Ziehl-Neelsen Staining
This technique allows differentiation of most bacteria into two groups corresponding to cell wall type. Gram positive cells contain thick peptidoglycan with numerous techoic-acid cross-linkages which bind to the highly basic Crystal violet dye.
- sample
preparation - staining
protocol - microscopic
evaluation
Sample Preparation
Vortex the specimen - concentrated sediment, unconcentrated sputum, other purulent material, or stool. Aspirate 0.1-0.2 ml with a Pasteur pipet and place 2-3 drops on the slide. Using the pipette or a sterile applicator, slowly spread the liquid uniformly to make a thin smear. For CSF do somehting else...
Fix the smear at 80C for 15 minutes, or for 2 hours at 65-75 C on an electric hot plate.
Staining Protocol
After heat fixing, flood the smear with carbofuchsin stain and steam the slides gently for 1 min, using a flaming above a rack. Do not allow slides to boil or dry out.
Allow stain to remain another 4-5 minutes without heat.
Rinse slides with deionized water and tilt to drain.
Decolorize with 3.0% acid-alcohol for 2 minutes. Rinse with deionized water and tilt to drain. Flood slides with methylene blue for 1 minute.
Rinse with deionized water and allow to dry.
From another source:
- Primary Staining: Add Ziehl-Neelsen carbol fuchsin to the slide for 5-10 minutes while applying heat.
- Follow with a gentle wash with water to cool the slide.
- Decolorising: Can be done with 20% sulfuric acid and Alcohol separately or with acid alcohol (which contains hydrochloric acid and ethanol).
- Wash the slide in water.
- Counterstaining: Usually done with methylene blue or malachite green.
- The acid-fast bacteria retain the red color, and therefore look hot pink or red. Non acid-fast bacteria will not be red.
online resources:
http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/ZIEHL.PDF
Using oil immersion (1000x)
Mycobacterium spp will appear red or have a red-blue, beaded appearance. Nonmycobacteria will appear blue.