Overview
Corynebacterium diphtheriae, also known as the Klebs-LΓΆffler bacillus after its discoverers Edwin Klebs and Friedrich LΓΆffler in 1884, is the causative agent of diphtheria β an acute, contagious infection characterised by pseudomembrane formation around the tonsils and throat.
Corynebacteria are Gram-positive, club-shaped, non-acid-fast rods. This lesson covers their morphology, staining characteristics, toxin production, clinical features of diphtheria, laboratory diagnosis including the Elek test, and the concept of non-pathogenic diphtheroids.
Learning Objectives
After this lesson you will be able toβ¦- Describe the morphological characteristics of Corynebacterium diphtheriae.
- Explain the clinical features of diphtheria.
- Discuss the laboratory diagnosis of Corynebacterium diphtheriae, including virulence testing.
- Explain the disease spectrum caused by Corynebacterium diphtheriae and differentiate it from diphtheroids.
Clinical Story
Why This MattersAn unvaccinated 7-year-old presents with a sore throat, low-grade fever and a greyish membrane covering both tonsils that bleeds when scraped. The paediatrician suspects diphtheria. The lab technologist collects a throat swab, performs Albert staining to visualise the characteristic metachromatic granules, and sets up an Elek test to confirm toxin production β a race against time, since cardiac and neurological complications can follow within days.
Core Concepts
Corynebacteria are Gram-positive, non-acid-fast, non-motile, pleomorphic rods with irregularly stained segments and granules, frequently showing club-shaped swellings (coryne = club). They are non-sporing and non-capsulated, tending to decolourise easily on Gram stain. On Albert staining, the bacilli appear green with black-staining metachromatic (polymetaphosphate) granules β a hallmark diagnostic feature.
Four subspecies are recognised β C. diphtheriae mitis, intermedius, gravis, and belfanti β differing slightly in colonial morphology and biochemistry, but all can be toxigenic or non-toxigenic. Humans are the sole reservoir; transmission is chiefly by droplet infection, less often via contaminated objects. Virulent, toxigenic strains are lysogenic, carrying the diphtheria toxin gene on a Ξ²-prophage (bacteriophage), not on the bacterial chromosome itself.
Growth on ordinary media is scanty; enrichment with blood, serum or egg is necessary. Optimum temperature is 37Β°C (range 15β40Β°C), optimum pH 7.2. The bacillus is an aerobe and facultative anaerobe. Loeffler's serum slope supports very rapid growth, with visible colonies in 6β8 hours β long before other bacteria. Tellurite blood agar is also used, and virulent strains ferment glucose, galactose, maltose and dextrin (acid, no gas) but not lactose, mannitol or sucrose.
Virulent strains produce a powerful exotoxin composed of two polypeptide fragments, A and B β both necessary for toxic effect. Fragment A carries the enzymatic activity (masked until activation), while fragment B binds the toxin to the host cell. Almost all gravis and intermedius strains (95β99%) are toxigenic, compared to only 80β85% of mitis strains.
Every diphtheria isolate must be tested for toxigenicity to complete bacteriological diagnosis. In vivo testing uses subcutaneous or intradermal inoculation of guinea pigs. In vitro testing includes the Elek gel precipitation test β a filter paper strip soaked in antitoxin is placed on serum agar, streaks of the test strain are made at right angles, and arrow-head lines of precipitation form where toxin meets antitoxin of optimal concentration β and the tissue culture test, where toxin diffusing from bacterial growth kills a cell monolayer.
After a 2β4 day incubation, patients develop upper respiratory symptoms with pseudomembrane formation on the tonsils. Cardiac toxicity (arrhythmias, CHF) typically appears 1β2 weeks after onset; neurological symptoms (bulbar palsy, later skeletal muscle paralysis) can appear within weeks; cutaneous ulcers occur in tropical climates. Diphtheria is preventable by immunisation with diphtheria toxoid, usually given as the trivalent DPT (diphtheria-pertussis-tetanus) vaccine.
Laboratory Principle
The Elek gel precipitation test exploits the principle of immunodiffusion: diphtheria exotoxin secreted by a toxigenic strain diffuses radially through the agar, while antitoxin diffuses from an impregnated filter paper strip. Where the two diffusion fronts meet at an optimal (equivalence) concentration ratio, they form an insoluble antigen-antibody precipitin complex visible as a white arrowhead line β a direct, low-cost demonstration of toxin production that remains the gold-standard in-vitro confirmatory test for toxigenic Corynebacterium diphtheriae.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Loeffler's Serum Slope | Coagulated serum medium | Rapid enrichment culture (6β8 hr colonies) | 2β8Β°C |
| Potassium Tellurite Blood Agar | Selective/differential | Selective culture β black colonies | 2β8Β°C, protect from light |
| Albert Stain (I & II) | Toluidine blue, malachite green, iodine | Demonstrates metachromatic granules | Room temperature |
| Diphtheria Antitoxin | 1000 units/mL | Elek gel precipitation test | 2β8Β°C |
| 20% Normal Horse Serum Agar | Serum-enriched agar | Medium for Elek test | 2β8Β°C |
| Hiss's Serum Water Sugars | Glucose, maltose, sucrose, etc. | Carbohydrate fermentation testing | 2β8Β°C |
Step-by-Step Procedure
Collect two swabs from beneath the pseudomembrane at the lesion site, under direct vision.
Examine for slender, pleomorphic, club-shaped rods; Albert stain shows green bacilli with black metachromatic granules.
Inoculate Loeffler's serum slope (rapid growth, 6β8 hrs) and tellurite blood agar (selective, black colonies after 24β48 hrs).
Test fermentation of glucose, maltose, galactose, dextrin (positive) versus lactose, mannitol, sucrose (negative) using Hiss's serum water.
Streak the isolate at right angles to an antitoxin-impregnated filter paper strip on serum agar; incubate 24β48 hours and examine for arrow-head precipitin lines.
Confirm toxigenic strains by a positive Elek test (or in-vivo/tissue culture test) before finalising the diagnosis of diphtheria.
Flow Diagram
Quality Control
Run known toxigenic and non-toxigenic C. diphtheriae control strains with every Elek test batch to confirm the antitoxin strip is active and precipitin lines form correctly. Verify tellurite agar selectivity monthly using a non-corynebacterial control that should be inhibited.
Participate in national reference laboratory proficiency schemes for diphtheria toxin testing, since accurate toxigenicity confirmation directly affects public health outbreak response and vaccination policy decisions.
Reference Values
Key Diagnostic Parametersβ οΈ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Club-shaped, Albert-positive bacilli with arrow-head Elek precipitin line | Confirms toxigenic Corynebacterium diphtheriae | Report as toxigenic; notify public health authority, begin antitoxin/antibiotics |
| Club-shaped bacilli, Elek test negative | Non-toxigenic C. diphtheriae strain or diphtheroid | Correlate clinically; non-toxigenic strains do not cause classic diphtheria |
| Uniformly staining bacilli in palisade arrangement, no metachromatic granules | Diphtheroid (commensal Corynebacterium) | Report as normal flora/diphtheroid, not C. diphtheriae |
| Pseudomembrane with bleeding on removal, positive culture | Classic diphtheria β risk of airway obstruction and systemic toxin effects | Immediate antitoxin administration plus antibiotics; monitor cardiac/neuro status |
Common Errors & How to Avoid Them
Cause: A positive culture alone does not confirm disease-causing potential, since non-toxigenic strains exist.
Prevention: Always perform the Elek test (or equivalent) to confirm toxigenicity before reporting a case as confirmed diphtheria.
Cause: Diphtheroids are normal skin/throat commensals that resemble C. diphtheriae morphologically.
Prevention: Differentiate using staining pattern (uniform staining, palisade arrangement, few granules in diphtheroids) and confirm with culture and biochemical/toxin testing.
Cause: Waiting for full laboratory confirmation before starting antitoxin can allow toxin-mediated complications (cardiac, neurological) to progress.
Prevention: Begin antitoxin therapy based on strong clinical suspicion; do not wait for laboratory confirmation in classic presentations.
Laboratory Tips from the Bench
Always collect two throat swabs β one for immediate Gram/Albert staining and direct microscopy, and one for culture β so a rapid presumptive report can be issued while culture and Elek testing proceed.
Loeffler's serum slope grows C. diphtheriae faster than almost any other organism (6β8 hours) β use it as your first-line rapid enrichment medium when diphtheria is clinically suspected.
Remember 'AB' for diphtheria toxin: fragment A = Active/enzymatic, fragment B = Binding to host cell β both fragments are required for toxicity.
Important Notes
Diphtheria antitoxin must be given as soon as the diagnosis is clinically suspected β it neutralises only free toxin, not toxin already bound to tissue, so early administration is critical to prevent cardiac and neurological complications.
Unusually, the diphtheria toxin gene is carried by a bacteriophage (Ξ²-prophage) integrated into toxigenic strains, not on the bacterial chromosome β a classic example of phage-mediated (lysogenic) conversion.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your Knowledge2-day history of sore throat, low-grade fever and malaise. On examination, a thick greyish-white pseudomembrane covers both tonsils and bleeds when the physician attempts to swab it off.
The clinical picture of a bleeding pseudomembrane in an unvaccinated child, combined with club-shaped bacilli showing metachromatic granules and a positive Elek test, confirms toxigenic Corynebacterium diphtheriae infection β classic diphtheria requiring urgent antitoxin and antibiotic therapy.
- βVaccination status is a key clinical clue β diphtheria is now largely a disease of the unvaccinated.
- βThe Elek test is essential to confirm toxigenicity, not just organism identity.
- βAntitoxin must be given promptly, based on clinical suspicion, without waiting for full lab confirmation.
Frequently Asked Questions
Only toxigenic strains cause classic diphtheria with pseudomembrane formation and systemic complications; non-toxigenic strains can colonise the throat without causing the disease, so toxin testing determines clinical significance.
Diphtheroids are non-pathogenic Corynebacterium species that are normal commensals of the throat, skin and conjunctiva; they stain more uniformly, have few or no metachromatic granules, and arrange in parallel (palisade) rows rather than the cuneiform pattern of C. diphtheriae.
Vaccination with diphtheria toxoid primarily protects against the toxin's effects rather than preventing colonisation entirely, so breakthrough mild infections can rarely occur, but severe disease is markedly reduced in vaccinated populations.
Quick Revision
10-Minute ReviewKey Takeaways
- Corynebacterium diphtheriae causes diphtheria through a powerful exotoxin encoded by a bacteriophage.
- Albert staining and the Elek gel precipitation test are the two most important laboratory tools for diagnosis.
- Only toxigenic strains cause classic disease β toxin testing is essential, not just organism identification.
- Prompt antitoxin administration is critical, as it can only neutralise free, not tissue-bound, toxin.
- Diphtheroids are harmless commensals that must be distinguished from true C. diphtheriae.
Competency Checklist
Track Your MasteryReferences
- National Institute of Open Schooling. Microbiology β Lesson 19: Corynebacterium.
- Ananthanarayan R, Paniker CKJ. Textbook of Microbiology. Universities Press.
- World Health Organization. Diphtheria vaccine position paper.