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
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 the distilled water completely. Filter and store in the refrigerator.
C. Homogenized 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 a 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.
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 sterilize it).
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.
- 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 %.
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).
- Remove the condensed moisture before inoculation.
- Inoculate each slope with 0.2-0.4 ml (2-4 drops or 2-4 loopful) of the centrifuged sediment, distributed over the surface.
- Incubate the cultures at 35-37°C until growth is observed or discarded as negative after eight weeks.
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
- after one week to detect rapidly growing mycobacteria which may be mistaken for M. tuberculosis
- after 3-4 weeks to 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 to detect very slow-growing mycobacteria, including M. tuberculosis, before judging the culture to be negative
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.
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
- Laboratory services in Tuberculosis Control, Part III: Culture, World Health Organization
- Image source: Thermo-scientific