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

Levinthal’s medium is a type of enriched culture medium that was developed by Samuel Levinthal in 1938. It is used for the isolation and identification of Haemophilus species, which are gram-negative bacteria that cause respiratory infections such as pneumonia, meningitis, and otitis media. Haemophilus species require certain growth factors that are not present in ordinary media, such as blood agar or chocolate agar. These growth factors are hemin (also known as factor X) and nicotinamide adenine dinucleotide (NAD) (also known as factor V). Levinthal’s medium contains these growth factors in the form of rabbit or human blood, which is added to a base medium consisting of peptone, beef extract, sodium chloride, and bacitracin. Bacitracin is an antibiotic that inhibits the growth of normal flora, such as staphylococci and streptococci, that may contaminate the specimens or interfere with the identification of Haemophilus species. By using Levinthal’s medium, Haemophilus species can be differentiated based on their ability to grow on the medium with or without added blood, and their hemolytic reactions on the agar surface. Levinthal’s medium is also useful for cultivating other fastidious organisms that require similar growth factors, such as Neisseria and Bordetella species. Levinthal’s medium is one of the classic media in microbiology that has contributed to the advancement of bacterial diagnosis and taxonomy.

• Peptic digest of animal tissue: This provides nitrogenous compounds, amino acids, peptides, and other growth factors for the bacteria.
• Beef extract: This provides organic nitrogen, carbon, and vitamins for the bacteria.
• Sodium chloride: This helps to maintain the osmotic balance of the medium and the bacterial cells.
• Bacitracin: This is an antibiotic that inhibits the growth of normal flora and other contaminants, which enhances the isolation and recovery of Haemophilus species.
• Rabbit or human blood: This provides two essential factors for the growth of Haemophilus species, namely factor X and factor V. Factor X is a heat-stable substance that is derived from hemin, which is associated with hemoglobin. Factor V is a heat-labile coenzyme that is also known as nicotinamide adenine dinucleotide (NAD). These factors are required by some strains of Haemophilus species to synthesize nucleic acids and other cellular components.

The final pH of the medium at 25°C is 7.6±0.2, which is suitable for the growth of Haemophilus species. The medium is prepared by suspending 45 grams of the dehydrated powder in 1000 ml of distilled water, heating to boiling to dissolve the medium completely, autoclaving at 121°C for 15 minutes, cooling to 50°C, adding 5 ml of sterile rabbit or human blood to 100 ml of medium, heating the mixture in a boiling water bath, allowing the deposits to settle, and dispensing the clear supernatant into sterile petri plates. The medium can be stored at 2-8°C for up to one month.

The composition of Levinthal’s medium makes it a selective and differential medium for Haemophilus species. The selective property is due to the presence of bacitracin, which inhibits other bacteria. The differential property is due to the presence of blood, which allows the identification of Haemophilus species based on their growth requirements for factor X and factor V and their hemolytic reactions on the agar. For example, Haemophilus influenzae requires both factor X and factor V for growth and produces non-hemolytic colonies. Haemophilus parainfluenzae requires only factor V for growth and produces non-hemolytic colonies. Haemophilus haemolyticus requires both factor X and factor V for growth and produces hemolytic colonies.

Levinthal’s medium is a selective and differential medium for the isolation and identification of Haemophilus species, which are gram-negative bacteria that cause respiratory infections such as pneumonia, bronchitis, and meningitis. Haemophilus species require certain growth factors that are not present in ordinary media, such as factor X (hemin) and factor V (NAD). These factors are found in the blood of animals or humans, and can be added to the medium to support the growth of Haemophilus species.

Levinthal’s medium contains a peptic digest of animal tissue and beef extract as sources of nitrogen and other nutrients, sodium chloride to maintain the osmotic balance, and bacitracin to inhibit the growth of normal flora that may interfere with the isolation of Haemophilus species. The medium also contains agar as a solidifying agent. The pH of the medium is adjusted to 7.6, which is optimal for the growth of Haemophilus species.

The blood added to the medium provides both factor X and factor V, as well as hemoglobin, which can be used to differentiate Haemophilus species based on their hemolytic reactions. Some Haemophilus species produce hemolysins that can lyse the red blood cells in the medium, resulting in clear zones around the colonies. These are called beta-hemolytic Haemophilus species. Other Haemophilus species do not produce hemolysins, but can use the hemoglobin released from the lysed red blood cells by other bacteria. These are called alpha-hemolytic Haemophilus species, and they produce greenish or brownish zones around the colonies. Some Haemophilus species do not have any hemolytic activity, and they produce non-hemolytic or gamma-hemolytic colonies.

By observing the growth and hemolytic reactions of Haemophilus species on Levinthal’s medium, one can presumptively identify them based on their requirements for factor X and factor V. For example, Haemophilus influenzae requires both factor X and factor V for growth, and is usually non-hemolytic or alpha-hemolytic. Haemophilus parainfluenzae requires only factor V for growth, and is usually non-hemolytic or beta-hemolytic. Haemophilus ducreyi requires only factor X for growth, and is usually non-hemolytic or beta-hemolytic. However, these results are not conclusive, and further biochemical, immunological, molecular, or mass spectrometry testing should be performed on pure cultures for complete identification.

To prepare Levinthal’s Medium, you will need the following materials and equipment:

• Levinthal’s Medium Base powder
• Distilled water
• Measuring cylinder
• Weighing balance
• Flask
• Autoclave
• Sterile rabbit or human blood
• Water bath
• Petri plates

The steps for preparing Levinthal’s Medium are as follows:

1. Measure 1000 ml of distilled water using a measuring cylinder and pour it into a flask.
2. Weigh 45 grams of Levinthal’s Medium Base powder using a weighing balance and add it to the flask.
3. Heat the flask to boiling to dissolve the medium completely. You may need to swirl the flask gently to mix the contents.
4. Dispense the medium in 100 ml amounts into smaller flasks or bottles and label them.
5. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
6. Cool the medium to 50°C in a water bath or on a benchtop.
7. Add 5 ml of sterile rabbit or human blood to each 100 ml of medium and mix well.
8. Heat the mixture in a boiling water bath for a few minutes to lyse the red blood cells and release the X and V factors.
9. Allow the deposits to settle and dispense the clear supernatant into sterile petri plates.
10. Store the plates in a refrigerator until use.

You have now prepared Levinthal’s Medium for cultivating Haemophilus species. Make sure to use proper aseptic techniques when handling the medium and the cultures.😊

The growth of Haemophilus species on Levinthal’s medium depends on their requirement for X and V factors. Different species can be differentiated by their growth patterns on the medium with or without the addition of these factors. The following table summarizes the results:

Some Haemophilus species also produce hemolysis on the medium, which can be observed as a clear zone around the colonies. The hemolytic reactions are as follows:

Therefore, by using Levinthal’s medium, one can presumptively identify the Haemophilus species based on their growth and hemolysis characteristics. However, further confirmation tests are required for definitive identification.

Levinthal’s medium is mainly used for the cultivation and identification of Haemophilus species, which are gram-negative bacteria that cause various infections in humans and animals. Haemophilus species require specific growth factors, such as X factor (hemin) and V factor (NAD), which are provided by the blood added to the medium. The medium also contains bacitracin, which inhibits the growth of normal flora and enhances the recovery of Haemophilus species.

Levinthal’s medium can be used to presumptively identify pathogenic Haemophilus species by determining their in vitro growth requirements for X and V factors and by observing their hemolytic reactions on the agar. For example, Haemophilus influenzae requires both X and V factors and produces non-hemolytic colonies, whereas Haemophilus parainfluenzae requires only V factor and produces hemolytic colonies. Other Haemophilus species may have different growth and hemolysis patterns.

Levinthal’s medium can also be used for cultivating a variety of fastidious organisms, such as Neisseria species, Streptococcus species, Listeria species, Brucella species, Bordetella species, and Campylobacter species. These organisms may have different nutritional or environmental requirements that are met by the components of Levinthal’s medium. For example, Neisseria species require a moist atmosphere with increased carbon dioxide, which can be achieved by incubating the plates in a candle jar. Streptococcus species require a lower pH than other bacteria, which can be adjusted by adding acid to the medium. Listeria species require cold enrichment, which can be done by incubating the plates at 4°C for several days.

Levinthal’s medium is a useful tool for the isolation and identification of various bacteria that cause human and animal diseases. However, it is not sufficient for the complete identification of these organisms, and further biochemical, immunological, molecular, or mass spectrometry testing should be performed on colonies from pure culture.

Levinthal’s Medium is a useful medium for cultivating Haemophilus species and other fastidious organisms, but it also has some limitations that should be considered. Some of these limitations are:

• Levinthal’s Medium is not selective for Haemophilus species, as it can support the growth of other bacteria that require X and V factors, such as some Neisseria and Pasteurella species. Therefore, it is important to use other methods to confirm the identity of the isolates, such as biochemical tests, immunological tests, molecular tests, or mass spectrometry.
• Levinthal’s Medium is not differential for Haemophilus species, as it does not show any distinctive reactions or color changes based on the presence or absence of X and V factors. Therefore, it is necessary to use other media, such as chocolate agar or Mueller-Hinton agar with X and V factors added separately, to determine the growth requirements of the isolates.
• Levinthal’s Medium requires the addition of fresh blood, which can be a source of contamination and variability. The quality and quantity of blood can affect the performance of the medium, as different blood sources may have different levels of X and V factors. Moreover, blood can introduce unwanted microorganisms or substances that may interfere with the growth or identification of Haemophilus species.
• Levinthal’s Medium has a short shelf life, as it can lose its potency and clarity over time. The medium should be used within one week after preparation and stored at 2-8°C. The medium should also be protected from light and moisture, as these can affect its stability and pH.

These are some of the limitations of Levinthal’s Medium that should be taken into account when using it for cultivation and identification of Haemophilus species. Despite these limitations, Levinthal’s Medium is still a valuable medium for isolating and studying these fastidious organisms.

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