Laboratory techniques for mycobacteria of Mycobacterium Infections
Laboratory techniques for mycobacteria
The commoner species of mycobacteria are classified into two groups: (1) the typical tubercle bacilli i.e. M, tuberculosis and M. bovis, and (2) the atypical mycobacteria. Mycobacterium are difficult to stain but once stained, they resist decolourisation with acid and alcohol. Three main methods are used to detect tubercle bacilli in sputum and other materials.
Staining
Typically, the human tubercle bacilli are slender, straight or slightly curved rods. They are non-motile, non-sporing and uncapsulated. They remain uncoloured with simple stains but show acid-fast staining with warm carbol fuchsin followed by 20% H2SO4 or by 3%HCl in 95% ethanol (Ziehl-Neelsen method). Bovine tubercle bacilli tend to be shorter and thicker than the human type.
Culture
The tubercle bacillus is an obligate aerobe and grows at temperatures from 30-41oC, optimally at 35-37 oC. Lowenstein-Jensen media with glycerol or sodium pyruvate is generally used for isolation. The specimen is incubated on slopes of L-J medium at 37 oC. Growth is slow so that colonies only appear after 2-3 weeks. The slopes should be incubated for a total of 6-8 weeks before discarding. M. tuberculosis colonies on L-J media are rough, buff to yellowish in colour, and tough when picked off. Dispersed uniform growth can be obtained by subculturing two or three times in Dubos and Davis liquid medium continuing Tween 80. The addition of glcerol to L-J medium improves the growth of M. tuberculosis, but not that of M. bovis. Sodium pyruvate, on the other hand increases the growth of M. bovis and some strains of drug-resistant M. tuberculosis.
Tubercle bacilli are killed by heat at 60oC for 15-20 min. They survive many weeks in moist conditions in the dark. They are killed rapidly by sunlight. They are relatively resistant to chemical disinfectants. The tubercle bacilli are catalase positive and do not produce acid in sugar-containing media. Biochemical tests are generally not used in their confirmation. Several phage groups of M. tuberculosis had been identified and it is now possible to use phage typing to demonstrate case to case spread of infection.
Guinea pig
The guinea pig is highly susceptible to infection by both M. tuberculosis and M. bovis. After subcautaneous injection of an infected specimen, a local swelling appears within a few days which proceeds to caseate and finally ulcerate. The organism spreads to the lymph nodes, spleen, liver, and peritoneum. The animal dies within 6-15 weeks. Because modern methods of culture are so efficient, inoculation of sputum into animals is rarely indicated.
Specimens
1. Sputum – sputum should be examined microscopically firstly in a direct smear made from the untreated specimen and then again in a smear made from the centrifuged, concentrated deposit that has been treated and homogenised.
2. BAL, Laryngeal swab and gastric lavage – where sputum is absent, material for culture may be obtained from a BAL, laryngeal swab, or gastric lavage.
3. Urine – special caution must be exercised in interpreting the finding of mycobacteria in urine. Samples may be contaminated by M. smegmatis from the urethral orifice.
4. Pleural and peritoneal fluid – as tubercle bacilli are usually scanty, a large specimen e.g. 50-100 ml should be obtained.
5. CSF – as large as possible a specimen should be obtained.
6. Pus – examine with direct smears and Z-N stain.
7. Tissue – tissue should be homogenised.
Sensitivity tests
Sensitivity tests are done on slopes of L-J glycerol medium each containing a series of concentrations of a particular antibiotic. After incubation for 3 weeks at 37oC, the slopes are examined for growth and the lowest concentration of antibiotic showing no more than 0-20 colonies is taken as the end-point, or nominal “MIC”. The conventional methods of sensitivity testing involve a delay of 3 weeks before results are available. Rapid methods are available which measure the evolution of 14CO2 from a radio-labelled substrate in the medium e.g. Bactec.
Identification tests
Identification tests should be carried out at the same time as sensitivity tests. The following five tests are routinely carried out for the purpose of identification:-
1. L-J slope with para-nitrobenzoic acid (PNB) 500 mg/l – all tubercle bacilli are sensitive to PNB and thus should fail to grow.
2. L-J slope with thiophen-2-carboxylic acid hydrazide (TCH) – of the strains which are sensitive to PNB, only strains of M. tuberculosis are resistant to TCH and can grow on the TCH medium.
3. Dubos and Davis medium for the niacin test – M. tuberculosis produce niacin and so differs from M. bovis and BCG which do not.
4. L-J slope for incubation in an incubator with light for pigment production – atypical tuberculoid bacteria can be divided in (1) photochromogens, (2) scotochromogens, and (3) non-chromogens. (see below)
5. Sauton agar with 0.2% picric acid – rapidly growing mycobacteria can be distinguished from slowly growing mycobacteria by their ability to grow on Sauton agar containing 0.2% picric acid. Typically, rapid growing mycobacteria will show good growth within 5 days at 37oC
Photochromogens are slowly growing mycobacteria that form yellow or orange pigment when their cultures are exposed to light, but not when they are kept in the dark. In the standard test for photochromogenesis, two L-J slopes are seeded, one is wrapped in aluminium foil to exclude light from it and both are incubated for 14 days at 37oC in a light incubator. Scotochromogens are slowly growing mycobacteria that form pigment in both light and dark. Non-chromogens are those that do not form pigment at all, of which the most important members are those of the MAC complex.
Newer methods
HPLC is now routinely used in large laboratories for the identification of mycobacteria, as is the case of nucleic acid probes on culture. The role of PCR in the routine diagnosis of mycobacteria infection has yet to be fully established.
Tuberculin test
Antibodies to mycobacteria in patient’s serum can be demonstrated by various serological techniques such as HAI and EIA. However, the presence or absence of antibodies, or their titres when present, has shown little correlation with the clinical state of the patient. The principal immunological response in tuberculosis is the development of cell-mediated immunity. It is demonstrated by the delayed hypersensitivity reaction which follows the intradermal inoculation of tuberculin. A positive test in a person who has not been vaccinated with BCG indicates that tuberculous infection has taken place in the recent or distant past, but is not necessarily a sign of active disease. In the absence of BCG vaccination, a positive reaction is valuable confirmatory evidence. The test is also useful in screening children who have been in contact with an open case of disease. However, the test may be negative in advanced or miliary infection. Two methods of testing are currently in use: Mantoux test and heaf test.
Tuberculin test – this is the standard method by which all other methods are compared. A test dose of 0.1 ml of Purified Protein Derivative (PPD) containing 5 Tuberculin Units is injected intracutaneously into the skin of the forearm. The development of an area of palpable firm induration greater than 10 mm in diameter is recorded as positive. The extent of the accompanying erythema is irrelevant. If the reaction is completely negative, the test may be repeated by giving an injection of 100 Tuberculin Units.
Heaf test – this test is done with a multiple puncture apparatus with 6 needles that prick tuberculin 1-2 mm deep into the skin. A drop of undiluted PPD is spread onto the area of the skin selected for inoculation, the instrument is pressed against this are of skin and the needles are released. The site is inspected 72 h later. When a reaction comprising the presence of erythema and oedema or induration around at least 4 of the punctures is regarded as positive. Because of its ease of performance, the Heaf test is principally used in epidemiological surveys and as a test for immunity before BCG vaccination. For diagnostic purposes, the more accurate Mantoux test should be used. A fresh apparatus must be used for each person tested in order to prevent the spread of HIV and HBV infections.
Some Characteristics of Nontuberculous Mycobacteria Commonly Encountered in Human Material
Optimal
Growth Catalase
Clinical Temperature Tween Iron Growth
Species Significance* (oC) Pigment 25‑38oC 68oC Nitrate Hydrolysis Urease Uptake Rate
M. avium‑intracellulare + 37 ‑ or S Weak + - - - Slow
M kansasii + 37 P Strong + + + + Slow
M xenopi + 42 S Weak + ‑ - - Slow
M. scrofulaceum + 37 S Strong + ‑ - + Slow
M. simiae + 37 P(weak) Strong + ‑ - + Slow
M szulgai + 37 S/P Strong + + ± + Slow
M. gordonae ± 30‑37 S Strong + ‑ + - Slow
M flavescens ± 30‑37 S Strong + + + + - Intermediate
M. terrae‑trivale ± 30‑37 - Strong + + + - Slow
M. gastri - 30‑37 - Weak ‑ ‑ + + Slow
M marinum + 30 p Weak + ‑ + + Intermediate
M. fortuitum + 30‑37 - Strong + + ± + + Fast
M. chelonae + 30‑37 - Strong + ‑ - + - Fast
M. smegmatis - 25‑45 - Strong + + + + + Fast
+ May be pathogenic for hosts without general immune suppression
± documented pathogen only for hosts with abnormal local and/or general defense mechanisms
P = photochromogenic, S = scotochromogenic,
S/P Scotochromogenic when grown at 37oC but variably photochronnogenic at 25oC.
TABLE 35‑2. Nontuberculous Mycobacterial Diseases of Man
Disease Common Associated Species Other Associated Species
Chronic cavitary lung disease in adults MAI, M. kans. M. xenopi, szulgai, simiae, malmoense
scrof,, fort.
Local lymphadenitis in children MAI, M. scrof M. kans., M. fort.
Arthritis, tenosynovitis, and osteomyelitis, MAI, M. kans. M. fort., M. terrae, M. marinum,
including hand infection M. xenopi
Bursitis M. kans. M. szulgai
Skin nodules and abscesses M. marinum, M. haemophilum, M. fort. M. kans., MAI, M. fort., M. szulgai
Buruli or Bairnsdale ulcer M, ulcerans
Disseminated disease MAI, M. kans. M. scrof., M. fort.
Leprosy M leprae
Abbreviations: MAJ, M. avium‑intracellulare, M. fort., M. fortuitum‑chelonao,. M. scrof, M. scrofulaceum, M. kans., M. kansasn.