Löwenstein–Jensen (LJ) Medium: Preparation, Uses

By Acharya Tankeshwar •  Updated: 05/03/22 •  5 min read

M. tuberculosis requires aerobic conditions and a protein-enriched medium for culture. Löwenstein–Jensen (LJ) slopes are the main solid media used to culture Mycobacteria. It contains inspissated eggs, malachite green, and glycerol (or pyruvate). LJ medium containing glycerol favors the growth of M. tuberculosis while LJ medium without glycerol but containing pyruvate encourages the growth of M. bovis.

M. tuberculosis grows slowly (generation time of 16 to 24 hours)  and takes 3-6 weeks or longer to give visible colonies. It produces raised, dry, cream (buff) colored colonies in Löwenstein–Jensen media.

Preparation of Löwenstein–Jensen Media

Ingredients

A.  Mineral salt solution

Dissolve the ingredients in order in the distilled water by heating. Autoclave at 121°C for 30 minutes to sterilize. Cool to room temperature. This solution keeps indefinitely and may be stored in suitable amounts in the refrigerator.

B. Malachite green solution 2%

C. Homogenized whole eggs

Preparation of complete medium
Aseptically pool the following reagents in a large, sterile flask and mixed well:

The complete egg medium is distributed in 6-8ml volumes in sterile universal containers or culture bottles  (14 ml or 28 ml) and the caps tightly closed and inspissated without delay to prevent sedimentation of heavier ingredients.

Cultures are usually made in bottles rather than in Petri dishes because of the long incubation time required. Use of bottle limits both chances of contamination and drying of the culture media (if the caps are tightly closed).

Coagulation of medium

The quality of egg media deteriorates when coagulation is done at too high a temperature or for too long. Discoloration of the coagulated medium may be due to excessive temperature. The appearance of little holes or bubbles on the surface of the medium also indicates faulty coagulation procedures.

Sterility check

Lowenstein Jensen Medium
Lowenstein Jensen Medium

Storage
The LJ medium should be dated and stored with the batch number in the refrigerator and can keep for up to 4 weeks if the caps are tightly closed to prevent drying of the medium.

Inoculation and Incubation

Two slopes of LJ medium should be inoculated per specimen (an additional one slope with pyruvate in M. bovis endemic areas).

Examination Schedule

All cultures should be examined 72 hours after inoculation to check that liquid has completely evaporated, to tighten the caps in order to prevent drying out of media and to detect contaminants. Thereafter, cultures are examined weekly, or if this is not operationally feasible, on at least three occasions, viz

Results:

Visible colonies are usually produced 2-3 weeks after incubation (M. tuberculosis is a SLOW GROWERS,  do not grow in primary culture in less than one week and may take 3-4 weeks to give visible growth), but cultures should be incubated for up to 8 weeks before being discarded.

When cultured on Lowenstein Jensen (LJ) medium at 35-37°C, M.tuberculosis produces rough (having the appearance of bread crumbs or cauliflower), raised, dry, non-pigmented (cream/buff colored) colonies.

Close view of Mycobacterium tuberculosis colonies
Close view of Mycobacterium tuberculosis colonies
(Image source: CDC/ Dr. George Kubica)

With doubtful cultures or when less experienced staff read cultures, the acid fastness of the isolate should be confirmed by Ziehl-Neelsen (ZN) staining.

Mycobacterium tuberculosis can be identified presumptively on the basis of its colony characteristics. Though there is not a completely reliable single test that will differentiate M. tuberculosis from other mycobacteria.

The following tests when used in combination help to identify the M.tuberculosis strains.

References and Further Reading

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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One response to “Atypical Mycobacterial Infections”

  1. Basant Joshi says:

    Thank you sir. These notes will be very helpful. Could you please also post some MCQS on this topic?

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