Identification is achieved by detecting the microorganism or its products or the patient’s immune response

Laboratory tests are carried out:

Specimen handling and interpretation of results is based upon a knowledge of normal flora and contaminants

Specimens intended for cultivation of microorganisms can be divided into two types:

A thorough knowledge of the microorganisms normally isolated from specimens from non-sterile sites, and the common contaminants of specimens collected from sterile sites, is important to ensure that specimens are properly handled and the results are correctly interpreted. Some specimens from sites that should be sterile (e.g. bladder urine, sputum from the lower respiratory tract) are usually collected after passage through orifices that have a normal flora, which may contaminate the specimens. This needs to be considered when interpreting the culture results of these specimens.

In an ideal world, each specimen arriving in the laboratory would be considered in turn together with the information provided about the patient on the request form so that the microbiologist could assess the pathogens likely to be present and devise an ‘individualized’ processing plan. However, in reality, this approach is not practicable because of constraints on time and money. Thus, specimens tend to be processed by type (e.g. urine, blood, faeces) and the microbiologist looks for easily cultivated pathogens known to be associated with each sample type. However, if the laboratory is provided with suitable information, such as a statement of possible aetiology, more fastidious or unusual pathogens can be sought and relevant antibiotic susceptibilities assessed. To obtain a test result that correctly identifies the infection, it is important to collect an appropriate specimen, to use the appropriate transport conditions and to deliver specimens rapidly to the laboratory. These conditions all affect the accuracy of the laboratory report, and therefore its value to the clinician and ultimately to the patient. Key points to remember about specimen collection are summarized in Box 31.2.

Routine culture takes at least 18    h to produce a result

Time is a key factor because the conventional methods of microbiologic diagnosis depend upon growth and identification of the pathogen. Results of routine culture cannot be achieved in    <       18    h and may take much longer (e.g. several weeks) for a minority of pathogens such as the mycobacteria, which grow very slowly. Thus, specimen processing can be categorized according to the time required to achieve a result and the method – cultural or non-cultural. An alternative route to the diagnosis of an infection is an immunologic one, relying on the detection of an antibody response to the putative pathogen in the patient’s blood. These diagnostic routes are summarized in Figure 31.1, but rapid technologies (e.g. PCR, nucleic-acid probes, microarrays, etc.) have had a major influence on this process.

Figure 31.1 Route from patient to microbiologic diagnosis. This scheme shows a general overview of key steps in specimen processing. Some tests can be performed on the specimen immediately and yield ‘same day’ results. Culture of specimens usually involves a minimum of 18    h incubation before colonies are visible and can be identified. Antibiotic susceptibility tests involve a further incubation period. Alternatively, the diagnosis may be based on the detection of specific antibodies in serum samples: cerebrospinal fluid (CSF), genomic sequences, etc.

Non-cultural techniques do not require microorganism multiplication before detection

Although medical microbiology has long been synonymous with the cultivation of microorganisms from patients’ specimens, these techniques are labour-intensive and slow to produce results (days rather than hours) because replication of organisms is a necessary, but rate-limiting, step. In addition, some microorganisms cannot be cultured in artificial media, and viable organisms may be difficult to recover from specimens of patients who have received antimicrobial therapy. Non-cultural techniques do not require multiplication of the microorganism before its detection. Some techniques, such as microscopy and detection of microbial antigens in specimens, can provide very rapid results (i.e. within 2    h). Other non-cultural methods such as the use of DNA probes and amplification of DNA by the polymerase chain reaction (PCR) may also provide a rapid answer in a matter of hours.

Microscopy is an important first step in the examination of specimens

Microscopy plays a fundamental role in microbiology. Although microorganisms show a wide range in size (see Ch. 1) they are too small to be seen individually by the naked eye, and therefore a microscope is an essential tool in microbiology. The various types of microscopy are summarized in Figure 31.2. The light microscope magnifies objects and therefore improves the resolving power of the naked eye from about 100 000    nm (0.1    mm) to 200    nm; the electron microscope can improve this to 0.1 to 1.0    nm.

Figure 31.2 Applications of microscopy to microbiology. The scheme shows the different uses of light and electron microscopy (EM) for looking at microbes, although the latter is much less frequently used.

Bright field microscopy is used to examine specimens and cultures as wet or stained preparations

Wet preparations are used to demonstrate:

Dyes are used to stain cells so that they can be seen more easily. Stains are usually applied to dried material that has been fixed (by heat or alcohol) onto the microscope slide. Samples from specimens themselves, or pure cultures, can be stained. The slide can then be viewed in the light microscope with an oil immersion lens, which improves the resolving power of the microscope.

The most important differential staining technique in bacteriology is the ‘Gram’ stain

Differential staining procedures exploit the fact that cells with different properties stain differently and thus can be distinguished. Based on their reaction to Gram’s stain (Fig. 31.3), bacteria are divided into two broad groups:

This difference is related to differences in the structure of the cell walls of the two groups (see Ch. 2).

Figure 31.3 The Gram stain is the most important stain for studying bacteria. The combination of the violet dye (crystal violet) and iodine (acting as a mordant) binds to the cell wall. Gram-positive cells retain the stain when challenged with acetone and remain purple. Gram-negative cells lose the purple stain and appear colourless until stained with a pink counterstain (neutral red or safranin). Examination of Gram-stained films also allows the shape of the cells to be noted. Some examples are shown: (A) Gram-positive cocci in chains (streptococci); (B) Gram-positive rods (Listeria); (C) Gram-negative rods (E. coli); (D) Gram-negative cocci (Neisseria).

Acid-fast stains are used to detect mycobacteria

Some organisms, particularly mycobacteria, which have waxy cell walls, do not readily take up the Gram stain. To demonstrate their presence, special staining techniques are used which rely on the ability of such organisms to retain the stain in the presence of ‘decolourizing’ agents such as acid and alcohol. The Ziehl–Neelsen stain (see Fig. 19.20) is a classic differential staining procedure that uses heat to drive the fuchsin stain into the cells; mycobacteria stained with fuchsin withstand decolourization with acid and alcohol and are therefore known as ‘acid-’ and ‘alcohol-fast’, whereas other bacteria lose the stain after acid and alcohol treatment. Alternatively, many laboratories use the fluorescent dye auramine, which has a strong affinity for the waxy cell wall of mycobacteria, to demonstrate these organisms by fluorescence microscopy (Fig. 31.4).

Figure 31.4 Fluorochrome stain of Mycobacterium tuberculosis with a mixture of auramine O and rhodamine B. Mycobacteria appear fluorescent under ultraviolet light.

(Courtesy of D.K. Banerjee.)

Other staining techniques can be used to demonstrate particular features of cells

Examples of such features to aid identification include the volutin (polyphosphate) storage granules in Corynebacterium spp. and lipid in Bacillus spp. (Fig. 31.5).

Figure 31.5 Special staining techniques can be used to demonstrate particular features of bacterial cells. (A) Corynebacteria stained to demonstrate polymetaphosphate storage granules (volutin granules), which appear as dark spots in blue-green cells (Albert’s stain). (B) Lipid storage granules in Bacillus cereus stained with Sudan black (black lipid against red cells).

Dark field (dark ground) microscopy is useful for observing motility and thin cells such as spirochetes

The light microscope may be adapted by modifying the condenser so that the object appears brightly lit against a dark background. Living organisms can be examined by dark field microscopy and thus motility can be observed. The method is also used for visualizing very thin cells such as spirochetes because the light reflected from the surface of the cells makes them appear larger and therefore more easily visible than when examined by bright field microscopy (Fig. 31.6).

Figure 31.6 Spirochetes visualized by dark ground microscopy. Spirochetes and leptospires are much thinner than most bacterial cells (approximately 0.1    μm in diameter compared with 1    μm for E. coli), but they appear larger when viewed by dark ground illumination.

Phase contrast microscopy increases the contrast of an image

This technique enhances the very small differences in refractive index and density between living cells and the fluid in which they are suspended and therefore produces an image with a higher degree of contrast than that achieved by bright field microscopy.

Fluorescence microscopy is used for substances that are either naturally fluorescent or have been stained with fluorescent dyes

If light of one wavelength shines on a fluorescent object, it emits light of a different wavelength. Some biological substances are naturally fluorescent; others can be stained with fluorescent dyes and viewed in a microscope with an ultraviolet light source instead of white light (see Fig. 31.4).

Fluorescence microscopy is widely used in microbiology and immunology and has been developed to detect microbial antigens in specimens and tissues by ‘staining’ with specific antibodies tagged with fluorescent dyes (immunofluorescence). The method can be made more sensitive or can be adapted to the detection of antibody by labelling a second antibody in an indirect test (Fig. 31.7).

Figure 31.7 The fluorescent antibody test for detection and identification of microbial (or tissue) antigens or antibodies directed against them. In the direct test, antibody labelled with a fluorescent dye is applied to a tissue section bearing the antigen, unbound antibody is washed away, and the bound antibody showing the presence and location of the antigen is visualized by fluorescence microscopy. In the indirect test, antigen is revealed by successive treatments with unlabelled antigen-specific antibody and then fluorescent-labelled anti-immunoglobulin which amplifies the signal (thus if the first antibody is human, the labelled antibody will be an anti-human Ig).

The specimen needs to be cut into thin sections for electron microscopy

The electron microscope uses a beam of electrons instead of light, and magnets are used to focus the beam instead of the lenses used in a light microscope. The whole system is operated under a high vacuum. Electron beams penetrate poorly, and a single microbial cell is too thick to be viewed directly. To overcome this, the specimen is fixed and mounted in plastic and cut into thin sections, which are examined individually. Electron-dense stains such as osmium tetroxide, uranyl acetate or glutaraldehyde are applied to the specimen to improve contrast. The electrons pass through the section and produce an image on a fluorescent screen. Images are photographed and enlarged so that the original specimen is magnified many thousandfold (Fig. 31.8).

Figure 31.8 Electron micrograph of papillomavirus, the human wart virus.

(Courtesy of the Regional Virus Laboratory, Birmingham, UK.)

Although not routinely used in the clinical laboratory, electron microscopy can aid in the identification of virus particles

Direct examination of specimens allows rapid identification of virus particles and detection of viruses that are difficult or impossible to cultivate (e.g. rotaviruses). Fluid for examination is dried onto a copper grid and examined. About one million virus particles per millilitre are needed if they are to be detectable. The sensitivity can be increased by reacting the fluid with antiviral antibody so that clumps of virus particles are visible. This is known as immunoelectron microscopy, a technique analogous to immunofluorescence in light microscopy.