In the District Tuberculosis Programme (DTP) the diagnosis is based on sputum microscopy. Majority of health institutions in the district are provided with microscopes for this purpose. In the Peripheral Health Institutions, the programme activities have to be carried out by its staff after a short period of training given by District TB Centre personnel on the spot. So the microscopy work in the PHIs is likely to be carried out by any paramedical personnel and not necessarily by a qualified laboratory technician. It is therefore, necessary to know whether the standard of microscopy carried out by these paramedical personnel after a short training will be upto the mark. To assess the efficiency of smear examination done by these individuals, a study was conducted in Bangalore district covering nine microscopy centres in various types of health institutions, a few months after the implementation of the programme. Under the DTP a spot specimen is collected from every chest symptomatic attending the health institutions and a smear is made and examined for the presence of AFB and all positive cases are put under treatment. The sputum specimens and the smears examined in these nine centres were brought to National TB Institute laboratory. The smears were examined by an experienced laboratory technician. Duplicate smears were also prepared from these specimens and their results compared with results of re examination and centre's examination. All specimens were cultured by swab method and all positive cultures were subjected to sensitivity and identification tests.

Analysis of the results based on culture showed that barring a few centres where the performance was poor, the standard of examination was fairly good. The under and over diagnosis based on culture were 38.2% and 2.6% respectively, and these were within the limits observed generally. Comparison of results on re examination of centre smears and duplicate smears indicated that both reading variation and defective smear preparations and staining could have influenced under diagnosis in these centres. The study has also thrown some light on methodology of assessment of sputum examination that could be adopted wherever a tuberculosis control programme is functioning.

Laboratory diagnosis of pulmonary tuberculosis is based on the presence of tubercle bacilli in sputum by direct microscopy, culture and/or animal inoculation. Culture examination, followed by tests for identifying the bacilli, is recognized as the most accurate and reliable method. Its efficacy depends on the laboratory techniques employed and its use in different practical situations such as epidemiological surveys, active community Case-finding, organization of diagnostic services and evaluation of diagnosis and treatment in tuberculosis control programmes. But the practicability of culture method in developing countries must be studied. The present paper deals with a systematic study of data from four investigations designed to elucidate the influence of certain operational factors on the utility of the culture method.

STUDY I: is a longitudinal survey in a randomly selected population in 134 villages in the three sub-divisions of Bangalore district. The analysis is based on the material from the first round, when two samples of sputum, (spot and overnight) were collected at intervals of 24-48 hours from persons aged 5 years and above having abnormal x ray shadows. The specimens were collected in house to house visits, stored after collection in insulated box with ice container and transported to the main laboratory at the National Tuberculosis Institute (NTI). The interval between collection of specimens in the field and culture in the laboratory was 1-7 days. A smear was stained and examined first by fluorescence microscopy and then by Ziehl-Neelsen (ZN) method. Each specimen was cultured on two slopes of Lowenstein-Jensen medium. All positive cultures were submitted to further identification tests; i.e., growth at room temperature, rate of growth at 37%C, pigment production in the dark and exposure to light, catalase and peroxidase reactions, niacin production, and sensitivity to INH, SM and PAS. STUDY II: relates to a mass Case-finding programme in Tumkur district when two specimens (spot and overnight) were collected from individuals aged 20 years and above with symptoms suggestive of pulmonary tuberculosis and from positive tuberculin reactors below 20 years voluntarily reporting with symptoms. The specimens were then treated in the same way as in Study I. STUDY III: pertains to the technical assessment of microscopy using Ziehl-Neelsen method performed by the auxiliary health staff of Peripheral Health Institutions in Bangalore district. A spot specimen was collected daily by auxiliary staff at each health facility from patients who were symptomatics. All smears were examined by ZN method at each centre and the corresponding sputum specimens were transported to NTI laboratory twice weekly. Duplicate smears were made and reexamined and culture was also done at NTI. All positive cultures were identified as in Study I. No refrigeration facilities were available in these centres and specimens were not transported in an insulated box. Rest of the procedures were followed as in previous studies. STUDY IV: is connected with operational and technical assessment of the District Tuberculosis Programme in Anantapur district one year after its commencement. A sample was taken from all patients who started treatment during a particular period but did not collect their drugs. Spot specimens were collected in the field, stored without any refrigeration and transported to NTI laboratory, thereafter the same procedure was followed as above.

An analysis of these four studies brought out certain operational factors affecting the culture method. (1) The results showed that an interval of 7 days between collection of sputum in the field and its processing in the laboratory did not affect the yield of positive cultures, even though the specimens were stored and transported under field conditions. (2) A higher proportion of positive cases were detected by culture than by direct microscopy but the magnitude of additional yield was dependant upon the procedure of selecting persons for sputum examination. (3) In service programmes restricted to persons with symptoms who attend diagnostic centres, the increase in yield is too small, to justify the introduction of culture examination.

Of all the available methods for the diagnosis of pulmonary tuberculosis, bacteriological examination is the most reliable. Diagnosis of pulmonary tuberculosis is chiefly done by sputum microscopy and culture. This paper discusses sputum microscopy from various points of view. Sputum, which forms the basis of bacteriological diagnosis, is a variable source material. Type of specimen, its quality, quantity, bacterial content and viability of organism considerably influence the sensitivity and the specificity of the methods; and these in turn would vary under different diagnostic situations. One of the reasons for the observed variations could be the different criteria adopted for examination; another might be due to the observed range of diagnostic situations varying from an epidemiological survey situation at the one extreme to the other where cases seek treatment in a comparatively backward community with poor tuberculosis diagnostic services.

In epidemiological community survey (ICMR 1968), it has been found that culture positives that were also smear positives varied from 73% to 87%, whereas among patients attending rural general health institutions for diagnosis, about 82% of the infectious cases found by culture could also be discovered by microscopy of single spot specimens (Rao, 1966). Sikand (1965) from New Delhi Tuberculosis Centre, could get 67% of culture positives by microscopy, whereas Mitchison (1967) found that 35% were smear positive among the sputum positive patients reporting for the first time. In the longitudinal epidemiological study carried out in the Bangalore rural area, it was found that about 40%-48% were positive by both direct smear and culture and the rest by culture only. Reasons for these variations could be (i) different criteria adopted for examination (ii) different situations from where the sputum specimens were collected (iii) sensitivity and specificity of sputum microscopy technique adopted and (iv)the experience of the trained technician. It was observed that over diagnosis by the trained auxiliary staff in the general health institutions (1.9%) compares favourably with the over diagnosis of 1.3% by experienced technicians indicating simplicity of smear examination. Besides these aspects, other factors like the quality of sputum smear, time spent on examination, type of sputum specimen, the use of multiple smears, etc., also influence the results. The cost of bacteriological examination have also been studied, and the cost ratio between microscopy and culture have been worked out to be 1:6.6. Cost can become an important factor in deciding the suitability of bacteriological methods for diagnosis of pulmonary tuberculosis in various countries and in different diagnostic situations.