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 cultivable.
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.
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..
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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 companies 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..
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 autoclaving..
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 the 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 classical 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..
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 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 over a 30 minute period. 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, HtwoSW4 5% in the presence of the phenol red indicator until it reaches a neutral pH that produces a salmon color.
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..
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..
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 no bacteria are observed, the decontamination step is not necessary, nor is the neutralization step.
In this case the sample can be sown directly using the sediment. If there are bacteria, proceed to decontaminate and neutralize as described above..
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.
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.
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 can be seeded containing Stonebrink medium for the purpose of isolating Mycobacterium bovis and other species that do not grow in the Löwenstein-Jensen medium.
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.
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 neoformans ATCC 32045
Excellent development is expected for the first three species mentioned, for M. fortuitum growth must be good, while for M. bovis little or no growth is expected. Whereas, species other than the genus Mycobacterium must be fully inhibited.
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 egg, can be easily contaminated if it is not handled aseptically. Can be dissolved if contaminated with proteolytic bacteria.
The cultivation and manipulation of bacteria of the genus Mycobacterium requires qualified personnel, who are aware of the biosafety 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..
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