What is Levinthal’s Medium?

• Levinthal’s Medium is a specialized culture medium used for the cultivation of Haemophilus species. Haemophilus is a genus that comprises several species capable of causing various infections, all of which share common morphology and have a dependency on blood-derived factors for their growth.
• Among the Haemophilus species, Haemophilus influenzae is the most virulent pathogen, often leading to bloodstream invasion and meningitis in children under the age of two. Although other Haemophilus species cause diseases less frequently, they still require specific conditions for growth. These gram-negative rods can be cultured on blood agar, a type of agar that contains blood.
• The blood present in Levinthal’s Medium provides two essential factors necessary for the growth of many Haemophilus species: factor-X and factor-V. These factors are crucial for the survival and proliferation of the bacteria. Factor-X is a heat-stable substance known as hemin, which is associated with hemoglobin. Factor-V, on the other hand, is a heat-labile coenzyme called Nicotinamide Adenine Dinucleotide (NAD).
• To create Levinthal’s Medium, a base medium is used, which is supplemented with blood. The blood used can be from rabbits or humans and serves as a source of factor-X and factor-V. Other nutrients required for bacterial growth, such as nitrogen compounds, are provided by incorporating peptic digest of animal tissue and beef extract into the medium. Additionally, sodium chloride is included to maintain the osmotic balance of the medium.
• Levinthal’s Medium plays a crucial role in the cultivation and identification of Haemophilus species. By determining the in vitro growth requirements for X and V factors and observing hemolytic reactions, pathogenic Haemophilus species can be presumptively identified. This medium provides the necessary conditions for the growth and study of these bacteria, contributing to our understanding and ability to diagnose and treat Haemophilus infections.

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

Principle of Levinthal’s Medium

The principle of Levinthal’s Medium revolves around providing the necessary growth factors and conditions for the cultivation and identification of Haemophilus species.

The whole blood of rabbits or humans is a key component of Levinthal’s Medium. It contains two essential factors known as factor-X and factor-V, which are crucial for the growth of the type species of Haemophilus influenzae. Factor-X is a heat-stable substance called hemin, which is associated with hemoglobin. On the other hand, factor-V is a heat-labile coenzyme known as Nicotinamide Adenine Dinucleotide (NAD).

In addition to the blood components, Levinthal’s Medium incorporates other nutrients required for bacterial growth. Nitrogen compounds are supplied by adding a peptic digest of animal tissue and beef extract to the medium. These nutrients support the metabolic processes of Haemophilus species.

To maintain the osmotic balance of the medium, sodium chloride is included. Osmotic balance is crucial for the bacteria’s growth and survival, as it ensures proper water and ion exchange within the cells.

Levinthal’s Medium also contains bacitracin, which inhibits the growth of normal flora. By suppressing the growth of other microorganisms present in the sample, the recovery of Haemophilus species is enhanced. This allows for better isolation and identification of the pathogenic Haemophilus strains.

One of the key applications of Levinthal’s Medium is the presumptive identification of pathogenic Haemophilus species. By observing the in vitro growth requirements for X and V factors, as well as analyzing the hemolytic reactions on the agar, it is possible to identify these bacteria. The growth of Haemophilus species on the medium and the specific reactions they exhibit can provide valuable information for diagnosing and understanding the pathogenicity of these organisms.

In summary, the principle of Levinthal’s Medium is based on providing the necessary growth factors, nutrients, and conditions for the cultivation and identification of Haemophilus species. By incorporating factors derived from whole blood, supplying essential nutrients, maintaining osmotic balance, and inhibiting normal flora, this medium facilitates the growth and study of these bacteria in the laboratory setting.

Preparation of Levinthal’s Medium

The preparation of Levinthal’s Medium involves the following steps:

1. Suspend 45 grams of the medium in 1000 ml of distilled water. This can be done by slowly adding the medium to the water while stirring continuously to ensure uniform dispersion.
2. Heat the suspension to boiling point, allowing the medium to dissolve completely. Boiling helps in the thorough dissolution of the components.
3. Once the medium is completely dissolved, it needs to be sterilized. Dispense the medium in 100 ml aliquots into suitable containers, such as glass bottles or flasks, that can withstand autoclaving.
4. Sterilize the medium by autoclaving at 15 pounds of pressure (121°C) for 15 minutes. Autoclaving is a common method used to sterilize media and equipment by subjecting them to high temperature and pressure.
5. After the autoclaving process, cool the medium to approximately 50°C. It’s important to allow the medium to cool down to a suitable temperature to avoid damaging any heat-sensitive components.
6. Add 5 ml of sterile rabbit or human blood to each 100 ml of the cooled medium. The blood serves as a source of essential growth factors, particularly factor-X and factor-V, necessary for the growth of Haemophilus species.
7. Heat the mixture in a boiling water bath. This step helps in achieving proper mixing and incorporation of the blood into the medium.
8. Allow the mixture to settle, typically by allowing it to stand undisturbed for a period of time. As a result, the deposits will settle at the bottom of the container.
9. Once the deposits have settled, carefully dispense the clear supernatant without disturbing the settled deposits. This ensures that the clear liquid portion, which contains the desired components, is collected for further use.
10. The final step is to pour the prepared medium into sterile petri plates. Petri plates are commonly used in microbiology laboratories for culturing microorganisms. Ensure the plates are sterile to prevent contamination.

By following these steps, one can prepare Levinthal’s Medium, which provides the necessary nutrients and growth factors for the cultivation of Haemophilus species.

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

Quality Control of Levinthal’s Medium

The quality control of Levinthal’s Medium involves several parameters that are assessed to ensure its performance and reliability. The following aspects are evaluated:

1. Appearance: The medium should have a cream to yellow homogeneous free-flowing powder consistency.
2. Gelling: After preparation, the medium should form a firm gel comparable to a 2.0% agar gel. This property is important for providing a solid surface for bacterial growth.
3. Colour and Clarity of Prepared Medium: The basal medium, before the addition of blood and heating, should appear as a light yellow-colored, clear to slightly opalescent gel. After the addition of blood and heating, the medium should turn into a chocolate brown-colored, opaque gel in the Petri plates.
4. Reaction: The pH of a 4.5% w/v aqueous solution of Levinthal’s Medium should be within the range of 7.6±0.2 at 25°C. This pH range is critical for supporting the growth of specific microorganisms.
5. pH: The pH of the prepared medium should fall within the range of 7.40-7.80. Maintaining the appropriate pH ensures optimal conditions for bacterial growth.
6. Cultural Response: The cultural response of Levinthal’s Medium is evaluated using specific organisms under controlled conditions. After incubation at 35-37°C for 18-24 hours in an environment of 5-10% CO2 and 70% humidity, the growth and recovery of the organisms are assessed.
   • Haemophilus influenzae (ATCC 35056): The medium should support luxuriant growth, with 50-100 colony-forming units (CFU) observed, and the recovery should be equal to or greater than 70%.
   • Staphylococcus aureus (ATCC 25923): Similar to H. influenzae, the medium should show luxuriant growth with 50-100 CFU and a recovery rate of equal to or greater than 70%.
   • Streptococcus pyogenes (ATCC 19615): Again, luxuriant growth of 50-100 CFU with a recovery rate of equal to or greater than 70% should be observed.

By evaluating these parameters, the quality control of Levinthal’s Medium ensures that it meets the necessary specifications and provides a suitable environment for the growth and identification of target microorganisms.

Uses of Levinthal’s Medium

• Levinthal’s Medium finds primary use in the cultivation of Haemophilus species when supplemented with blood. The addition of blood provides the necessary growth factors, such as factor-X and factor-V, required for the growth and survival of Haemophilus bacteria. This medium serves as a specific and conducive environment for the cultivation of Haemophilus species.
• In addition to its specific use for Haemophilus, Levinthal’s Medium can also be employed for cultivating various other fastidious organisms. Fastidious organisms are those that have strict and specific nutritional requirements, making them difficult to cultivate on standard media. The enriched composition of Levinthal’s Medium, with its inclusion of blood and other essential nutrients, meets the specific nutritional needs of these fastidious organisms, allowing for their successful cultivation.
• By providing the necessary growth factors and nutrients, Levinthal’s Medium enables the successful growth and isolation of Haemophilus species and other fastidious organisms. This medium serves as a valuable tool in microbiology laboratories for studying these organisms, identifying pathogenic strains, and conducting research on their characteristics and behaviors.

Limitation of Levinthal’s Medium

• One limitation of Levinthal’s Medium is that it alone is not sufficient for the complete identification of microorganisms. While the medium provides a suitable environment for the growth and isolation of Haemophilus species and other fastidious organisms, further testing is required for their complete identification.
• To achieve comprehensive identification, additional tests such as biochemical, immunological, molecular, or mass spectrometry testing should be performed on the colonies obtained from pure cultures. These tests help to characterize the specific properties, metabolic activities, and genetic profiles of the microorganisms.
• Biochemical tests involve examining the enzymatic activities, metabolic pathways, and utilization of specific substrates by the microorganisms. Immunological tests focus on the detection of specific antigens or antibodies associated with the microorganisms, providing information about their immunological properties.
• Molecular testing, such as polymerase chain reaction (PCR), can identify specific DNA sequences or genes unique to the microorganisms, aiding in their precise identification. Mass spectrometry analysis can provide detailed information about the molecular composition and profiles of the microorganisms, assisting in their differentiation and identification.
• By combining these additional tests with the growth obtained on Levinthal’s Medium, a more accurate and complete identification of the microorganisms can be achieved. It is important to employ a range of testing methods to ensure reliable and conclusive results, especially in the case of microbial identification for clinical, research, or epidemiological purposes.

FAQ

References

• https://www.himedialabs.com/media/TD/M472.pdf
• https://pgblazer.com/best-culture-medium-for-primary-isolation-of-h-influenzae/