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.
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.
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:
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.