Löwenstein–Jensen (LJ) Medium

Preparation of Media

Ingredients
A.  Mineral salt solution

• Potassium dihydrogen phosphate anhydrous (KH2PO4): 2.4 g
• Magnesium sulphate anhydrous: (MgSo4.7H20): 0.24 g
• Magnesium citrate: 0.6 g
• Asparagine: 3.6 g
• Glycerol (reagent grade): 12 ml
• Distilled water: 600 ml

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%

• Malachite green dye: 2.0 g
• Distilled water: 100 ml
  Dissolve the dye in distilled water completely. Filter and store in refrigerator.

C. Homogenised whole eggs

• Get a fresh (those are not more than seven days old), hen’s eggs
• Clean the eggs by scrubbing thoroughly with a hand brush in water and soap.
• Let the eggs soak for 30 minutes in soap solution.
• Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 minutes.
• Before handling the clean dry eggs scrub and wash the hands with a disinfectant.
• Crack the eggs with the edge of the beaker into a sterile flask and beat them in a sterile blender for 30 seconds to one minute.

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

• Mineral salt solution: 600ml
• Malachite green: 20 ml
• Homogenised eggs (25-30 eggs, depending on size): 1000ml

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

• Heat the inspissator to 85°C to quicken the build-up of the temperature before loading.
• Place the bottles in a slanted position in the inspissator and coagulate the medium for 50 minutes at 85°C (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilise it).

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

• 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 picked up randomly and continued for incubation for 14 days to check for fungal sterility.
• In both the cases the contamination rate should not be > 10 %.

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.