Lowenstein Jensen medium : Principle , Composition & Cultural Characteristics

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Lowenstein Jensen medium is recommended for the isolation and cultivation of Mycobacterium species. It was originally formulated by Lowenstein, which included dyes such as congo red and malachite green. Jensen altered Lowenstein’s medium by changing the citrate and phosphate contents, removing the Congo red dye, and increasing the malachite green concentration. Gruft enhanced the selectivity of L. J. Medium by adding two antimicrobics.

Principle of Lowenstein Jensen medium

  • The combination of penicillin, nalidixic acid, and malachite green inhibits the growth of the majority of contaminants that survive decontamination of the specimen while encouraging the earliest possible growth of Mycobacteria.
  • RNA works as a stimulant, assisting in the isolation of Mycobacteria. If bovine or other glycerophobic strains are to be grown, do not add glycerol to the medium.
  • Malachite green is both an inhibitor and a pH indicator. The formation of a blue zone shows a reduction in pH caused by gram-positive contaminants (e.g. Streptococci) while the formation of a yellow zone indicates dye degradation caused by gram-negative bacilli. Proteolytic contaminants cause localized or total medium digestion.

Composition of Lowenstein Jensen medium

IngredientsGms / Litre
L-Asparagine3.600
Potassium dihydrogen phosphate2.400
Magnesium sulphate0.240
Magnesium citrate0.600
Potato starch, soluble30.000
Malachite green0.400
Composition of Lowenstein jensen medium

Type of specimen

  • Sputum

Preparation procedure of Lowenstein Jensen medium

  • Suspend 37.24 grams in 600 mL purified / distilled water containing 12 mL glycerol (for bovine bacteria or other glycerophobic organisms additions of glycerol is not desirable).
  • If necessary, heat the medium to completely dissolve it.
  • Sterilize by autoclaving at 15 pounds of pressure (121 degrees Celsius) for 15 minutes.
  • Cool to 45-50°C. Meanwhile, make 1000 mL of aseptically collected whole egg emulsion.
  • Aseptically add and gently mix egg emulsion base and Gruft Mycobacterial Supplement (if desired) to obtain a uniform mixture.
  • Distribute in sterile screw-capped tubes.
  • Arrange the tubes in a slanted style.
  • Coagulate and inspissate the medium in an inspissator water bath or autoclave at 85°C for 45 minutes.

Quality Control of Lowenstein Jensen medium

AppearanceGreenish blue to peacock blue homogeneous free flowing powder
Color and Clarity of prepared mediumThe mixture of sterile basal medium and whole egg emulsion, when inspissated, coagulates to yield pale bluish green coloured, opaque smooth slants
ReactionReaction of 3.7% w/v aqueous solution at 25°C. pH : 7.4±0.2
Cultural ResponseCultural characteristics observed in presence of 5-10% CO2, with added egg emulsion base, after an incubation at 35-37ºC for 2-4 weeks.
Quality Control of Lowenstein Jensen medium
  • Appearance of Lowenstein Jensen medium

Cultural Response on Lowenstein Jensen medium

Mycobacterium aviumluxuriantsmooth, non-pigmented colonies
Mycobacterium gordonaeluxuriantsmooth, yellow, orange colonies
Mycobacterium kansasiiluxuriantphotochromogenic, smooth to rough
Mycobacterium smegmatisluxuriantwrinkled,creamy white colonies
M. tuberculosisluxuriantgranular, rough, warty, dry friable colonies
Cultural Response on Lowenstein Jensen medium

Limitations of Lowenstein Jensen medium

  • This medium is intended for general use and may not support the growth of fastidious organisms.
  • The medium may support the growth of gram-positive contaminants (e.g., streptococci) as well as gram-negative bacilli.
  • Some Saprophytes may grow on the medium as well.
  • Proteolytic contaminants cause localized or complete medium digestion.

Storage and Shelf Life

  • In a tightly closed container, store below 10-30°C, and the prepared medium at 2-8°C. Use before the expiration date printed on the label.
  • Due to the hygroscopic nature of the product, the product should be stored dry after opening and tightly capping the bottle to prevent lump formation.
  • Improper product storage may result in the formation of lumps.
  • Store in a dry, ventilated area away from extremes of temperature and ignition sources. After use, tightly close the container.
  • Use before the expiration date printed on the label.
  • When used within the specified expiry period, the product performs best.

Reference

  1. A.V. Hardy, et al, Am. J. Publ. Hlth. 48(1), 754 (1958)
  2. Boisvert H., 1960, Ann. Inst. Pasteur, 99:600.
  3. Cernoch P., Enns R., Saubolle M. and Wallace R., 1994, Cumitech, 16A, Laboratory Diagnosis of the Mycobacterioses coord , Ed., Weissfeld , ASM, Washington, D. C

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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