microbeonline

Löwenstein–Jensen (LJ) Medium: Prepartion and Uses

M. tuberculosis requires aerobic condition and a protein enriched medium for culture. Löwenstein–Jensen (LJ) slopes are main solid media used to culture Mycobacteria. It contains inspissated eggs, malachite green and glycerol (or pyruvate). LJ medium containing glycerol favours 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. Homogenised 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.

Note:  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

Note: The quality of egg media deteriorates when coagulation is done at too high a temperature or for too long. Discolouration 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

Storage
The LJ medium should be dated and stored with the batch number in the refrigerator and can keep for upto 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:

Lowenstein Jensen Medium

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.

Note: 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 completely reliable single test that will differentiate M. tuberculosis from other mycobacteria, following tests when used in combination help to identify the M.tuberculosis strains.

References and Further Reading