| Technique | ACID FAST STAIN |
| Method | Ziehl-Neelson Procedure Acridine Orange Procedure Kinyoun Procedure |
| Positive control | Mycobacterium smegmatis
(ATCC 19420) Mycobacterium phlei (ATCC 354) |
| Negative control | Esherichia coli (ATCC 11229 |
| Technique | ACID FAST STAIN Ziehl-Neelson |
| Principle | |
| Cautions | Do not let the carbol fuchsin stain
dry on the slide but add additional stain if necessary Decolourization should not be attempted in one stage; there should be intermittent washings in water and re-application of acid alcohol Do not blot dry Do not use a common staining jar or dish |
| Method | 1: Heat fix the smear 2:Cover the slide with a strip of filter paper 3: Flood the covered smear with carbol fuchsin solution 4: Heat slowly to steaming point 5: let slide sit for 5 min 6 remove the filter paper strip and wash the slide thoroughly under running water 4: Decolourize with acid alcohol solution until all traces of red have disappeared from the film. 5: Rinse well with water when decolourization is complete. 6: Counterstain with methylene blue or 0.05% malachite green for 1 min. 7: Rinse with water and stand on end to drain. |
| Results | Acid Fast organisms are red, other organisms are blue or green. |
| Reagents | Carbol Fuchsin Solution Solution A Basic fuchsin 0.3 gm; ethanol (95%) 10 ml Solution B Phenol 5 gm; distilled water 95 ml Mix equal volumes of solution A and Solution B Acid Alcohol Solution Methylene Blue
Solution Loeffler's
Alkaline Methylene Blue Alcoholic
Methylene Blue |
| Reference | Manual of Clinical
Microbiology 1985 E. H. Lennette, A. Balows, W.J. Hausler
Jr, H. J. Shadomy American Society for Microbiology
Washington DC pp 225-226 Cowan & Steel 1970 Manual of Microbiological Methods by the Society of Americam Bacteriologists 1957 McGraw Hill New York pp 13-16 Ziehl, F. 1882 Zur Farbung des Tuberkelbacillus. Deut. med Wochschr., 8:451 Neelson, F. 1883 Ein casuistischer Beitrag zur Lehre von der Tuberkulose. Centre. Med. Wisse., 21: 497-501 (see page 500) |
| Technique | Kinyoun acid Fast Stain |
| Principle | |
| Cautions | |
| Method | 1: Heat fix the smear 2:Cover the slide with a strip of filter paper 3: Flood the covered smear with carbol fuchsin solution 4: Heat slowly to steaming point 5: let slide sit for 5 min 6 remove the filter paper strip and wash the slide thoroughly under running water 4: Decolourize with acid alcohol solution until all traces of red have disappeared from the film. 5: Rinse well with water when decolourization is complete. 6: Counterstain with methylene blue or 0.05% malachite green for 1 min. 7: Rinse with water and stand on end to drain. |
| Results | Acid Fast organisms are red, other organisms are blue or green. |
| Reagents | Carbol Fuchsin Solution Solution A Basic fuchsin 0.3 gm; ethanol (95%) 10 ml Solution B Phenol 5 gm; distilled water 95 ml Mix equal volumes of solution A and Solution B Acid Alcohol Solution Methylene Blue
Solution Loeffler's
Alkaline Methylene Blue Alcoholic
Methylene Blue |
| Reference | Manual of Clinical
Microbiology 1985 E. H. Lennette, A. Balows, W.J. Hausler
Jr, H. J. Shadomy American Society for Microbiology
Washington DC pp 225-226 Cowan & Steel 1970 Manual of Microbiological Methods by the Society of Americam Bacteriologists 1957 McGraw Hill New York pp 13-16 |