Chapter 6 Diagnostic microbiology and laboratory methods
Diagnostic microbiology involves the study of specimens taken from patients suspected of having infections. The end result is a report that should assist the clinician in reaching a definitive diagnosis and a decision on antimicrobial therapy. Hence, clinicians should be acquainted with the techniques of taking specimens, and understand the principles and techniques behind laboratory analysis.
The diagnosis of an infectious disease entails a number of decisions and actions by many people. The diagnostic cycle begins when the clinician takes a microbiological sample and ends when the clinician receives the laboratory report and uses the information to manage the condition (Fig. 6.1). The steps in the diagnostic cycle are:
The appropriate tests for each specimen have to be selected by the microbiologist according to clinical information given in the accompanying request form. Hence, information such as age, main clinical condition, date of onset of illness, recent/current antibiotic therapy, antibiotic allergies and history of previous specimens are all important for the rationalization of investigations and should be supplied with the specimen.
Specimens should be as fresh as possible: many organisms (e.g. anaerobes, most viruses) do not survive for long in specimens at room temperature. Others, such as coliforms and staphylococci, may multiply at room temperature, and subsequent analysis of such specimens will give misleading results.
Transport specimens in an appropriate medium (see below), otherwise dehydration and/or exposure of organisms to aerobic conditions occurs, with the resultant death and reduction in their numbers. The transport medium should be compatible with the organisms that are believed to be present in the clinical sample (e.g. virus specimens should be transported in viral transport medium, which is not suitable for bacteriological samples). Transport specimens in safe, robust containers to avoid contamination.
A wide array of specimens are received and analyzed by a number of methods in diagnostic microbiology laboratories. The analytical process of a pus specimen from a dental abscess is given below, as an illustration (Fig. 6.2):
While interpretation of most microbiology reports may be straightforward, there are situations in which the clinician should contact the microbiologist, e.g. for guidance in relation to antibiotic therapy and the necessity for further sampling. Good collaboration between the clinician and the microbiologist is essential to achieve optimal therapy.
Routinely used in diagnostic microbiology, stained smears from lesions are examined with the oil immersion objective (×100) using the ×10 eyepiece, yielding a magnification of ×1000. Wet films are examined with a dry objective (×40) (e.g. to demonstrate motility of bacteria).
The specimen is illuminated obliquely by a special condenser so that the light rays do not enter the objective directly. Instead, the organisms appear bright, as the light rays hit them, against the dark background.
Fluorescence techniques are widely used, especially in immunology. This method employs the principle of emission of a different wavelength of light when light of one wavelength strikes a fluorescent object. Ultraviolet light is normally used, and the bacteria or cells are stained with fluorescent dyes such as auramine; for example, to detect microbial antigens in a specimen, the latter is ‘stained’ with specific antibodies tagged with fluorescent dyes (immunofluorescence; see below).
In electron microscopy, light waves are replaced by a beam of electrons, which allows resolution of extremely small organisms such as virions, e.g. 0.001 µm. Electron microscopy can be used in diagnostic virology, for instance, for direct examination of specimens (e.g. rotavirus, hepatitis A virus). Approximately 1 million virus particles are needed for such visualization. Clumps of such viral particles can be obtained by reacting the sample with antiviral antibody – immunoelectron microscopy.
Some bacteria, such as tubercle bacilli, are difficult to stain by the Gram method because they possess a thick, waxy outer cell wall. Instead, the Ziehl–Neelsen technique is used. The organisms are exposed to hot, concentrated carbolfuchsin for about 5 min, decolourized with acid and alcohol (hence, the term acid- and alcohol-fast bacilli), and finally counterstained with methylene blue or malachite green. The bacilli will stain red against a blue background.
Very small bacterial numbers (10–100) in patient specimens can be detected using the standard polymerase chain reaction (PCR) techniques (Chapter 3), while more sophisticated techniques can detect one human immunodeficiency virus (HIV) proviral DNA sequence in 106 cells. The main advantage of this method is its rapidity (a few hours compared with many days for conventional cultural techniques). However, PCR reactions may yield non-specific data and hence judicial selection of primers and careful conduct of the assays (to prevent contaminants giving rise to false-positive results) are important. For these reasons, PCR techniques are not common in the diagnostic laboratory, but with new developments such as microarray technology and nested PCR, it is only a matter of time before this technique becomes more popular.
In this technique, a labelled, single-stranded nucleic acid molecule is used to detect a complementary sequence of DNA of the pathogen in the patient sample, by hybridizing to it. The probes are obtained in the first instance from naturally occurring DNA by cloning DNA fragments into appropriate plasmid vectors and then isolating the cloned DNA. However, if the sequence of the target gene (in the pathogen) is known, oligonucleotide probes can be synthesized and labelled with a radioactive isotope or with compounds that give colour reactions under appropriate conditions.
This technique is not sensitive for detecting small numbers of organisms (i.e. few copy numbers of the gene) in clinical samples. However, a combination of the PCR technique (to produce high copy numbers) and hybridization with an oligonucleotide probe is likely to be the method of choice in identifying organisms that are slow or difficult to grow in the laboratory.
Bacteria grow well on artificial media, unlike viruses that require live cells for growth. Blood agar is the most widely used bacterial culture medium. It is an example of a non-selective medium as many organisms can grow on it. However, when chemicals are incorporated into media to prevent the growth of certain bacterial species and to promote the growth of others, selective media can be developed (e.g. the addition of bile salts helps the isolation of enterobacteria from a stool sample by suppressing the growth of most gut commensals). Some examples of selective media and their use are given in Table 6.1.
When all the necessary ingredients have been added to the molten agar, it is dispensed, while still warm, into plastic or glass Petri dishes. The agar will gradually cool and set at room temperature, yielding a plate ready for inoculation of the specimen.
The objective of inoculating the specimen or a culture of bacteria on to a solid medium is to obtain discrete colonies of organisms after appropriate incubation. Hence, a standard technique (Fig. 6.3) should be used. Solid media are more useful than liquid media as they facilitate:
|Nutrient agar||Nutrient broth, agar||General purpose|
|Blood agar||Nutrient agar, 5–10% horse or sheep blood||Very popular, general use|
|Chocolate agar||Heated blood agar||Isolation of Haemophilus and Neisseria spp.|
|CLED agar||Peptone, l-cystine, lactose, etc.||Culture of coliforms|
|Antibiotic sensitivity||Peptone and a semisynthetic medium||Antibiotic sensitivity tests|
|Peptone||Peptone, sodium chloride, water||General use; base for sugar fermentation tests|
|Nutrient broth||Peptone water, meat extract||General culture|
|Robertson’s meat medium||Nutrient broth, minced meat||Mainly to culture anaerobes|
|Selenite F broth||Peptone, water, sodium selenite||Enrichment medium for Salmonella and Shigella spp.|
When the infectious agent is circulating in blood (e.g. in septicaemia, endocarditis, pneumonia), the latter has to be aseptically withdrawn by venepuncture and cultured. Blood culture has to be performed on special liquid media, under both aerobic and anaerobic conditions. The blood is aseptically transferred to a rich growth medium (e.g. brain–heart infusion broth) containing anticoagulants (Fig. 6.4). Cultures are checked for turbidity and gas production daily, up to a week (in many laboratories, this process is now automated and machines are used to detect bacterial growth). Positive cultures are sampled, and the organisms are isolated and identified.