How to Identify Corynebacterium Diphtheriae from Throat and Mouth Specimens

Corynebacterium diphtheriae is the causative organism for diphtheria. It can be identified and isolated in the medical laboratory by following the procedures described below. Remember that all samples and specimens must be collected aseptically to avoid cross-contamination which may lead to false positive or false negative result.

1. Culture the Specimen

Blood agar

• Innoculate the swab specimen collected from a patient on a plate of blood agar. Use the loop to make also a few stabs in the agar (well area). Colonies of S. pyogenes growing below the surface will show more distinct zones of haemolysis because of the aerobic conditions provided.

• When a swab is received in silica gel (e.g. from a health centre), moisten it first with sterile nutrient broth and then inoculate the plate.

• Add a 0.05 unit bacitracin disc (Reagent No. 15) to the plate. This will help in the identification of S. pyogenes. Some workers also add a co-trimoxazole disc (as used for sensitivity testing) which prevents the growth of other bacteria, making it easier to see beta-haemolytic S. pyogenes colonies.

• Incubate the plate preferably anaerobically or, when this is not possible, in a carbon dioxide enriched atmosphere overnight at 35 – 37^o Candle jar incubation will detect most beta-heamolytic streptococci.

Note: Beta-haemolytic streptococci produce larger zones of haemolysis when incubated anaerobically. A minority of Group A Streptococcus strains will only grow anaerobically.

Immunological detection of S. pyogenes antigen in specimens Immunochromatic tests, dip sticks and other simple to perform technologies have been developed to detect S. pyogenes antigen directly in specimens. 

Culture of Specimen when Diphtheria is Suspected

When diphtheria is suspected and culture is specifically requested, inoculate the swab on Tinsdale medium or tellurite blood agar. Incubate the plate aerobically for growth after overnight incubation.

2. Examine the Specimen Microscopically

Gram Smear

Make an evenly spread of the specimen on a slide. Allow the smear to air-dry in a safe place. Fix and stain by the Gram technique. Use dilute carbol fuchsin (1 in 10 dilution) as the counterstain in preference to safranin or neutral red (stains Vincent’s organisms better).

Examine the smear for pus cells and Vincent’s organisms:

Vincent’s organisms: These are seen as Gram negative spirochaetes (B. vincenti) and Gram negative fusiform rods.

Other bacteria: No attempt should be made to report routinely other bacteria in a Gram stained smear from a throat swab because the throat contains a wide variety of commensals that cannot be distinguished morphologically from pathogens.

When thrush is suspected, look for Gram positive Candida yeast cells.

Albert Stained Smear when Diphtheria is Suspected

Prepare the smear as described previously under Gram smear. Fix with alcohol. Examine the smear for bacteria that could be Corynebacterium diphtheriae.

Look for pleomorphic rods containing dark-staining volution granules. The pleomorphic rods tend to join together at angles giving the appearance of Chinese letters. Pleomorphic and granule formation are best seen in smears from a Loeffler serum or Dorset egg medium culture. Smears directly from specimens may not show these features.

Volutin Granules

Although the presence of volutin granules is characteristic of Corynebacterium diphtheriae, especially when the organisms when the organisms are pleomorphic, some virulent strains of the gravis biovar (biotype) may contain few or no volutin granules. It is also possible for commensal diphtheroids to contain volutin granules but the commensals are not pleomorphic like Corynebacterium diphtheriae.

When C. diphtheria is cultured on terullite blood agar and modified Tinsdale medium, granule formulation is usually restricted.

Note: In Grams stained smears, C. diphtheria stains variably and weakly Gram positive, whereas commensal diphtheroids appear strongly Gram positive.

3. Examine and Report the Cultures

Blood Agar Culture

Look for beta-haemolytic colonies that could be Streptococcus pyogenes (Lancefield Group A Streptococcus). Most strains are sensitive to bacitracin as shown in colour Plate 26. However, bacitracin sensitivity cannot be completely relied on to identify S. pyogenes. The organism should be tested serologically to confirm that it belongs to Lancefield Group A or tested biochemically using the PYR test

Lancefield Grouping of Beta-Haemolytic Streptococci

Beta-haemolytic streptococci are grouped by their specific cell wall polysaccharide antigens (C substance) using specific anti-serum. S. pyogenes belongs to Group A. Simple to use latex and co-agglutination slide test kits are available for grouping beta-haemolytic streptococci. 

PYR test for the presumptive identification of S. pyogenes

The cultural features of C. diphtheria on Tinsdale medium and tellurite blood agar (TBA) are described in subunit 7.18.7. When colonies suspected of being C. diphtheria are isolated, identify as follows:

• Examine a Gram stained smear for variable staining pleomorphic rods 

• Subinoculate two slopes of Dorset egg medium (see No. 34) or Loeffler serum agar. Incubate at 35 – 37^oC for 6 hours or until sufficient growth is obtained.

• Examine an Albert stained smear of the subculture for pleomorphic rods containing volutin granules (see colour Plate 32). Examine a Gram stained smear to check that the subculture is a pure growth.

• Identify the isolate biochemically 

• Using the growth from the other subculture, test the strain for toxin production using the Elek precipitation technique 

Antimicrobial Sensitivity Testing

The World Health Organization, WHO, in its publication Basic Laboratory Procedures in Clinical Bacteriology² advises that routine susceptibility tests on throat or pharyngeal isolates are most often not required, and may even be misleading. The major pathogens involved in bacterial pharyngitis are S. pyogenes and C. diphtheria. Benzylpenicillin and erythromycin are considered as the antibiotics of choice to treat both types of infection. In cases of diphtheria, treatment with antitoxin is also indicated.