Albert’s staining method
ACID FAST STAIN
- Differential stain
- Distinguishes organisms with waxy (mycolic acid) cell walls that can resist decolourisation with acid alcohol.
METHODS OF AFB STAIN
1. Ziehl-Neelsen stain (Hot stain)
2. Kinyoun stain (cold stain)
3. Flurochrome stain
Acid Fast Organisms & Structures
1. Mycobacterium Species
2. Nocardia Species
3. Rhodococcus Species
4. Legionella Micodadei
5. Oocyst of Cryptospora Parvum & Isospora belli & Cyclospora species
6. Head of Sperm
7. Spores of Bacteria
8. Eggs of Tinea Saginata
9. Helicobacter Pylori
1. Ziehl Neelsen Staining (ZN Staining)
- Ehrlich – 1882 discovered the AF nature
- Franz Ziehl in1882 modified.
- F. Neelsen in 1883 modified.
- ZN Carbol Fuchsin 1%(primary stain)
- Basic Fuchsin 3 gm
- Ethanol 90-95 % 10 ml
- Phenol 5 % 90 ml
- Sulphuric Acid( 20 % decolouriser )
- Concentrated H2SO4 ( 98 % ) 250 ml
- Distilled Water 1 Lit
- Alcohol 95%(secondary decolouriser )
- Ethanol 95 ml
- Distilled Water 100 ml or
- Industrial Methylated spirit – 3 % HCL in 95 % Alcohol
- Acid Alcohol (decolouriser )
- Concentrated HCL 3 ml
- Industrial Methylated Spirit 97 ml
- Loeffler’s Methylene Blue 1% (counter stain)
- Methylene blue powder 1 gm
- Distilled water 100 ml
- Malachite Green 1 %
- Malachite Green powder 5 gm
- Distilled water 500 ml
Stock solution 1% 40 ml + Distilled water 360 ml
Smear Preparation – SPUTUM
Direct or concentration smears of sputum are examined
Various concentration methods:
- Petroffs method – widely used
- N-acetyl-L-cystein with sodium Hydroxide
- Dilute acids (5% oxalic acid, 3% hydrochloric acid or 6% sulphuric acid)
Smear preparation should be done near a flame in safety hood
1. Pick up large, yellow, purulent or blood stained portion of sputum with broomstick or inoculation loop
2. Smear the specimen in the middle of wax free, clean, labeled new glass slide. Slide should not be reused
3. Spread evenly over 1 x 2 cm or 2 x 3 cm area. Print should be readable through the wet smear
4. Allow the smear to air dry completely
5. Fix the slide by passing over flame 2 – 3 times for 2-3 seconds each.
- Place the slides in serial order on the leveled staining rack. Leave enough space between slides to prevent the transfer of material or staining solution from one smear to another.
- Cover the entire surface of the slide with 1 % filtered carbol fuchsin solution.
- Heat the slide with Bunsen burner or alcohol soaked cotton swab till steam arises. Let the slide stand for 5 min. Do not allow the stain to dry on the slide. If necessary put more carbol fuchsin. - Rinse the slide with tap water. Drain off excess water.
- Decolorize with 20% H2SO4 for 2-4 min. wash the slide with water. again pour fresh acid. Repeat several times till the stain is faint pink. This requires a total time of at least 10 min.
- Counter stain with 1% methylene blue & let stand for 30 sec to 1 min.
- Gently rinse with tap water. Allow the slide to dry or blot with filter paper.
- Examine the slides under the microscope oil immersion lens.
- Mycobacteria appear Acid fast (red), long, slender, slightly curved with barred or beaded appearance.