Lowenstein Jensen (LJ) Media- Composition, Principle, Preparation, Results, Uses

Mycobacterium tuberculosis is the causative agent of tuberculosis, a chronic infectious disease that affects mainly the lungs but can also involve other organs and tissues. Tuberculosis is one of the leading causes of death from infectious diseases worldwide, especially in low- and middle-income countries.

The diagnosis of tuberculosis relies on the detection and identification of Mycobacterium tuberculosis from clinical specimens, such as sputum, urine, blood, cerebrospinal fluid, etc. However, Mycobacterium tuberculosis is a slow-growing bacterium that requires special culture conditions and techniques to grow and isolate.

Lowenstein Jensen (LJ) Media is a selective medium commonly used for Mycobacterium cultivation and isolation of species from clinical specimens, most notably Mycobacterium tuberculosis. LJ medium was originally formulated by Lowenstein, who incorporated congo red and malachite green to inhibit unwanted bacteria. The present formulation comprises a glycerate egg-based medium and is based upon Jensens modification. Jensen modified Lowensteins medium by altering the citrate and phosphate contents, eliminating the congo red dye, and Increasing the concentration of malachite green. Gruft improved the selectivity of L. J. Medium by adding penicillin, nalidixic acid, and ribonucleic acid (RNA).

LJ A wide range of Mycobacteria can grow in this medium, which can also be used for niacin testing. When grown on LJ medium, M. tuberculosis appears as brown, granular colonies (sometimes called "buff, rough and tough"). The medium must be incubated for a significant length of time, usually four weeks, due to the slow doubling time of M. tuberculosis (15–20 hours) compared with other bacteria.

LJ medium is one of the most popular means of culturing mycobacteria, as recommended by the International Union against Tuberculosis. It is widely used for the diagnosis of mycobacterial infections, testing antibiotic susceptibility of isolates, and differentiating different species of Mycobacterium (by colony morphology, growth rate, biochemical characteristics, and microscopy).

In this article, we will discuss the composition, principle, preparation, results interpretation, uses, and limitations of the LJ medium in detail.

Lowenstein Jensen (LJ) media is a type of egg-based medium that is used for the cultivation and isolation of Mycobacterium species, especially Mycobacterium tuberculosis. The medium was developed by Lowenstein, who incorporated congo red and malachite green to inhibit unwanted bacteria. The present formulation, based on Jensen`s modification, eliminates congo red and uses a moderate concentration of malachite green.

The composition of LJ media as applicable to M. tuberculosis is:

• Potato Flour (Potato Starch): 30.0 g

• L-Asparagine: 3.6 g

• Monopotassium Phosphate: 2.4 g

• Magnesium Citrate: 0.6 g

• Malachite Green: 0.4 g

• Magnesium Sulfate: 0.24 g

• Glycerol: 12 ml

• Egg suspension: 1000 ml

• Distilled Water: 600 ml

For the cultivation of M. bovis, glycerol is omitted, and sodium pyruvate is added.

The ingredients of LJ media have different roles in supporting the growth of mycobacteria and inhibiting the growth of contaminants. These roles will be explained in the next point.

Lowenstein Jensen, Gruft Modification is a variant of LJ Medium that contains some additional ingredients to increase the selectivity and sensitivity of the medium for the isolation and cultivation of mycobacteria. The additional ingredients are:

• Penicillin G: This is an antibiotic that inhibits the growth of gram-positive bacteria that may contaminate the clinical specimens.

• Nalidixic acid: This is another antibiotic that inhibits the growth of some gram-negative bacteria that may also contaminate the clinical specimens.

• Ribonucleic acid (RNA): This is a nucleic acid that acts as a growth stimulant for mycobacteria and contributes to an increase in the isolation rate of mycobacterium tuberculosis and other species.

These ingredients are added to the standard LJ Medium along with malachite green, which prevents the growth of most other contaminants surviving specimen decontamination while encouraging mycobacterial growth. The composition of Lowenstein Jensen, Gruft Modification is as follows:

| Ingredient | Amount per liter |

| --- | --- |

| Potato Flour | 30.0 g |

| Malachite Green | 0.4 g |

| Asparagine | 3.6 g |

| Magnesium Sulfate | 0.24 g |

| Monopotassium Phosphate | 2.4 g |

| Whole Eggs | 1000.0 ml |

| Magnesium Citrate | 0.6 g |

| Glycerol | 12.0 ml |

| Penicillin G | 80,000 U |

| Nalidixic Acid | 56.0 mg |

| Ribonucleic Acid | 80.0 μg |

Lowenstein Jensen, Gruft Modification is used for the diagnosis of Mycobacterial infections, especially in specimens with a high bacterial load or a low number of mycobacteria. It is also used for testing the antibiotic susceptibility of isolates.

Lowenstein Jensen (LJ) Media is based on the principle that Mycobacteria require certain nutrients and environmental conditions for their growth and differentiation. The media contains various ingredients that serve different purposes in supporting the growth of Mycobacteria and inhibiting the growth of other contaminants.

• L-Asparagine and Potato Flour are sources of nitrogen and vitamins for the Mycobacteria. L-Asparagine is an essential amino acid that is involved in protein synthesis and metabolism. Potato Flour provides starch, which can be hydrolyzed by some Mycobacteria to produce acid.

• Monopotassium Phosphate and Magnesium Sulfate enhance organism growth and act as buffers. Monopotassium Phosphate provides phosphorus, which is a component of nucleic acids and phospholipids. Magnesium Sulfate provides magnesium, which is a cofactor for many enzymes. Both compounds also help to maintain the pH of the media within the optimal range for Mycobacteria (6.6 to 7.0).

• Malachite Green inhibits the growth of the majority of contaminants that survive specimen decontamination while encouraging the growth of Mycobacteria. Malachite Green is a dye that has bacteriostatic and bactericidal properties against many Gram-positive and Gram-negative bacteria. However, it has little effect on Mycobacteria, which have a thick and waxy cell wall that protects them from the dye. Malachite Green also gives the media a distinctive green color, which helps to differentiate Mycobacterial colonies from other organisms.

• Egg Suspension provides fatty acids and protein required for the metabolism of Mycobacteria. Egg Suspension is prepared from fresh eggs under aseptic conditions and contains egg albumin and egg yolk. Egg albumin provides protein, which is a source of amino acids and nitrogen. Egg yolk provides fatty acids, which are essential for the synthesis of mycolic acids, a characteristic component of the Mycobacterial cell wall. When heated, the egg albumin coagulates, thus providing a solid surface for inoculation.

• Glycerol serves as a carbon source and is favorable to the growth of the human-type tubercle bacillus while being unfavorable to the bovine type. Glycerol is a simple sugar alcohol that can be utilized by some Mycobacteria as an energy source. Glycerol also affects the virulence and pathogenicity of Mycobacteria by influencing their lipid metabolism and cell wall composition. Glycerol favors the growth of human-type tubercle bacillus (Mycobacterium tuberculosis), which causes tuberculosis in humans while inhibiting the growth of bovine type (Mycobacterium bovis), which causes tuberculosis in cattle.

In addition to these ingredients, Lowenstein Jensen Gruft Modification contains three additional components that increase the selectivity of the media:

• Penicillin and Nalidixic Acid, along with malachite green, prevent the growth of the majority of contaminants surviving decontamination of the specimen while encouraging the earliest possible growth of Mycobacteria. Penicillin is an antibiotic that inhibits the synthesis of peptidoglycan, a component of bacterial cell walls. Nalidixic Acid is an antibacterial agent that inhibits DNA replication in bacteria. Both agents are effective against many Gram-positive and Gram-negative bacteria but not against Mycobacteria, which have different cell wall structures and DNA replication mechanisms.

• RNA acts as a stimulant and helps to increase the isolation rate of Mycobacteria. RNA is a nucleic acid that is involved in protein synthesis and gene expression. RNA can stimulate the growth of Mycobacteria by providing nucleotides and enhancing their metabolic activity.

To prepare Lowenstein Jensen (LJ) Media, you will need the following ingredients and equipment:

• Potato flour

• L-asparagine

• Monopotassium phosphate

• Magnesium citrate

• Malachite green

• Magnesium sulfate

• Glycerol

• Egg base

• Nalidixic acid (optional)

• Penicillin (optional)

• RNA (optional)

• Purified water

• Autoclave

• Blender

• Flask

• Beaker

• Test tubes or bottles

• Insipissator

The steps for preparing Lowenstein Jensen (LJ) Media are as follows:

1. Dissolve 37.3 g of potato flour, 3.6 g of L-asparagine, 2.4 g of monopotassium phosphate, 0.6 g of magnesium citrate, 0.4 g of malachite green, and 0.24 g of magnesium sulfate in 600 mL of purified water containing 12 mL of glycerol. Heat the solution with frequent agitation until it dissolves completely.

1. Sterilize the solution by autoclaving it at 121°C for 15 minutes. Cool it to room temperature and store it in the refrigerator until needed.

1. Prepare a uniform suspension of fresh eggs under aseptic conditions. Scrub the eggs thoroughly with a hand brush in water and soap, then soak them in a soap solution for 30 minutes. Rinse the eggs well in running water and soak them in 70% ethanol for 15 minutes. Crack the eggs into a sterile flask and blend them for 30 seconds to one minute. You will need about 1000 mL of egg suspension for one batch of medium.

1. Aseptically mix the egg suspension with the sterile mineral salt solution (600 mL) in a large sterile flask, avoiding air bubbles. If you want to make the Gruft modification, add 56 mg of nalidixic acid, 52.8 mg of penicillin, and 0.05 mg of RNA to the mixture.

1. Dispense the finished medium into sterile screw-cap test tubes or bottles in 6-8 mL volumes. Close the caps tightly and inspissate the medium without delay to prevent sedimentation of heavier ingredients.

1. Coagulate the medium by heating it at 85°C for 45 minutes in an inspissator. Place the tubes or bottles in a slanted position to create a slope for inoculation.

1. Store the prepared medium in a dark place at room temperature until use.

• Cultures should be read within 5 to 7 days after inoculation and once a week thereafter for up to 8 weeks.

• The typical colonies of Mycobacterium tuberculosis are non-pigmented, rough, dry, and raised on LJ medium.

• The green color of the medium is due to the presence of malachite green, which is one of the selective agents to prevent the growth of most other contaminants.

• Rapid growers have mature colonies within 7 days; slow growers require more than 7 days for mature colony forms.

• Pigment production can be observed by exposing the cultures to light for 5 minutes. Pigmented colonies can be classified as:

• White, cream or buff = Nonchromogenic (NC)

• Lemon, yellow, orange, red = Chromogenic (Ch)

• Some species of Mycobacterium can produce iron uptake, which results in rusty brown colonies on LJ medium with iron. This can be used as a differentiation criterion between slow and rapid growers.

• Niacin test can also be performed on LJ medium by adding cyanogen bromide reagent to the colonies. A positive result is indicated by a yellow color change due to the presence of niacin in the culture. This test can help identify M. tuberculosis from other mycobacteria.

Lowenstein Jensen (LJ) Media has several uses in the diagnosis and testing of Mycobacterium species, especially Mycobacterium tuberculosis, the causative agent of tuberculosis. Some of the main uses are:

• Isolation and cultivation of Mycobacterium species from clinical specimens. LJ media is a selective medium that inhibits the growth of most other bacteria by the presence of malachite green, penicillin, and nalidixic acid. It also provides nutrients and conditions that favor the growth of mycobacteria, such as glycerol, asparagine, egg suspension, and CO2 atmosphere.

• Testing antibiotic susceptibility of isolates. LJ media can be used to perform drug susceptibility testing (DST) of Mycobacterium species by incorporating different concentrations of antibiotics into the medium and observing the growth or inhibition of colonies. This can help to determine the appropriate treatment for patients with tuberculosis or other mycobacterial infections.

• Differentiating different species of Mycobacterium by colony morphology, growth rate, biochemical characteristics, and microscopy. LJ media can help to identify and classify different Mycobacterium species based on their appearance, growth rate, pigment production, niacin accumulation, catalase activity, and acid-fast staining on the medium. For example, M. tuberculosis typically produces non-pigmented, rough, dry colonies that grow slowly and are niacin-positive and catalase negative.

LJ media is one of the most widely used media for the diagnosis and testing of Mycobacterium species. However, it also has some limitations that will be discussed in the next point.

• LJ media is intended for general use and may not support the growth of fastidious organisms.

• LJ media may support the growth of gram-positive contaminants (e.g., streptococci) as well as gram-negative bacilli. Some saprophytes may also grow on the medium.

• Proteolytic contaminants may cause localized or complete medium digestion.

• Biochemical and/or serological tests are recommended for the complete identification of colonies from a pure culture.

• LJ media requires incubation in a 5-10% CO2 atmosphere to recover mycobacteria. Mycobacteria are not recovered well from candle extinction jars for unknown reasons.

• The media should be protected from all sources of light, as malachite green is very photosensitive.

• Negative culture results do not rule out active infection by mycobacteria.

• Due to nutritional variation, some strains may grow poorly or fail to grow on this medium. Further tests are necessary for confirmation of Mycobacterium spp.

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