Part III: Culture
media

The definitive diagnosis of tuberculosis demands that M. tuberculosis be recovered on culture media and identified using differential in vitro tests. Many different media have been devised for cultivating tubercle bacilli and three main groups can be identified, viz egg-based media, agar-based media and liquid media.

The ideal medium for isolation of tubercle bacilli should (a) be economical and simple to prepare from readily available ingredients, (b) inhibit the growth of contaminants, (c) support luxuriant growth of small numbers of bacilli and (d) permit preliminary differentiation of isolates on the basis of colony morphology. For the culture of sputum specimens, egg-based media should be the first choice, since they meet all these requirements. These is increasing evidence that liquid media may give better results with other specimens. While cost prevents their routine use with sputum specimens, it is recommended that both egg-based and liquid medium be used for non-repeatable specimens, eg. cerebrospinal fluid and biopsy material.

It is recommended that all sputum specimens submitted for culture also undergo microscopic examination as outlined in the Technical Series on Microscopy.

Advantages and disadvantages of egg-based media
Advantages

• it is easy to prepare
• it is the least expensive of all media available and supports good growth of tubercle bacilli
• it may be stored in the refrigerator for several weeks provided it was made from fresh eggs and culture bottle caps are tightly closed to minimise drying by evaporation
• contamination during preparation is  limited because it is inspissated after being placed in bottles. In addition, the  malachite green added to the media suppresses the growth of nonmycobacterial organisms

Disadvantages

• it may take as long as eight weeks before  cultures become positive, especially if specimens contain few bacilli or if  decontamination procedures have been overly harsh
• when contamination does occur, it often  involves the total surface of the medium and the culture is usually lost

Precautions during media preparation
For media of the best quality, chemicals of certified purity, clean glassware and freshly distilled and sterilised water should be used. Directions for preparing media must be followed precisely and without modification. A few general points to obtain good quality media and avoid contamination of reagents and media are as follows:

• Keep the environment as clean as possible. Swab the work surface with a suitable disinfectant (eg.  5% methylated spirits) before dispensing sterile reagents and media. Clean the floor with a wet mop to limit dust
• Use sterile  glassware and equipment
• Use reagent  grade chemicals and reagents unless otherwise specified
• Check the  temperature of inspissators and hot air ovens
• Follow strict  aseptic techniques when preparing media, eg. flaming flasks and tubes
• When preparing egg-based media, carefully clean egg shells before breaking
• Do not overheat medium during inspissation
• Do not leave prepared media exposed to light (including ultra-violet light), but store in the refrigerator in the dark when not in use
• Do not skimp on the volume of medium. Place 6-8ml of egg medium in each bottle or 20ml into each test tube

Preparation of egg-based media

LÖWENSTEIN-JENSEN MEDIUM
Löwenstein-Jensen (LJ) medium is most widely used for tuberculosis culture. The modification of the International Union Against Tuberculosis and Lung Disease (IUATLD) is recommended and will be described in detail. LJ medium containing glycerol favours the growth of M. tuberculosis while LJ medium without glycerol but containing pyruvate encourages the growth of M. bovis. Both should be used in countries or regions where patients may be infected with either organism.

Ingredients
Mineral salt solution
Potassium dihydrogen phosphate anhydrous (KH₂PO₄) 2.4g
Magnesium sulphate (MgSO₄. 7H₂O) 0.24g
Magnesium citrate 0.6g
Asparagine 3.6g
Glycerol (reagent grade) 12ml
Distilled water 600ml

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

Malachite green solution, 2%
Malachite green dye 2.0g
Sterile distilled water 100ml

Using aseptic techniques dissolve the dye in sterile distilled water by placing the solution in the incubator for 1-2 hours. This solution will not store indefinitely and may precipitate or change to a less-deeply coloured solution. In either case discard and prepare a fresh solution.

Homogenised whole eggs
Fresh hens' eggs, not more than seven days old, are cleaned by scrubbing thoroughly with a hand brush in warm water and a plain alkaline soap. Let the eggs soak for 30 minutes in the soap solution. Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 minutes. Before handling the clean dry eggs scrub the hands and wash them. Crack the eggs with a sterile knife into a sterile flask and beat them with a sterile egg whisk or in a sterile blender.

Preparation of complete medium
The following ingredients are aseptically pooled in a large, sterile flask and mixed well:

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

The complete egg medium is distributed in 6-8ml volumes in sterile 14ml or 28ml McCartney bottles or in 20ml volumes in 20 x 150mm screw-capped test tubes, and the tops are securely fastened.

Inspissate the medium within 15 minutes of distribution to prevent sedimentation of the heavier ingredients.

Coagulation of medium
Before loading, heat the inspissator to 80EC to quicken the build-up of the temperature. Place the bottles in a slanted position in the inspissator and coagulate the medium for 45 minutes at 80E-85EC (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilise it). Heating for a second or third time has a detrimental effect on the quality of the medium.

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.

Poor quality media should be discarded.

Sterility check
After inspissation, the whole media batch or a representative sample of culture bottles should be incubated at 35E-37EC for 24 hours as a check of sterility.

Storage
The LJ medium should be dated and stored in the refrigerator and can keep for several weeks if the caps are tightly closed to prevent drying out of the medium. For optimal isolation from specimens, LJ medium should not be older than 4 weeks.

For the cultivation of M. bovis, LJ medium is enriched with 0,5% sodium pyruvate. Glycerol is omitted and 8.0g sodium pyruvate is added to the mineral solution.

OGAWA MEDIUM
This medium is cheaper than Löwenstein-Jensen because it is made without asparagine.

Ingredients
Mineral salt solution
Potassium dihydrogen phosphate anhydrous (KH₂PO₄) 3.0g
Sodium glutamate 3.0g
Distilled water 300ml

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

Malachite green solution, 2%
Malachite green dye 2.0g
Sterile distilled water 100ml

Using aseptic techniques dissolve the dye in sterile distilled water by placing the solution in the incubator for 1-2 hours. This solution will not store indefinitely and may precipitate or change to a less-deeply coloured solution. In either case discard and prepare a fresh solution.

Homogenised whole eggs

Fresh hens' eggs, not more than seven days old, are cleaned by scrubbing thoroughly with a hand brush in warm water and a plain alkaline soap. Let the eggs soak for 30 minutes in the soap solution. Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 minutes. Before handling the clean dry eggs scrub the hands and wash them. Crack the eggs with a sterile knife into a sterile flask and beat them with a sterile egg whisk or in a sterile blender.

Preparation of complete medium
The following ingredients are aseptically pooled in a large, sterile flask and mixed well:

Mineral salt solution 300ml
Malachite green solution 18ml
Whole hens? eggs (12-16 eggs, depending on size) 600ml
Glycerol 18ml

The resulting pH of the medium is 6.8. The medium is mixed well and distributed in 6-8ml volumes in sterile 14ml or 28ml McCartney bottles or in 20ml volumes in 20x150mm screw-capped test tubes.

Coagulation of medium
Before loading, heat the inspissator to 80EC to quicken the build-up of the temperature. Place the bottles in a slanted position in the inspissator and coagulate the medium for 45 minutes at 80E-85EC (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilise it). Heating for a second or third time has a detrimental effect on the quality of the medium.

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.

Poor quality media should be discarded.

Sterility check
After inspissation, the whole media batch or a representative sample of culture bottles should be incubated at 37EC for 24 hours as a check of sterility.

Storage
The medium should be dated and stored in the refrigerator and can keep for several weeks if the caps are tightly closed to prevent drying out.

In laboratories where centrifuges are not available, a simple culture technique could be employed as follows: Sputum specimens are decontaminated with equal volumes of 4% NaOH and inoculated directly onto modified or acid-buffered Ogawa medium. This technique shows a fairly comparable oase yield when compared with concentrated culture techniques.

ACID-BUFFERED OGAWA MEDIUM
Ingredients
Modified Ogawa Acid-buffered Ogawa
Potassium dihydrogen phosphate 2g 3g
Magnesium citrate (KH₂PO₄) 0.1g -
Sodiumglutamate 0.5g 1.0g
Glycerol 4ml 6ml
Distilled water 100ml 100ml
Homogenised whole eggs 200ml 200ml
2% Malachite green solution 4ml 6ml
Final pH 6.4 6.2

Preparation
Dissolve the ingredients in the distilled water and boil for 30 minutes. Cool to room temperature and add the homogenised eggs and malachite green solution. Transfer 6-8ml volumes to suitable bottles and inspissate at 85EC for 45-60 minutes.

A variety of more expensive and labour intensive culture methods¹are available to countries with the required financial and human resources. Some of these will be discussed briefly:

¹ Kent PT, Kubica GP. Public health mycobacteriology: Guide for the Level III Laboratory. US Department of Health and Human Services, Centres for Disease Control, USA, 1985.        

OPTION RADIOMETRIC METHOD FOR TUBERCULOSIS CULTURE Recent development in the diagnosis of tuberculosis include an automated system for detecting early growth of mycobacteria by a radiometric method (BACTEC: Beckton Dickinson). Sputum or other homogenates are decontaminated as necessary  and added to vials containing Middlebrook 7H12 medium, an antibiotic mixture (to avoid the  growth of other organisms) and 14C-labelled palmitic acid. The medium is  prepared commercially (BACTEC 12B: Beckton Dickinson) in rubber-sealed bottles  and inoculated with a syringe and hypodermic needle. If mycobacterial growth occurs, 14C  palmitic acid is utilised and 14CO2 is produced. The air space above  the medium in each bottle is sampled automatically by the BACTEC machine at fixed  intervals and the amount of radioactive gas is estimated and recorded. Infectious aerosols  are contained in the apparatus and captured in HEPA filters before the air is exhausted. Growth of mycobacteria may be detected within 5-7 days, but positive results require further testing to distinguish between tubercle bacilli and other  mycobacteria. In the BACTEC machine, p-nitro-a-acetylamino-ß-priophenone (NAP) is used and tubercle  bacilli can be differentiated within five days. NAP inhibits the growth of M. tuberculosis and usually does not affect the growth of MOTT bacilli. Comparative tests have shown that the method is very successful and  reliable and that confirmatory results for M. tuberculosis can be obtained within  two weeks. However, the BACTEC machine is very expensive to purchase and to operate. In addition, two hazards must be considered if the machine is to be used for routine tuberculosis bacteriology: the use of hypodermic needles for the inoculation of media  carries the risk of needle-stick injury, while the culture media is radioactive and presents a problem in terms of waste disposal. In summary, the BACTEC method is invaluable for the detection of tubercle bacilli in material such as cerebrospinal fluid where rapid results are crucial in the management of the patient. However, the high cost of both the apparatus and the  radio-labelled medium prohibits its routine use in most high tuberculosis prevalence countries.