6: Diagnostic microbiology and laboratory methods

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:

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):

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).

Laboratory methods

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

Microscopic methods

Light microscopy and stains

In light microscopy, bacterial stains are used:

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

Detection of microbes by probing for their genes

Polymerase chain reaction

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.

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.

Liquid media

Liquid media are used in microbiology to:

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.

CLED, cystine–lactose–electrolyte-deficient.

Jan 4, 2015 | Posted by in General Dentistry | Comments Off on 6: Diagnostic microbiology and laboratory methods
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