Introduction of Lowenstein-Jensen (LJ) Medium

Lowenstein-Jensen (LJ) Medium is named after the surnames of  Austrian pathologist Ernst Lowenstein (1878–1950) and the Danish chemist Kai Arne Jensen (1908–1992). The etiological agent of tuberculosis is Mycobacterium tuberculosis. It requires aerobic conditions and a protein-enriched medium for culture. The short name of Lowenstein–Jensen medium is LJ medium. LJ  medium slopes are the main solid media used to cultivate Mycobacterium spp. (especially Mycobacterium tuberculosis) that contains inspissated eggs, malachite green, and glycerol.

This medium containing glycerol favors the growth of M. tuberculosis while LJ medium with pyruvate (in place of glycerol) encourages the growth of Mycobacterium bovis. The bacterium, M. tuberculosis grows slowly (generation time of 16 to 24 hours) and it takes 3-6 weeks or longer to give visible colonies. It produces granular, raised, dry, cream (buff) colored colonies in Lowenstein–Jensen medium as shown above footage. LJ medium uses for the diagnosis of mycobacterial infections, antimicrobial susceptibility testing (AST) of isolates, and differentiating different Mycobacterium spp. by colony morphology, growth rate, biochemical characteristics, and microscopic detections.

Principle of Lowenstein-Jensen (LJ) Medium

L-asparagine and potato flour supply nitrogen and vitamins for the organisms (mycobacteria) while monopotassium phosphate and magnesium sulfate enhance bacterial growth and also act as buffers. Malachite green, prevent the growth of the majority of contaminants surviving decontamination of the specimens while encouraging the growth of mycobacteria and it also serves as a pH indicator. Egg suspension provides fatty acids and protein required for the metabolism of Mycobacterium spp. Heating the egg albumin coagulates gives a solid surface for inoculation. Glycerol serves as a carbon source and it is favorable to the growth of the human-type tubercle bacillus (Mycobacterium tuberculosis) while being unfavorable to the bovine type (Mycobacterium bovis).

Composition and Preparation of L- J Medium

Lowenstein Jensen medium is recommended for the isolation and cultivation of Mycobacterium spp. ( especially M. tuberculosis). This medium can be prepared in the laboratory as follows-

Ingredients	Volume
Homogenized whole eggs	275 ml (6-8 fresh hen’s  eggs are used)
Hydrochloric acid (1N)	8 ml
Salt glycerol solution	153 ml
Malachite green 2% solution	2.75 ml

Clean the outside of the eggshell and wipe it with the spirit swab. Break the eggs and collect them in the sterile breaker. Homogenize using the sterile homogenizer. Separately prepare the other ingredients.

Salt glycerol solution Preparation

Ingredients	Amount
Potassium dihydrogen phosphate	6.3 gm
Magnesium sulfate	0.3 gm
Glycerol	12 ml
Distilled water	600 ml

• Mix all the ingredients with water and dissolve properly.

Malachite green(2%) Preparation

Ingredients	Amount
Malachite green	1 gm
Distilled water	50 ml

• Dissolve and autoclave and keep ready for use.

1. Mix all the above solutions aseptically and dispense in 4 ml (depending on the container) amounts in sterile screw-capped bottles.
2. Arrange them in the slant in a special tray provided for inspissation and then inspissate at 80 C° for 1 hour. This will sterilize the medium.
3. Note: The slope of the medium should end about 1 cm away from the neck of the bottle. This LJ slope will become solid, and light green in color. Label and store in the refrigerator with tight lids. The LJ slopes can be stored for 8 weeks.

Test Requirements for LJ  Medium

• Test sample
• Biological safety cabinet (BSC)
• Centrifuge
• Inoculating loop/ dropper
• Plastic tubes
• LJ medium slope
• Waste bin
• Control strain (Mycobacterium smegmatis ATCC 14468)
• BOD Incubator

Test Procedure of Lowenstein Jensen Medium

1. Remove the condensed moisture before inoculation putting the LJ slope in the BOD incubator for half an hour.
2. Inoculate slope with 0.2-0.4 ml or 2-4 drops or 2-4 loopful of the centrifuged sediment, distributed over the surface.
3. Incubate LJ slope at 35-37°C  until growth is observed or discarded as negative after 8  weeks.
4. Examine culture weekly if possible otherwise on at least three occasions. After a week to detect rapidly growing Mycobacterium species which may be mistaken for M. tuberculosis. After 3-4 weeks detect positive cultures of M. tuberculosis as well as other slow-growing mycobacteria which may be either harmless saprophytes or potential pathogens. After 8 weeks detect very slow-growing mycobacteria, including M. tuberculosis, before judging the culture to be negative.

Result Interpretation of Lowenstein-Jensen (LJ) Medium

Result	Interpretation
No growth after 8 weeks	Negative
Growth	Positive (a typical colony of rough, buff, and tough)
Control strain (Mycobacterium smegmatis ATCC 14468)	wrinkled, creamy white colonies

Limitations of LJ Medium

• For confirmation biochemical and/or serological tests are recommended from the pure colonies of the culture.
• LJ medium requires a capnophilic environment i.e. incubation in a 5-10% CO2 atmosphere in order to recover mycobacteria. Mycobacteria, for unknown reasons, are not recovered well from candle jars.
• Negative culture results may not rule out active infection by mycobacteria.
• Because of nutritional variation, some strains may be encountered that grow poorly or fail to grow on the LJ medium. Further investigations are necessary for confirmation of Mycobacterium spp.

Keynotes on Lowenstein-Jensen (LJ) Medium

1. Cultures are usually made in bottles or test tubes rather than in Petri plates because of the long incubation time required. The use of bottles or test tubes limits both chances of contamination and drying of the culture media (if the caps are tightly closed).
2. Two slopes of LJ media are preferred per specimen while an additional slope with pyruvate in Mycobacterium bovis endemic areas should be included.
3. With doubtful cultures or lacking trained staff read cultures, the acid fastness of the isolate should be confirmed by acid-fast/Ziehl-Neelsen staining.
4. Sterility check: After inspissation, the whole media batch of the media bottles should be incubated at 35°C-37°C for 24 hours as a check for bacterial sterility. After 24 hours 5% of the slopes should be picked up randomly and continued for incubation for 14 days to check for fungal sterility. In both cases, the contamination rate should not be > 10 %.
5. Niacin, nitrate reduction, and catalase tests are used to identify Tuberculosis.
6. Since the organism, M. tuberculosis is risk group III and therefore contaminated materials must be sterilized by autoclaving before discarding.
7. Culture activity and biochemical or serological tests from the culture should only be performed in the biological safety cabinet (BSC II).

Acid Fast bacilli (AFB) in Ziehl-Neelsen or acid-fast stained smear microscopy

Acid Fast bacilli (AFB) of Mycobacterium tuberculosis in counter stain Malachite green at a high magnification

Acid-fast bacilli in Fluorochrome staining, Auramine phenol

Urease-positive Mycobacterium tuberculosis

Further Readings

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045330/
2. http://legacy.bd.com/ds/technicalCenter/inserts/L007464(11).pdf
3. https://en.wikipedia.org/wiki/L%C3%B6wenstein%E2%80%93Jensen_medium
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552209/
5. http://apps.who.int/iris/bitstream/handle/10665/65942/WHO_TB_98.258_(part3).pdf;jsessionid=6AB07F40A95AF0B1EE5589E577F8659C?sequence=3
6.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
7. https://himedialabs.com/td/m162.pdf
8.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.