A.J. Pathan and
A.R.Arain
Institute of Chest Disease,
Sind, Kotri, Pakistan.
Methods
Duplicate smears were
prepared from 652 sputum specimens, one for Ziehl-Neelsen staining and other for
Cold Staining. Both were air dried. No heat fixation was performed for the
latter.
Ziehl-Neelsen staining was
performed following the technical guide of IUALTD (1978). The slides for cold
staining were placed on a slide-rack and were covered by staining solution
(basic fuchsine 1 G, 95% alcohol 10 ml, phenol 3 G, distilled water 100 ml) which
was left for 25 minutes before being poured off. The slide was then covered by decolorizing
cumcounterstain solution (10% Nacl in water solution 10 ml,
Methanol 90 ml + Methylene blue 100 mg) for 30 seconds, and then washed in a
gentle stream of water. The second step was repeated once again until the color of the smear became pale blue. The slide was dried in air.
The
slides for both the staining method were examined independently under the microscope
and the results using scores 0, doubtful (+), +, ++, +++ were
recorded.
Results and
observations
The
652 pairs of slides were examined in parallel and Table I was compiled form the
results. Identical results are shown on the diagonal. Scores higher by
Ziehl-Neelsen technique are shown above the diagonal, and those higher by the
cold technique blow it.
Although scores were
definitely higher by the Ziehl-Neelsen technique, the total yield of positive
results from 652 specimens was only slightly higher by this technique i.e.607
(93.9%) positive, as against 583 (89%) positive by the cold technique.
Disregarding the scores, 628 (583 positive and 45 negative or doubtful) of the
652 pairs of smears gave identical results i.e. there was 95% agreement or 4%
disagreement.
It
may be seen from Table I that when cold technique gave the result 0 or
(+), in only 24 of 69 instances (35 %) did the Ziehl-Neelsen technique
yield a positive result (+, ++, or +++); in other words, there was agreement
between the two techniques on negative results in 45 of 69 (65 %). On the other
hand, if the cold technique reported a positive result , the probability of
agreement with the Ziehl-Nesslen technique was complete i.e. 100%.
The
greatest extent of disagreement occurred when the results of the cold technique
reported were doubtful (+). The results of the Ziehl-Neelsen technique
disagreed in 33 of 35 instances, being positive (+) in 17 cases and negative in
16.
Discussion
Sputum smear microscopy is
of adequate sensitivity, adequate specificity and low cost when applied to
symptomatic tuberculosis cases. About 75 % of sputum-positive patients.
Table I. Correlation between
conventional Ziehl-Neelsen technique and the cold technique: results of
examining 652 pairs of smears independently by both methods.
Will be detected at the
first examination, and if two or even three sputum smear examinations are done
for each patient, the sensitivity of sputum smear microscopy may reach that of a
single culture [1].
The
Ziehl-Neelsen method certainly gives better results than the cold method as is
evident from Table I. However taking the results in their entirety, an agreement
of 96 % highly encouraging.
The
staining effect by the cold method is weaker then by the Ziehl-Neelsen method.
Although the acid-fast bacilli stain less intensely and look sharp and thinner
by this method, their delectability is good enough for clinical purposes. Better
results are obtained by increasing the time rather than the concentration of the
basic fuchsine [2].
Under field conditions, the
whole process of sputum collection and smear preparation can be rendered quite
simple: one can use water-proof paper, newspaper or tree leaves for collection
of sputum specimens in place of plastic cups. Also, wooden sticks or tree twigs
can be used instead of platinum loops for smear preparation. As the name
indicates, no heating device is needed for the cold method as against the
heating required for the Ziehl-Neelsen method. It may be pointed out that though
the technique is good, further comparisons with the Ziehl-Neelsen method are required in
relation to culture positively and negativity. Also, further technical
improvements in this technique would be highly desirable, and this matter is
presently under our consideration.
References
1- Nag Paul, D.R., et al. In
Proceedings of the 9th Eastern Region Tuberculosis and Chest
Diseases, Delhi, November 1974. Delhi, Indian Tuberculosis Association
1975.
2- Tan Thiam Hok. A simple and
rapid cold staining method for acid fast bacteria. Amer Rev Resp Dis 1962; 85:
753-4.