Levinthal’s Medium- Composition, Principle, Preparation, Results, Uses

Levinthal’s Medium is used for the cultivation of Haemophilus species. The genus Haemophilus includes a number of species that cause a wide variety of infections but share a common morphology and a requirement for blood-derived factors during growth that has given the genus its name. The Haemophilus genus represents a large group of gram-negative rods that grow on agar-containing blood. The blood provides two factors, which many Haemophilus species require for growth: factor-X and factor-V.

Levinthal’s Medium

Composition of Levinthal’s Medium

Peptic digest of animal tissue10.000
Beef extract10.000
Sodium chloride5.000

Final pH (at 25°C): 7.6±0.2

Principle of Levinthal’s Medium

  • Whole blood of rabbit or human blood contains two important factors viz factor-X and factor-V, which are necessary for the growth of type species of influenzae.
  • Factor-X is a heat stable substance, the hemin associated with hemoglobin, whereas factor-V is a heat labile coenzyme Nicotinamide Adenine Dinucleotide (NAD).
  • Other nutrients such as nitrogen compounds are supplied by a peptic digest of animal tissue and beef extract incorporated in the medium.
  • Sodium chloride helps to maintain the osmotic balance of the medium.
  • The bacitracin added inhibits normal flora, which enhances the recovery of Haemophilus species.
  • Pathogenic Haemophilus species may be presumptively identified by determining in vitro growth requirements for X and V factors and by hemolytic reactions on the agar.

Preparation of Levinthal’s Medium

  1. Suspend 45 grams in 1000 ml distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Dispense in 100 ml amounts and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. Cool to 50°C and add 5 ml sterile rabbit or human blood to 100 ml medium.
  5. Heat the mixture in boiling water bath.
  6. Allow the deposits to settle and dispense clear supernatant.
  7. Pour into sterile petriplates.

Result Interpretation on Levinthal’s Medium

Haemophilus influenzaLuxuriant growth; capsulated strains show translucent colonies with distinctive iridescence; non-capsulated strains are transparent, bluish, and non-iridescent.
Staphylococcus aureusLuxuriant growth
Streptococcus pyogenesLuxuriant growth

 Uses of Levinthal’s Medium

  • Levinthal’s Medium Base with the addition of blood is used for cultivation of Haemophilus species.
  • It can be used for cultivating a variety of fastidious organisms.

Limitation of Levinthal’s Medium

  • It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.


  1. http://www.himedialabs.com/TD/M472.pdf
  2. https://www.ncbi.nlm.nih.gov/pubmed/8508821
  3. https://apps.szu.cz/cnctc/media/MEDIUM%20FORMULATIONS.pdf
  4. http://www.himedialabs.com/intl/en/products/Microbiology/Dehydrated-Culture-Media-Diagnostic-Animal-based-Media-Bacterial/Levinthals-Medium-Base-M472
  5. https://www.thomassci.com/Laboratory-Supplies/Microbiological-Media/_/Levinthals-Medium-Base
  6. https://books.google.com.np/books?id=MXMazr0LRDsC&pg=PA334&lpg=PA334&dq=levinthal+medium+hardy&source=bl&ots=0vMefp0BuW&sig=ACfU3U2QEEbA9EpScn5Xou8GEwH5uDTbog&hl=en&sa=X&ved=2ahUKEwj245Tj1aLhAhVOX30KHWBEBoEQ6AEwBHoECAgQAQ#v=onepage&q&f=false
  7. https://www.jcdr.net/article_fulltext.asp?id=2065
  8. https://books.google.com.np/books?id=GS_FDAAAQBAJ&pg=PA257&lpg=PA257&dq=levinthal+agar+with+bacitracin&source=bl&ots=oOqVi6HMcO&sig=ACfU3U0VHaXc6eGjdzksCNgp8Qa5GyVD-w&hl=en&sa=X&ved=2ahUKEwiTn5KLyafhAhWEto8KHdupDFIQ6AEwB3oECAkQAQ#v=onepage&q=levinthal%20agar%20with%20bacitracin&f=false
  9. https://jcp.bmj.com/content/jclinpath/13/6/519.full.pdf

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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