LÖWENSTEIN-JENSEN MEDIUM: FOUNDATION, PREPARATION AND USE - BIOLOGY - 2024

The Löwenstein-Jensen medium is a selective solid medium for the isolation and development of bacteria of the genus Mycobacterium, such as Mycobacterium tuberculosis, M. avium, among others, with the exception of the leprae species, which is not culturable.

Bacteria of the genus Mycobacterium do not grow in conventional culture media, therefore it was necessary to design a special medium for their isolation. The original medium was created by Löwenstein and later modified by Jensen.

Löwenstein-Jensen medium with colonies of Mycobacterium tuberculosis. Source: Agarwal et al / BioMed Central Ltd., courtesy of the Biology Image Library

The modification consisted in the elimination of the Congo red dye, replacing it with a higher concentration of malachite green. It also altered the concentrations of magnesium citrate and monopotassium phosphate.

Löwenstein-Jensen medium currently contains potato starch, asparagine, magnesium citrate, monopotassium phosphate, magnesium sulfate, malachite green, nalidixic acid, cycloheximide, lincomycin, beaten eggs, glycerin, and water.

Mycobacteria are normally isolated from sites that are not sterile, such as sputum, urine, abscesses, among others. This means that most of the samples will contain the usual microbiota of the area, plus the pathogen.

That is why the Löwenstein-Jensen medium contains a series of inhibitors in its composition represented by malachite green, antibiotics and antifungals.

In addition, samples that come from non-sterile sites must be decontaminated and neutralized before being seeded in the Löwenstein-Jensen medium.

Basis

The presence of egg and glycerin in the Löwenstein-Jensen medium stimulates the growth of mycobacteria because they provide the fatty acids and proteins necessary for the development of these microorganisms.

Löwenstein-Jensen medium contains malachite green, which is an inhibitor of the accompanying microbiota. But it also contains nalidixic acid (35 µg / mL), which inhibits the Gram negative microbiota, cycloheximide (400 µg / mL), which inhibits saprophytic fungi and yeasts, and lincomycin (2 µ / mL), which inhibits the Gram positive microbiota.

Some commercial houses prefer to add the following combination of antibiotics: polymyxin B 200,000 units / L, amphotericin B 10 mg / L, carbenicillin 50 mg / L and trimethoprim 10 mg / L.

This medium does not contain agar, therefore the solidification of the medium occurs due to the coagulation of the albumin present in the egg during sterilization.

Preparation

Weigh 37.3 g of the dehydrated medium in 600 ml of distilled water to which 12 ml of glycerol has previously been added. The mixture is heated, stirring frequently until completely dissolved. Autoclave the medium at 121 ° C for 15 minutes.

On the other hand, a homogeneous suspension of 1000 ml of fresh eggs should be prepared under aseptic conditions. Add the egg suspension to the 600 ml of medium prepared at a temperature of 50 - 60 ° C, avoiding air bubbles.

Antibiotic solutions are also added after sterilization in the autoclave.

Pour the medium into sterile screw-capped test tubes. Heat the tubes at 85 ° C for 45 minutes in an inclined position.

The color of the prepared medium is aquamarine green and may present whitish spots due to the presence of egg lipids.

The pH of the medium must be 7.2 ± 0.2

Store tubes in a refrigerator and protected from direct light until use. Temper before sowing.

There is a modification of the medium called "Gruft modification of the Löwenstein Jensen". This contains the same compounds as the classic medium but RNA-5mg / 100 mL is added, and as inhibitors it contains malachite green 0.025 g / 100 mL, penicillin 50 U / mL and nalidixic acid 35 ug / mL.

Applications

Löwenstein-Jensen medium is used for the isolation of mycobacteria from various types of samples. A Ziehl-Neelsen stain is recommended for any specimen in which the presence of mycobacteria is suspected.

Some samples come from sterile sites but others do not. Non-sterile samples must be decontaminated as appropriate:

Sputum

Sputum samples should be decontaminated as follows: determine the amount of sputum sample in ml and add the same amount of 4% NaOH to the sample and incubate at 37 ° C.

Shake the mixture frequently within 30 minutes. Subsequently centrifuge at 3000 RPM for 30 minutes.

Discard the supernatant over a phenolic disinfectant solution. Use the sediment for sowing, but first the pH must be neutralized.

To neutralize the sediment, 5% H ₂ SO ₄ is used in the presence of the phenol red indicator until it reaches a neutral pH that causes a salmon color.

Gastric lavage, bronchial lavage, and bronchial aspirate

In this case, the sample must be centrifuged at 3000 RPM for 30 minutes. The supernatant is discarded and the pellet is used. To decontaminate the sediment, add 3 ml of 4% NaOH and stir frequently at 37 ° C for a period of half an hour.

Centrifuge again, the supernatant is discarded and the pellet is used. The latter must be neutralized as explained in the sputum sample.

Urine

Allow the sample to settle in the refrigerator for 24 hours. Separate the supernatant. The remaining pellet should be centrifuged for 30 minutes at 3000 RMP. Discard the supernatant again and reconstitute the pellet with 3 ml of sterile physiological solution.

Add 3 ml of 4% NaOH and proceed to decontamination and neutralization as described before.

Ascites fluid, pleural fluid, cerebrospinal fluid

In this type of sample it is centrifuged and the supernatant is discarded. Perform a Gram on the sediment or observe directly under the microscope; If bacteria are not observed, the decontamination step is not necessary, nor is the neutralization step.

In this case the sample can be seeded directly using the sediment. If there are bacteria, proceed to decontaminate and neutralize as described above.

Biopsies

To this type of sample, 5 ml of distilled water must be added to later centrifuge at 1500 RPM for 10 minutes. Discard the supernatant and re-centrifuge the pellet at 3500 RPM for 30 minutes. Use the sediment to sow the culture medium.

Laryngeal swab

The swab should be placed in a sterile tube containing equal parts distilled water and 4% NaOH. The swab must be pressed against the walls of the tube so that the sample is diluted in the liquid. Centrifuge and use the sediment. Neutralize the sediment as already described.

Sown

The Löwenstein-Jensen medium is inoculated by adding 0.5 ml of the sample on the surface of the medium. Rotate the tube to distribute the sample throughout the medium. Do not use platinum handle.

A second tube containing Stonebrink medium can be seeded in order to isolate Mycobacterium bovis and other species that do not grow on Löwenstein-Jensen medium.

Incubation

The inoculated tubes are incubated aerobically at 37 ° C, with the cap slightly loose and inclined at approximately 5 ° and protected from light. The environment can be enriched with 5–10% carbon dioxide. Check cultures twice a week until colonies appear.

When the sample has been absorbed, the caps are tightened. The maximum incubation time is 8 weeks, if after this time there is no growth, it is reported as negative.

QA

The following strains can be used as quality control:

Mycobacterium tuberculosis ATCC 27294, Mycobacterium kansasii ATCC 12478, Mycobacterium avium ATCC 19291, Mycobacterium bovis ATCC 19219, Mycobacterium fortuitum ATCC 6841, Escherichia coli ATCC 25922, Streptococcus pyogenes ATCC 19615, Cryptococcus 320of ATCC of 19615, Cryptococcus 320of ATCC of nematococcus 320of

Excellent development is expected for the first three species mentioned, for M. fortuitum growth should be good, while for M. bovis little or no growth is expected. Meanwhile, species other than the genus Mycobacterium must be fully inhibited.

Limitations

The prepared medium must be protected from light, prolonged exposure to light causes the medium to turn from green to blue, in this case the medium can no longer be used. This is because malachite green is photosensitive.

The medium, as it contains eggs, can be easily contaminated if it is not handled aseptically. It can be dissolved if it becomes contaminated with proteolytic bacteria.

The cultivation and handling of bacteria of the genus Mycobacterium requires qualified personnel, who are aware of the biosecurity measures that must be followed to avoid being contaminated or contaminating others.

HCl should not be used in the neutralization step due to the formation of sodium chloride, which can be toxic to Koch's bacillus.

Samples should be kept refrigerated and protected from light while not being processed.

Reference

1. Francisco Soria Melguizo Laboratories. 2009. Löwenstein-Jensen selective medium. Available at: f-soria.es
2. Britannia Laboratories. 2017. Löwenstein-Jensen medium. Available at: britanialab.com.
3. Neogen Laboratories. Löwenstein-Jensen medium. Available at: foodsafety.neogen.com.
4. "Löwenstein-Jensen medium." Wikipedia, The Free Encyclopedia. 20 Nov 2018, 15:15 UTC. 24 Apr 2019, 18:34. wikipedia.org
5. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
6. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
7. Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.