Urine Analysis (Urinary Tract Infection)

Spontaneously voided urine is regularly contaminated with germs from the external region of the urethra and partly also from the vulvar region. For this reason, only germ counts of 105 and more are regarded as an indication of urinary tract infection. Whether or not the results can be used depends therefore on the proper collection of the urine and, hence, on proper instructions for urine collection given to the patient.

The patient is instructed to spread apart the pudendal lips, clean the opening of the urethra, and discard the first part of the urine; only then may she use the container provided for urine collection. Contamination is always suspected when several types of germs are detected in high concentrations.

Another problem is that urine is an excellent culture medium for bacteria. During shipment of urine samples—with transportation times lasting up to several hours—bacteria rapidly multiply and thus simulate high germ counts. For this reason, immersion procedures have been developed (e.g., Urotube, Roche) in which a prefabricated special agar is dipped into the freshly voided urine; after incubation, the actual germ count is indicated fairly accurately.

One disadvantage is the fact that not all bacteria grow equally well, if at all, on these prefabricated culture media. If a urinary tract infection is suspected and the test culture is negative for bacteria (e.g., during pregnancy), the freshly voided urine must reach the laboratory as quickly as possible and be kept at 4 °C during transport. Even relatively low germ counts (< 10⁵/mL) may indicate a urinary tract infection, especially when the urine has been collected by means of suprapubic bladder puncture or catheter.

The first 10 mL of the spontaneously voided urine (first-stream urine) are used for the detection of chlamydiae in the urethra.

Blood Culture (Sepsis)

In cases of severe infections (even without fever, e.g., in the case of septic shock) and/or fever (> 38.5 °C), blood cultures should be collected because there is the possibility of sepsis. For this purpose, ready-to-use culture flasks are available into which 5–10 mL of venous blood are transferred—by means of a direct tube system, if possible. Thorough and repeated disinfection of the collection site is particularly important, otherwise only contaminating germs will be detected (e. g., Staphylococcus epidermidis). In addition, one should always prepare an aerobic culture, which is supplied with air, and an anaerobic culture, into which only blood is transferred. It is best to collect one aerobic and one anaerobic culture from each arm (minimum: three cultures, optimal: four cultures). The more often blood cultures are collected, the greater the chance that a pathogen is detected. If at all possible, blood samples should be collected while the fever is still rising. Large statistical studies have shown that pathogens will only grow in approximately 20% of the blood cultures. The results of the blood culture are expected to be available not before 20 hours, because the culture flask has to be incubated for eight hours and the content then transferred to an agar plate.

If, for example, the blood culture yields Staphylococcus epidermidis in only one culture, this indicates that one is dealing with a contaminating germ from the skin and not with the pathogen causing the sepsis.

Gardnerella vaginalis cannot be detected with the usual blood culture media, because the presence of heparin inhibits the growth of this pathogen.

Special Procedures

For culturing chlamydiae, epithelial cells from the site of infection (cervix, urethra, infundibulum of uterine tube) are picked up with a swab—or, even better, with a brush or sponge-and transferred to an appropriate transport medium.

Transport Medium

Without the use of a transport medium, only extremely resistant bacteria (e. g., enterococci) can still be cultured after a long period of transport (several hours, one to three days). After 24 hours in a moist swab without transport medium, staphylococci can still be cultured in the original numbers, whereas certain Bacteroides species will have declined (by a magnitude of) 10³–10⁵ (in steps of log₁₀) cfu (colony forming units) within hours and can no longer be cultured.

Since the introduction of transport media and the improvement of culture procedures, fastidious bacteria have been detected far more frequently. Gonococci are relatively sensitive as well; they can be cultured after 24 or 48 hours only if shipped in a transport medium.

Transport media are particularly important for anaerobic bacteria, some of which are extremely sensitive to oxygen and can only survive in appropriate media. A multitude of different transport media are commercially available (e.g., Port-A-Cult, Transystem).

Culturing chlamydiae requires transport media other than those used for the usual bacteriological diagnostic procedures. Like viruses, these bacteria require buffered solutions to which the infected cells can be transferred.

Culture Methods

Common Culture Methods

Bacteria are normally cultured on solid agar media containing the essential nutrients. Each bacterium that is able to multiply produces one colony. By smearing the swab onto the culture plate in a fractional way, a first impression can be gained of the amount of bacteria present. Accurate germ counts are obtained by first setting up dilution series of the starting material.

From each swab different culture plates are prepared because different species of bacteria have different nutritional needs, and also because one might want to check simultaneously for certain growth properties, such as hemolysis.

The use of selective culture media, which contain inhibitors (e. g., antibiotics) suppressing the growth of other contaminating germs, allows for easier recognition of certain pathogens. Ever since selective culture media for streptococci of group B have been introduced, there has been a sharp rise in the detection rate in smears from the vagina and cervix. Selective culture media are also required for pathogens occurring only at low germ counts, such as gonococci.

Culture plates for the detection of aerobic bacteria can be incubated in a normal incubator. Culture plates for the detection of anaerobic bacteria, which do not grow in the presence of oxygen, must be incubated in an anaerobic culture container from which the oxygen is removed by physical or chemical means after the plates have been placed.

Normally, agar plates can be inspected after 24 hours. However, incubation may take several days in the case of slow-growing bacteria (e. g., many anaerobes). This is the case when the initial microbiological report mentions only a “physiological flora” or “no growth of pathogens” and, several days later, a subsequent report mentions anaerobes or other bacteria that are difficult to culture.

Tubercle bacteria grow particularly slowly, with a doubling time of 24 hours. Culture results are therefore available only after four to six weeks.

Further identification of the bacteria is based on the microscopic evaluation of gram-stained samples, testing for metabolic activities (e. g., by means of color strip tests), and—in the case of gonococci—the additional detection of peroxidase.

Other methods of determination include gas chromatography for identifying specific metabolites and the determination of the GC content (base composition of the DNA). Serological methods may be used as well for individual pathogens.

Subsequently, agar plates are inoculated with bacteria from the pure culture. By placing small paper discs impregnated with antibiotics onto these cultures, the sensitivity of the pathogen against various antibiotics can be checked; this test is carried out by following a predetermined, standardized procedure. As the antibiotic from each test disc diffuses into the agar and creates a concentration gradient, a more or less extensive zone of inhibition is formed around the test disc, depending on the sensitivity of the pathogen.

Special Culture Methods

They are required for the following bacteria:

Many other bacteria can only be isolated when appropriate culture media are used, for example, actinomycetes. Also, Gardnerella vaginalis requires a special culture medium (a double-layered agar medium with different additives and inhibitors).

Experimental animals are rarely used for the isolation of bacteria today. In the past, they played a role in diagnosing tuberculosis. However, animals (rabbits) are still required for culturing Treponema pallidum (the pathogen of syphilis) in their testes, although the only use of this method is for manufacturing material used in diagnostic procedures.

Detection of Viruses

The isolation of viruses is an expensive method because it requires cell cultures. It should only be ordered in special cases where other methods do not yield results, or when the task is to detect and identify the pathogen directly.

Materials and Methods of Specimen Collection

The following materials are suitable for the detection of viruses:

In cases of lesions (erosions, ulcers) and small vesicles (e. g., genital herpes), a small sterile swab is vigorously rubbed through the ulcer or the fundus of the vesicle (better still, through several lesions). Unfortunately, this procedure is painful. It is recommended that the swab be moistened with transport medium prior to use because this increases absorbency. The swab is then rinsed out in the transport medium, squeezed, and discarded.

Puncture of a vesicle is only meaningful if it is relatively large; the vesicle content is then aspirated using a small syringe with a thin needle.

Transport Medium

With the exception of papillomaviruses or enteroviruses, viruses are sensitive to desiccation; the swab material must therefore be transferred to a transport medium (e. g., cell culture medium). If this is not available, physiological saline may be used for a brief transport. This applies to all swab material and vesicle contents.

Culture Methods

The isolation of viruses requires cell cultures. Depending on the virus, different cell types are needed because not every virus grows on every cell type. There are permanent cell lines and primary cell cultures, with the permanent cell lines now having gained acceptance. In the majority of cases, multiplication of a virus is recognized by its cytopathic effect (destruction of the mono-layer of cells). This may become visible already after 24–48 hours, if one is dealing with a fast-growing virus or if viral numbers were already high at the time of seeding (e. g., herpes simplex virus). With other viruses, it may take up to eight days (e. g., CMV). Sometimes subcultures are required. Some viruses do not destroy the monolayer of cells (e.g., rubella virus). In that case, virus multiplication is demonstrated by detection of the virus-specific hemagglutinin, which is able to bind erythrocytes to the surface of the virus.

Further identification of the virus is carried out by immunological means, i.e., by blocking the cytopathic effects in the next round of cell culture or, more rapidly, by means of immunofluorescence tests using monoclonal antibodies (e.g., HSV).

Polymerase Chain Reaction

Collection of Material

Swabs from the vaginal region should be collected, if possible, by vigorous rubbing on the vaginal wall; especially in the case of an infection, fungi adhere with their cell walls and penetrate into the tissue.

As the distribution of fungi is usually not homogenous in the regions of vulva and vagina, it is essential for the detection of the pathogen that the swab material is collected at the proper location. Areas with reddening, and particularly those with floccular and often firmly adherent vaginal discharge, are especially suited. If the flakes for microscopic preparation are selectively picked up with the wooden handle of a swab and rubbed carefully into the methylene blue solution on the microscopic slide, chances for a direct detection are very high.

Transport Medium

This is not essential because fungi are very stable. However, the sample must not be allowed to dry out.

Culture Methods and Differentiation

Fungi are very modest in their growth requirements and thus grow on many media (e.g., Sabouraud 2% glucose agar; Kimmig agar, Merck).

Microscopic-morphological Differentiation