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138
A COLD STAINING METHOD FOR TUBERCLE BACILLI USING CHLOROFORM
K Padmanabha Rao, N Naganathan & SS Nair: Indian J TB 1966, 14, 3-9.

The difficulty in staining tubercle bacilli is believed to be related to the complex surface structure containing a large amount of unsaponifiable wax. Any staining technique which can counteract the influence of this wax could therefore be expected to give better results. The standard method in vogue is the application of heat which renders the bacilli permeable to aqueous dyes. Several attempts have been made to develop a cold staining method for tubercle bacilli as for other organisms. Since this wax is soluble in chloroform, a cold staining method using carbol fuchsin containing chloroform was developed and the results of staining by this new method have been compared with the conventional Ziehl-Neelsen (ZN) method in the present study. Triplicate smears were made from 186 specimens and these were stained by ZN, Cold Staining (CS) and Fluorescent Microscopy (FM) methods. In addition, single smears of 343 specimens previously examined by FM were randomly divided into two subgroups and restained by ZN and CS methods respectively.

The results of examination of duplicate smears by ZN and CS methods showed a high degree of correlation with 75%(140/186) showing identical grading and only 8 were positive by one and negative by the other method. Of the 8 smears positive by CS alone, 7 were confirmed by culture, whereas 3 were positive by culture out of the 8 positive by ZN method. This indicates that those positive by CS alone are likely to be real cases, whereas those positive by ZN alone may include some false positive cases. As far as false negatives are concerned, there was no difference between ZN and CS methods. The reliability of these methods was judged on the basis of culture results and agreement among themselves. The cold staining method was found to be as efficient as ZN method in detecting different gradings of culture positives. In addition, preparation of stain, training of personnel for CS was also found to be as simple as ZN method.

KEY WORDS: STAINING METHODS, COLD STAINING, TUBERCLE BACILLI, ZIEHL1-NEELSEN, FLUORESCENT.

143
AN INTER LABORATORY COMPARISON
N Naganathan: NTI Newsletter 1974, 11, 27-28.

The National Tuberculosis Institute (NTI), Bangalore was established in 1959 and its bacteriological laboratory started functioning from 1961. For the first few years WHO Experts were involved in the establishment and running of the laboratory but since many years the laboratory is being run only by the national staff.

The laboratory has been involved in research and training since its inception. In order to compare the standard of the various tests done in the NTI laboratory with that of a similar laboratory having some standing in tuberculosis research, a series of comparison studies were done between NTI and the laboratory of the Tuberculosis Chemotherapy Centre, Madras. The results were similar except for variations within normal limits. Besides, some cultures isolated in NTI laboratory were sent to the tuberculosis laboratory of the Centre for Disease Control (CDC), Atlanta (Georgia) U.S.A. for purpose of an inter-laboratory comparison. Forty seven cultures were sent to CDC, of which 38 were M.tuberculosis, 1 rapid grower, 1 H37 RV, 3 M.avium, 2 M.bovis, 1 B.C.G and 1 M.phlei. But for some minor variations in a few biochemical tests, the species classification compared well between the two laboratories. Taking all the results into account and making some allowance for unavoidable variations, it was observed that the standard of bacteriological investigations were similar between all the three laboratories.

KEY WORDS: LABORATORY, TUBERCLE BACILLI, SPECIES.

144
SOME GUIDELINES FOR ESTABLISHING A TUBERCULOSIS CULTURE LABORATORY
N Naganathan: NTI Newsletter 1974, 11, 32-34.

The issues to be addressed while establishing a tuberculosis culture laboratory are discussed in the paper. Primarily the following questions are to be considered: (1) Is it absolutely essential to have a tuberculosis culture laboratory? (2) Will it be big or small? (3) Are there adequate means to continue work in terms of finance, staff, equipment & specimens and (4) Is there a possibility of taking up any other type of bacteriological work, if necessary? Unlike other bacteriology laboratory, a tuberculosis laboratory has some unique features. Due to the slow growth of the organism, cultures need to be incubated for a long time i.e., 8 10 weeks. So an incubator room is required. More number of glassware are needed. Test tubes with cotton plugs are unsuitable as they are likely to dry up. Hence screw capped tubes or McCartney bottles are required to facilitate long incubation of cultures. For performing identification tests, incubators with varying temperatures, i.e., 23, 37, 44, are to be provided. Plenty of cold storage space is needed to stock cultures, media, etc.

The requirement of staff and organisation of work depends upon the number of specimens handled. If 50 specimens per day are likely to be processed, 5 lab technicians, 3 lab attendants, 1 sweeper and 1 bacteriologist are necessary. Once laboratory is opened, maximum benefit should be derived by getting adequate number of specimens.

KEY WORDS: GUIDELINES, TUBERCLE BACILLI, CULTURE LABORATORY.

145
COST OF ESTABLISHING AND OPERATING A TUBERCULOSIS BACTERIOLOGICAL LABORATORY
N Naganathan, K Padmanabha Rao & R Rajalakshmi: Indian J TB 1974, 21, 181-90.

This paper deals with the cost of establishing and running a bacteriological laboratory in State Tuberculosis Centres under the National Tuberculosis Programme, and the cost of various examinations to be undertaken in such a laboratory. A knowledge of the cost will enable proper planning and judicious utilization of the resources. Further, when services are rendered to private individuals or institutions, the charges for different examinations can be levied on a rational basis. The place of smear and culture examinations under the programme, the implications of establishing a culture laboratory, the limitations of cost worked out, have been discussed. A plan of the laboratory building is also provided.

The cost has been worked out presuming that about 12,000 specimens per year are likely to be received, of which 25% might turn out to be positives. Non recurring cost was estimated to be Rs.1,07,724 and annual recurring cost would be Rs.49,709. Factors that contribute to the cost structure are overheads, cost of material and labour. In addition, certain essential facilities like cold room, incubator room, gas supply, washing and sterilisation etc., add to the cost. (i) staff-bacteriologist-1, lab technicians-4, lab attendants-3 and registration clerk-1; their salaries, (ii) building-rent (iii) electricity (iv) furniture (v) equipment and supplies (vi) water charges had all been taken into consideration. The cost of one smear examination was estimated to be Rs.0.54 and that of culture and sensitivity test Rs.9.43.

KEY WORDS: COST, LABORATORY, TUBERCLE BACILLI, ESTABLISHMENT.

151
RECOVERY OF TUBERCLE BACILLI FROM URINE OF PULMONARY TUBERCULOSIS PATIENTS AND ITS COMPARISON WITH THE CORRESPONDING SPUTUM ISOLATES
VK Challu, B Mahadev, R Rajalakshmi & K Chaudhuri: Indian J TB 1989, 36, 107-11.

A study was done to compare (1) the filtration method with conventional centrifugation method for the recovery of tubercle bacilli from urine and (2) drug sensitivity profile, virulence for guinea pigs and phage type of the urine isolates with the corresponding isolates from the sputum of cases of bacillary pulmonary tuberculosis.

Urine specimens from 236 pulmonary tuberculosis patients were cultured by routine centrifugation method as well as filtration method. Filtration was done by passing urine through a 0.45 um membrane filter and treating the membrane with 5% oxalic acid for 15 minutes. LJ medium was used for culture in both the methods. Centrifugation yielded 27 positives (11.6%) whereas filtration gave 12 (12.6%) out of 95 specimens filtered. Contamination was more with filtration method. Comparison of the biological properties of M.tuberculosis isolated from urine and sputum of the same patients revealed difference in drug sensitivity profile or virulence for guineapigs for 13 of 25 (52%) of the pairs of isolates tested. Moreover 4 of 11 pairs subjected to phage typing were found to differ in both major and minor phage types. The significance of these findings in the light of the pathogenesis of tuberculosis is also discussed.

KEY WORDS: FILTRATION, CENTRIFUGATION, SENSITIVITY, VIRULENCE, SPECIFICITY, TUBERCLE BACILLI.
 
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