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 ~  Abstract
 ~  Materials and Me...
 ~  Discussion
 ~  Conclusion
 ~  Acknowledgements
 ~  References

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Year : 2001  |  Volume : 19  |  Issue : 3  |  Page : 163-165
 

Evaluation of a novel, two component, two step AFB cold staining method


Tulip Diagnostics (P) Ltd. Gitanjali, Tupil Block, Dr. Antonio Do Rego Bagh, Bambolim complex P.O., Bambolim, Goa - 403 202, India

Correspondence Address:
Tulip Diagnostics (P) Ltd. Gitanjali, Tupil Block, Dr. Antonio Do Rego Bagh, Bambolim complex P.O., Bambolim, Goa - 403 202, India

 ~ Abstract 

AFB smear is a clinically useful and a cost effective test for screening of sputum samples for Mycobacterium tuberculosis. Routinely, Ziehl Neelsen stain, using the traditional hot staining procedure is the mainstay of sputum smear AFB tests. We have evaluated a novel two components, two step cold AFB stain to establish its sensitivity, simplicity and reproducibility as compared to the traditional 'ZN Hot' stain still regarded as the standard AFB stain in routine laboratory practice. This study was conducted over a period of four months at the TB Hospital, Taleigao Goa. Comparatively the two components, two step AFB stain was easy to use, time, labour and cost saving. It gave reproducible results during the study period. The most important characteris tics were that it nearly doubled (~91%) the detection rate of AFB positive sputum in our hospital.

How to cite this article:
Tripathi D G, Desai M W, Mesquita A M. Evaluation of a novel, two component, two step AFB cold staining method. Indian J Med Microbiol 2001;19:163-5


How to cite this URL:
Tripathi D G, Desai M W, Mesquita A M. Evaluation of a novel, two component, two step AFB cold staining method. Indian J Med Microbiol [serial online] 2001 [cited 2019 Sep 18];19:163-5. Available from: http://www.ijmm.org/text.asp?2001/19/3/163/8157


Ziehl Neelsen, a carbol fuchsin based stain, using the three component hot process is a standard staining technique for staining sputum smears for the detection of Mycobacterium tuberculosis.
Though the technique yields excellent results, it is an involved method requiring considerable skill and judgement. Additionally the complete process from preparation of smears to processing, staining and observation takes well over 40 minutes. Modification of this traditional ZN stain such as the three component Kinyoun stain (ZN-cold method) have been developed to cut short the 'Processing Time' and increase Laboratory productivity. However further improvements and simplifications have been continuously sought to further simplify and speed up the AFB smear staining process.
We decided to evaluate a novel two component, two step cold AFB stain to assess its performance for sensitivity, simplicity and rapidity.

 ~ Materials and Methods Top

116 sputum specimens from patients visiting O.P.D at T.B. Hospital were screened, using a novel Two Step A.F.B cold staining procedure against the routinely adopted traditional Ziehl- Neelsen three component hot staining procedure. An ideal sputum smear (200 cubic mm) was prepared in duplicate for each sputum specimen. The duplicate smear series was stained using the novel Two Step A.F.B cold staining procedure and Ziehl - Neelsen hot staining procedure simultaneously. Both the smears were examined and observations reported by two experienced technicians. Each slide was screened for a minimum of 5 minutes (Minimum 20 fields) by each of the technicians.
Novel two component, two step A.F.B. cold staining procedure:
(a) Add stain 1 over the smear just to cover it, (approximately 0.5mL of stain is used).
(b) Keep for 6 mins. and rinse with plenty of distilled water, slowly to remove excess stain.
(c) Tilt the slide to drain and add stain 2, to cover it (approximately 0.5mL stain is used).
(d) Keep for 6 minutes and rinse the smear once more with distilled water.
(e) Air dry and observe under oil immersion (1000x).
Ziehl - Neelsen Hot staining procedure
(a) Add carbol fuchsin to cover the slide (approximately 3 - 4mL is used).
(b) Heat gently until steam rises.
(c) Continue to heat for 5 minutes so that steam is seen but without boiling.
Note: Do not allow the stain to boil or dry on the slide. If the stain starts to dry during heating add more carbol fuchsin.
(d) Cool the slide for 5 minutes.
(e) Discard the stain and rinse with water gently until the water that runs off is colourless.
(f) Tilt the slide to drain.
(g) Cover the slide with acid decolorising solution (approximately 3-4mL is used).
(h) After about a minute in the acid, wash the slide with water and pour on more decolouriser (approximately 3-4mL).
(i) Repeat this process several times until the film is only very faintly pink.
Note: Decolourisation generally requires contact with sulphuric acid for a total time of at least 10 minutes.
(j) Tilt the slide to drain.
(k) Counterstain with Loeffler's methylene blue stain for 15-20 seconds, (approximately 3-4mL is used).
(l) Discard the stain and rinse with water gently to remove excess stain.
(m) Air dry and observe under oil immersion (1000x).
The results were graded as follows:
[Table]
It was observed that the novel, Two step, two component cold stain as compared to the traditional Ziehl Neelsen Hot stain gave 10 additional positive AFB smears. To establish if these additional positives were indeed missed by the traditional ZN Hot stain, it was decided to follow up these patients over a period of three months to establish their clinical status. The results of the follow up showed that out of 10 patients negative by ZN hot stain and positive by two step AFB stain, 7 patients were clinically positive and 3 patients could not be traced. All of the seven clinically diagnosed patients were put on antitubercular therapy.

 ~ Discussion Top

The above data indicates that:
1. The novel two component, two step AFB cold stain improved the overall detection of AFB in sputum smears by 91%.
2. The traditional ZN cold stain missed 10 sputum smears, which could have been falsely reported as negative.
3. It was also observed that all the 10 sputum smears reported negative by ZN Hot method and positive by the novel two step stain were graded 1+ (scanty), indicating the novel two component two step stain achieved better sensitivity as compared to the traditional ZN Hot stain.
4. As compared to the traditional ZN Hot stain, the novel two step stain method was rapid and simple to perform.

 ~ Conclusion Top

The novel two component, two step AFB stain is an improvement over the traditional ZN Hot stain, as it does not require steaming, decolorisation steps and can be performed within 12 minutes. It can be routinely adopted for AFB sputum staining especially in clinical settings with high workloads as it improves turn around time.
Due to its simplicity and higher sensitivity the novel two step AFB stain is recommended for routine AFB sputum staining.

 ~ Acknowledgements Top

We are thankful to the staff and members of the T.B. Hospital, Panaji, Goa for co-operation extended through out these studies.
Our special thanks to Seema M.A of Tulip Diagnostics (P) Ltd. and Suneeta C.F of T.B. Hospital, Panaji, Goa for coordinating this project. 

 ~ References Top

1.K. Toman: Tuberculosis Case - Finding and Chemotherapy. WHO Publication 1979.  Back to cited text no. 1    
2.Larry I. Lutwick: Tuberculosis a Clinical Handbook.  Back to cited text no. 2    
3.Mackie and Mcartney: Practical Medical Microbiology, 13th Edition.  Back to cited text no. 3    
4.Kanai L. Mukherjee: Medical Laboratory Technology. A procedure manual for routine diagnostic tests. Vol. II.  Back to cited text no. 4    
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2004 - Indian Journal of Medical Microbiology
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