Elek Test: Principle, Procedure, Results

Elek test is an in vitro immunoprecipitation (immunodiffusion) test to determine whether or not a strain of Corynebacterium diphtheriae is toxigenic. A test strip of filter paper containing diphtheria antitoxin is placed in the center of the agar plate. Strains to be tested (patient’s isolate), known positive and negative toxigenic strains are also streaked on the agar’s surface in a line across the plate and at a right angle to the antitoxin paper strip.

Elek Test
                                         Fig 1: Elek Test

Antitoxin diffuses away from the strip of filter paper where as toxin produced by toxin-producing strains diffuse away from growth. At the zone of equivalence a precipitin line is formed.


  1. Mix a tube of melted nutrient agar. with 2 ml of sterile horse serum.
  2. Rotate the tube to mix the serum and agar. Do not shake the tube.
  3. Pour the mixture into a sterile petri dish.
  4. Using lightly flamed forceps, lay the strip of anti-toxin impregnated filter paper across the centre of the petri dish allowing it to sink beneath the agar surface.
  5. Allow the agar to set, then lift one corner of the lid and let the plate dry for 30-45 min in the incubator.
  6. When dry inoculate with a toxinogenic strain of C. diphtheriae by streaking a single line of inoculum across the plate and paper strip at right angles to the strip.
  7. Repeat this about 1 inch away from the C. diphtheriae inoculum with a test strain.
  8. Incubate the plate for 24 hrs and observe the results.


After 24 hours of incubation at 37°C, plate is examined with transmitted light for the presence of fine precipitin lines at 45 degree angle to the streaks. 

Positive  Test: Precipitin lines form at zone of equivalence, test organism is toxigenic.

Elek test for demonstration of toxin production by C. diphetheriae
Elek test for demonstration of toxin production by C. diphtheriae

If toxin is produced by the test strain, it diffuses sideways from the streak. The antitoxin diffuses from the filter paper and where the toxin and the antitoxin meet (at zone of equivalence) a precipitin line formed.

The control strain also will cause a precipitate to form which will coalesce with the precipitate of the test strain to form a line of identity

See the image (right side)  and try to interpret yourself before reading the explanation below:

  1. Line 1 is a negative control
  2. Line 2 is the positive control
  3. Line 3 is a test organism that is a nontoxigenic strain C. diphtheriae
  4. Line 4 is a test organism that is a toxigenic strain of C. diphtheriae


  1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology). Lippincott Williams & Wilkins
  2. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  3. Melnikov, V. G., Berger, A., & Sing, A. (2022). Detection of diphtheria toxin production by toxigenic corynebacteria using an optimized Elek test. Infection, 50(6), 1591–1595. https://doi.org/10.1007/s15010-022-01903-x

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

5 thoughts on “Elek Test: Principle, Procedure, Results

  1. Good day, i have a presentation tomorrow which will include elek’s precipitation test, i want to know the significance of the serum added to the molten medium. Your immediate reply will be appreciated. Thanks

  2. Hi Mr Acharya. I had my exam yesterday and a photograph of Elek’s test was kept as a spotter. One of the question asked was what other bacteria (other than corynebacterium diphtheria of course) can produce diphtheria toxin? Any answers? Thanks

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