- 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 is used for the cultivation of Haemophilus species.
Composition of Levinthal’s Medium
|Peptic digest of animal tissue||10.000|
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
- Suspend 45 grams in 1000 ml distilled water.
- Heat to boiling to dissolve the medium completely.
- Dispense in 100 ml amounts and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- Cool to 50°C and add 5 ml sterile rabbit or human blood to 100 ml medium.
- Heat the mixture in boiling water bath.
- Allow the deposits to settle and dispense clear supernatant.
- Pour into sterile petriplates.
Result Interpretation on Levinthal’s Medium
|Haemophilus influenza||Luxuriant growth; capsulated strains show translucent colonies with distinctive iridescence; non-capsulated strains are transparent, bluish and non-iridescent.|
|Staphylococcus aureus||Luxuriant growth|
|Streptococcus pyogenes||Luxuriant 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.
Limitations 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.