For the cultivation of Haemophilus, Levinthal’s Medium can be used. Although there are many species in the genus Haemophilus that can cause infections, they all share a common morphology. They also require blood-derived factors for growth. This is what gave the genus its name. The Haemophilus Genus is a large grouping of gram-negative rods, which can grow on agar-containing human blood. Two factors are required for Haemophilus species to grow: factor-X, and factor-V.
Principle of Levinthal’s Medium
Two factors are essential for the growth and development of influenzae type species. Factor-X, which is the hemin that is associated with hemoglobin is a heat stable compound, is factor-V, which is a heat labile COENZME Nicotinamide Adenine Dinucleotide. The medium also contains a peptic digest from animal tissue and beef extract, which provide additional nutrients like nitrogen compounds.
Sodium chloride is used to maintain the medium’s osmotic equilibrium. The addition of bacitracin inhibits normal flora and enhances recovery of Haemophilus spp. Presumably, pathogenic Haemophilus species can be identified by in vitro growth requirements and hemolytic reactions on the Agar.
Preparation of Levinthal’s Medium
- Take 45 grams and mix it with 1000 ml of distilled water.
- To dissolve the medium completely, heat to boiling
- Give 100 ml portions to your customers. After that, sterilize them by using an autoclave at 15 lbs pressure (121degC), for 15 minutes.
- Cool the mixture to 50°C. Add 5 ml of sterile rabbit blood or human blood to make 100 ml.
- In a boiling water bath, heat the mixture.
- Allow deposits to settle before you can dispense supernatant.
- Pour into sterile petriplates.
Composition of Levinthal’s Medium
| Ingredients | Gms/liter |
| Peptic digest of animal tissue | 10.000 |
| Beef extract | 10.000 |
| Sodium chloride | 5.000 |
| Bacitracin | 0.300 |
| Agar | 20.000 |
Final pH (at 25°C): 7.6±0.2
Result Interpretation on Levinthal’s Medium
| Organisms | Growth |
| 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
- For cultivation of Haemophilus species, Levinthal’s Medium base with blood is used.
- It can be used to cultivate a variety fastidious organisms.
Limitation of Levinthal’s Medium
- For complete identification, it is recommended that colonies grown from pure culture be subject to biochemical, immunological and molecular testing.
