Lowenstein-Jensen (LJ) Medium : Principle, Preparation and Colony Morphology

Lowenstein-Jensen (LJ) Medium : Principle, Preparation and Colony Morphology

Lowenstein-Jensen (LJ) Medium is used in the isolation and cultivation of Mycobacterium species.Solid media used for the isolation and cultivation of Mycobacteria are egg- based or agar- based.Egg- based media contain whole eggs or egg yolk, potato flour, salts and glycerol and are solidified by insipissation.Of the egg- based media, Lowenstein-Jensen (LJ) Medium is the most commonly used medium.It was was originally formulated by Lowenstein, containing red congo and green malachite dyes.Jensen modified the Lowensteins medium by altering the citrate and phosphate content, eliminating the congo red dye and increasing the malachite green concentration.Gruft further modified the L J medium with the addition of two antimicrobials to increase selectivity.This medium supports the growth of a wide range of mycobacteria and can also be used for niacin testing.

Principle of Lowenstein-Jensen (LJ) Medium

  • Penicillin and Nalidixic acid along with malachite green prevent the growth of the majority of contaminants that survive specimen decontamination while encouraging the earliest possible growth of Mycobacteria.
  • RNA acts as a stimulant and helps to increase the isolation rate of Mycobacteria.
  • Do not add glycerol to the medium if bovine or other glycerophobic strains are grown.
  • Malachite green acts as an inhibitor and also as a pH indicator.
  • Blue zone formation indicates a decrease in pH by gram- positive contaminants( e.g.Streptococci) and yellow dye destruction zones by gram- negative bacilli.
  • Proteolytic contaminants cause localized or complete medium digestion. Hardy et al. recommended that each specimen be inoculated and incubated in triplicate,so as to  identify saprophytes at room temperature (25°C),to identify presence or absence of pigmentation by photochromogens and scotochromogens at 35°C alternately in light and dark as per the type of organism.

Composition of Lowenstein-Jensen (LJ) Medium

IngredientsGms / 600 ml
L-Asparagine3.600
Monopotassium phosphate2.400
Magnesium sulphate0.240
Magnesium citrate0.600
Potato starch, soluble30.000
Malachite green0.400
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How to Prepare Lowenstein-Jensen (LJ) Medium

  1. Suspend 37.24 grams in 600 ml of distilled water containing 12 ml of glycerol( additions of glycerol are not desirable for bovine bacteria or other glycerophobic organisms). If necessary, heat to dissolve the medium completely.
  2. Sterilize at 15 lbs( 121 ° C) pressure for 15 minutes by autoclaving. In the meantime, prepare 1000 ml of whole egg emulsion collected aseptically.
  3. Add and mix the egg emulsion base and gruft mycobacterial supplement( if desired) gently to obtain a uniform mixture.
  4. Distribute in capped sterile screw tubes. Arrange tubes in a slanting position.
  5. Coagulate and inspissate the medium in an inspissator water bath or autoclave at 85°C for 45 minutes.

Appearance of Lowenstein-Jensen (LJ) Medium

It appears Greenish blue to peacock blue homogeneous.The mixture of sterile basal medium and whole egg emulsion, when inssipated, coagulates to produce pale bluish green, opaque smooth slants.

Lowenstein jensen medium
Lowenstein jensen medium

Cultural Response  and plate reading on  Lowenstein-Jensen (LJ) Medium

Cultural characteristics observed in the presence of 5- 10 percent carbon dioxide, with egg emulsion base added, after incubation at 35- 37oC for 2- 4 weeks.

OrganismGrowth Growth with Gruft SupplementColony Characteristic
Mycobacterium avium ATCC 25291 luxuriantgood-luxuriantsmooth, nonpigmented colonies
Mycobacterium gordonae ATCC 14470luxuriant good-luxuriant smooth, yellow, orange colonies
Mycobacterium kansasii ATCC 12478luxuriantgood-luxuriantphotochromogenic, smooth to rough
Mycobacterium smegmatis ATCC 14468luxuriantgood-luxuriantwrinkled,creamy white colonies
M. tuberculosis H37RV ATCC 25618luxuriantgood-luxuriantgranular, rough, warty, dry friable colonies

References

  • Murray P. R., Baron E. J., Jorgensen J. H., Pfaller M. A., Yolken R. H., (Eds.), 8th Ed., 2003, Manual of Clinical Microbiology, ASM, Washington, D.C.
  • Lowenstein E., 1931, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig., 120:127.
  • Jensen K. A., 1932, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig., 125:222.
  • Gruft, 1971, Health Lab. Sci., 8:79.
  • Gruft, 1963, Am. Rev. Respir. Dis., 88:412.
  • Boisvert H., 1960, Ann. Inst. Pasteur, 99:600.
  • MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
  • Boisvert H., 1960, Ann. Inst. Pasteur, 99:600. Kent P. T and Kubica G. P., 1985, Public Health Mycobacteriology: A Guide to the level III Laboratory, USDHHS, Centers for Disease Control, Atlanta, Ga.
  • Forbes B. A., Sahm A. S. and Weissfeld D. F., Bailey & Scotts Diagnostic Microbiology, 10th Ed., 1998, Mosby, Inc., St. Louis, Mo.
  • Cernoch P., Enns R., Saubolle M. and Wallace R., 1994, Cumitech, 16A, Laboratory Diagnosis of the Mycobacteriosis coord , Ed., Weissfeld , ASM, Washington, D. C.
  • Isenberg, (Ed.), 1992, Clinical Microbiology Procedures Handbook, Vol. I, ASM, Washington, D. C.
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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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