|A COLD STAINING METHOD FOR TUBERCLE BACILLI USING
|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.
|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
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.
|SOME GUIDELINES FOR ESTABLISHING A TUBERCULOSIS
|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
|KEY WORDS: GUIDELINES, TUBERCLE BACILLI, CULTURE
|COST OF ESTABLISHING AND OPERATING A TUBERCULOSIS
|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,
|RECOVERY OF TUBERCLE BACILLI FROM URINE OF PULMONARY
TUBERCULOSIS PATIENTS AND ITS COMPARISON WITH THE CORRESPONDING SPUTUM
|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
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.