The addition of para-aminobenzoic acid or catalase to Lowenstein-Jensen Medium and the effect of prolonged incubation in the culture of tubercle bacilli.
Subbaiah, T.V.; Selkon, J.B.; Bhatia, A.L.; Mitchison, D.A.; Radhakrishna, S.
Tubercle; 1960; 41; 334-340.
Fruhlinger and Bala (1953), Yegian, Budd and Bala (1955), Duerr (1955 a,b) and Duerr and Black (1956) have shown that, among patients under treatment with p-aminosalicyclic acid (PAS), enough PAS may be carried over in the sputum to prevent growth of tubercle bacilli in subsequent cultures. In their studies, sputum specimens were treated with sodium hydroxide and neutralised. The deposit was not washed or was washed with small volumes of diluent (often 1 ml.) and was then inoculated on to Hohn egg medium or oleic acid-albumin agar medium. Addition of p-aminobenzoic acid (PABA) , which antagonises the activity of PAS against tubercle bacilli, to the culture medium increased the incidence of positive cultures, especially from smear-positive specimens. However, Yegian, Budd and Bala (1955) found that PABA itself inhibited a small proportion of strains, which were drug-resistant to at least two of the three drugs, streptomycin, isoniazid and PAS.
Middlebrook (1957) has recommended that catalase be added to egg media to promote the growth of isoniazid-resistant, catalase-negative tubercle bacilli. These organisms are particularly susceptible to the presence of hydrogen peroxide in culture media, which may be formed during inspissation or subsequent incubation (Cohn and others, 1954; Barry and others, 1955).
In consequence, during the course of two controlled trials at the Tuberculosis Chemotherapy Centre, the method of culture on Lowenstein-Jensen medium was studied in comparison with culture on Lowenstein-Jensen medium to which PABA or catalase had been added, to see whether it was proving adequate both for culture of tubercle bacilli from patients receiving PAS and isoniazid, and also for culture of isoniazid-resistant tubercle bacilli from patients receiving either PAS and isoniazid, or isoniazid alone. In both these trials there was an appreciable incidence of smear-positive, culture-negative specimens, but evidence has been presented that it was justifiable to conclude that the bacilli in these specimens were non-viable (Tuberculosis Chemotherapy Centre 1959, 1960 b). The method of culture employed differed from that of Fruhlinger and Bala (1953), Yegian, Budd and Bala (1955), and Duerr (1957) in that, after treatment with sodium hydroxide, the centrifuged deposit was washed with a large volume of water before addition to the culture medium. In addition to these investigations a sample of the cultures which were negative after 8-9 weeks were incubated for a further period of 8 weeks (a total of 16-17 weeks incubation), to investigate whether any further positive cultures could be obtained, as has been suggested by Duerr (1957).
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