary stage. Laboratory diagnosis of syphilis is, therefore, essential in its recognition, treatment, and control.

Nonpathogenic species of Treponema are frequent members of the normal flora of the mouth and gums, and sometimes of the genital mucous membranes.

Borrelia: Borrelia species are pathogenic for humans and a wide variety of animals including rodents, birds, and cattle. They are transmitted by the bites of arthropods. Two important species for humans are Borrelia recurrentis, the agent of relapsing fever, and Borrelia burgdorferi, the agent of Lyme disease.

Relapsing fever is now primarily a tropical disease, and is transmitted by lice. As the name implies, the infection is characterized by repeated episodes of fever with afebrile intervals in between. Diagnosis is made primarily by seeing the organisms in the patient’s blood either in an unstained preparation viewed by dark-field microscopy or in a smear stained with routine dyes used in the hematology laboratory (e.g., Giemsa stain).

Lyme disease, transmitted by ticks, occurs in a number of countries. In the United States, it was first recognized in children living in Lyme, Connecticut. Although once thought to be confined to the eastern part of the United States, this disease is a growing problem in many parts of the country and throughout the world. The first sign of infection is a circular, rashlike lesion (called erythema migrans) that begins at the site of the tick bite. This lesion may remain localized or spread to other body areas. The rash may be accompanied by flulike or meningitis-like symptoms and, if untreated, many patients develop arthritis, chronic skin lesions, and nervous system abnormalities after many weeks or even years. Because the signs and symptoms mimic those of other infections, the correct diagnosis is often not suspected. Currently, diagnosis is best made by detecting antibodies against the spirochete in the patient’s serum but a history of tick bite provides an important clue.
Leptospira: This genus has been classified as having only one species, Leptospira interrogans, but molecular studies show several species are included in this group. Several different serological strains are pathogenic for animals (dogs, rodents) and one is associated with a human disease called icterohemorrhagia, or, more simply, leptospirosis. (It is also sometimes called Weil’s disease.) The spirochetes infect the liver and kidney, producing local hemorrhage and jaundice, hence the clinical term icterohemorrhagia. It can also cause meningitis.

The laboratory diagnosis of leptospirosis can sometimes be made by demonstrating the organism in dark-field preparations of blood or urine specimens (this spirochete is very tightly coiled, its ends are characteristically hooked, and it has a rapid, lashing motility). Culture of such specimens in serum medium (Fletcher’s) or animal inoculation can also lead to recovery of the spirochete. Serological diagnosis can be made by testing the patient’s serum for leptospiral antibodies.

In this experiment you will see the morphology of some spirochetes in prepared slides and demonstration material.

Purpose Demonstration of important spirochetes
Materials Prepared stained slides
Projection slides, if available

Examine the prepared material. From your observations and/or reading, illustrate the microscopic morphology of each spirochetal genus: