Chapter 6 Diagnostic microbiology and laboratory methods

Diagnostic microbiology

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:

Fig. 6.1 The cycle of important events in diagnostic microbiology, depicting the interaction between the clinician and the microbiology laboratory.

1. clinical request and provision of clinical information

2. collection and transport of appropriate specimen(s)

3. laboratory analysis

4. interpretation of the microbiology report and use of the information.

Clinical request

The first stage in the diagnostic cycle comprises the specimen and the accompanying request form. The following, which influence the quality of the specimen, should be noted:

• The clinical condition of the patient: if the patient is not suffering from a microbial infection then sampling for pathogens would be futile (e.g. tumours, trauma)

• Antibiotic therapy will alter the quality and quantity of the organisms. Hence, specimens should be collected before antibiotic therapy, if possible; exceptions are where the patient is seriously ill, immunologically compromised or not responding to a specific antibiotic, in which case the necessity of obtaining an interim report as a guide to further management justifies such action.

Provision of clinical information

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.

Collection and transport of specimens

Always collect appropriate specimens.

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.

Laboratory analysis

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):

Fig. 6.2 Laboratory analysis of a pus specimen illustrating the interactions between the laboratory and the clinician. AST, antibiotic sensitivity test.

1. Make a smear of the specimen, Gram stain and examine by microscopy. (A smear is made by spreading a small quantity of pus on a clean glass slide and heat-fixing.)

2. Inoculate the specimen on two blood agar plates for culture under aerobic and anaerobic conditions (these plates are referred to as the primary plates).

3. Incubate the blood agar plates for 2–3 days at 37°C (because most oral pathogens are slow-growing anaerobes; for isolating aerobes, an 18-h incubation period is adequate).

4. Inspect plates for growth. Note the shapes and size of different colony types for subculture. Infections can be due to one organism (monomicrobial) or more than one organism (polymicrobial), as in the case of the majority of dentoalveolar infections, where samples usually yield a mixture of two or three organisms.

5. Isolate the putative pathogen(s) by subculturing onto fresh blood agar plate(s) (single organism cultures) and incubating at 37°C for 24–48 h.

6. Harvest a pure culture of the pathogen and identify using biochemical reactions, selective media or specific antibody reactions (see below).

7. Antibiotic sensitivity tests can be performed on the mixed growth obtained from pus (primary antibiotic tests) or on the pure organism(s) obtained in step 6 (secondary antibiotic tests) (see below).

Finally, it should be noted that the microbiologist can issue a provisional report after 2 days but the final report may take longer (Fig. 6.2).

Interpretation of the microbiology report and use of information

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.

Laboratory methods

A number of methods and techniques are used in the laboratory diagnosis of infection; they can be broadly categorized into:

• Non-cultural methods. These are many and varied, and include:

microscopic methods (light microscopy, electron microscopy)

detection of microbes by probing for their genes using molecular tools

• Cultural methods. Classic methods of diagnosis, in which:

solid or liquid media are used for bacterial and fungal growth

cultured cells derived from animals and humans are used for viral growth

• Immunological methods. These are used to:

identify organisms

detect antibodies in a patient’s body fluids (e.g. serum, saliva), especially when the organism cannot be cultured in laboratory media.

Microscopic methods

Light microscopy

Light microscopy and stains

In light microscopy, bacterial stains are used:

• to visualize bacteria clearly

• to categorize them according to staining properties.

The most commonly used stain in diagnostic microbiology is the Gram stain.

Gram stain technique

1. After heat-fixing the dry film (by gently passing through a flame), flood with crystal violet for 15 s. Then wash the excess.

2. Flood with Lugol’s iodine for 30 s (to fix the stain); wash the excess.

3. Critical step. Decolourize with acetone or alcohol for about 5 s. When no blue colour comes off the smear, wash immediately with water.

4. Counterstain with dilute carbolfuchsin for 30 s (or neutral red for 2 min).

5. Wash with water and blot dry.

Staining characteristics

According to the results of Gram staining, bacteria may be either Gram-positive or Gram-negative (see Figs 13.2 and 18.1, respectively):

• Gram-positive bacteria retain the violet stain by resisting decolourization and are stained deep blue-black.

• Gram-negative bacteria lose the violet stain during decolourization and are therefore counterstained with pink, the colour of carbolfuchsin.

Ziehl–Neelsen technique

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.

Other stains

A number of other stains are used in microbiology to demonstrate flagella, capsules and granules, and for staining bacteria in tissue sections.

Detection of microbes by probing for their genes

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.

Nucleic acid probes

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.

Cultural methods

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.

Table 6.1 Some selective media used in routine microbiology

Bacteriological media

The main constituents of bacteriological media are:

• water

• agar: a carbohydrate obtained from seaweed (as agar melts at 90°C and solidifies at 40°C, heat-sensitive nutrients can be added to the agar base before the medium solidifies)

• growth-enriching constituents: e.g. yeast extract, meat extract (these contain carbohydrates, proteins, inorganic salts, vitamins, and growth factors for bacterial growth)

• blood: defibrinated horse blood or sheep blood.

Preparation of solid media and inoculation procedure

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:

Fig. 6.3 Method of inoculating an agar plate to obtain discrete colonies of bacteria (numbers indicate inoculation steps).

• discrete colony formation, allowing single, pure colonies to be picked from the primary plate for subculture on a secondary plate. The pure growth from the secondary culture can then be used for identification of the organism using biochemical tests, etc.

• observation of colonial characteristics helpful in identification of organisms

• quantification of organisms as colony-forming units (CFUs). This is valuable both in research and in diagnostic microbiology (e.g. if a urine specimen yields more than 10⁵ CFU/ml, the patient is deemed to have a urinary tract infection; a mixed saliva sample with more than 10⁶ CFU/ml of Streptococcus mutans indicates high cariogenic activity).

Liquid media

Liquid media are used in microbiology to:

• promote growth of small numbers of bacteria present in specimens contaminated with antibiotics. The antibiotic is diluted in the fluid medium, thereby promoting growth of the organism

• promote preferentially the growth of a specific bacterium while suppressing other bacterial commensals present in the sample. These are called enrichment media (e.g. selenite F broth used for stool cultures)

• test the biochemical activities of bacteria for identification purposes.

Some examples of solid and liquid media are given in Table 6.2.

Table 6.2 Constituents and uses of some commonly used solid and liquid media

Medium	Major ingredients	Use
Solid media
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
Liquid media
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.