The Ziehl-Neelsen stain was initially developed by Paul Ehrlich to reveal the presence of bacilli causing tuberculosis in clinical samples. This technique is able to differentiate Acid Fast bacteria. It forces the penetration of basic fuchsin into the cell wall by the combined action of phenol and heat, increasing the flowability of the layer of mycolic acids, the heat "melting" the wax component and the phenol act as solvents, allowing the passage of basic dye that binds to the negatively charged mycolic acids. When heat is applied and/or excess phenol is removed by washing with water, the wall recovers its waxiness, but fuchsin molecules are trapped in it.
With the Ziehl Neelsen stain thin, slightly curved, fuchsia red rods are observed standing out against the blue background commonly identified as M. tuberculosis.
Step 1: Sterilize the Slides
In order to have optimal conditions all the slides to be used must be sterilized with a fire source (typically a spirit lamp) applying the heat on both faces for a few seconds.
Step 2: Extract the Most Useful Parts of the Sputum Sample
Extract the purulent and bloody purulent parts of the sample, since they contain all the bacilli, and spread them on the slide making a thin uniform barely transparent smear with a wooden stick or a cotton hyssop. Once this is done fix the spread with a little heat from the spirit lamp applied (only) under the slide.
Step 3: Coloring the Sample
The reagents for the staining are: Carbol Fuchsin, AcidAlcohol and Methylene blue in the concentrations shown in the image attached to this step. It's important to previously filter the necessary amount of carbol fuchsin for all the slides that will be colored.
First we need to stain everything in the sample with the carbol fuchsin, so after setting the fixed slides on the support cover the entire surface with the fuchsin dispensing gently, without splashing and without touching the slide with the dropper or funnel.
Step 4: Apply Heat
In order to open the pores of the sample and penetrate the bacterial wall, heat must be applied with the spirit lamp under the slide with slow waving movements, until white fumes arise from the fuchsin which indicates the procedure is working. We must be careful to heat the slide not letting the fuchsin burn, dry or boil since the cell wall of the bacilli could be destroyed or improperly colored. This procedure must be repeated three times with a few seconds in between to let it cool. In case of spillage of the dye, replenish fuchsin. Do not heat with a lighter. Do not breathe the fumes.
Step 5: First Wash
Wash the surface of the slide carefully with a low pressure stream of water to completely eliminate the fuchsin solution. Tilt the slides to remove water excess and avoid diluting reagents to be used next.
Step 6: Discoloration
Cover the entire smear with the acid alcohol solution and leave for about 1 minute or a bit longer to ensure a proper discoloring. Make sure the smear loses its color (the thickest parts will retain a light pink appearance). In case red clusters are still present on the smear, the bleaching solution must be applied once more for the same period of time. Since M. tuberculosis is an acid fast bacteria it will not be affected by the bleaching, but everything around it will.
Note: a 20% Sulfuric Acid, 80% medicinal ethanol can also be used as decolorizer.
Step 7: Second Wash
Wash the surface of the slide carefully with a low pressure stream of water to completely eliminate the bleaching solution. Tilt the slides to remove water excess and avoid diluting reagents to be used next.
Step 8: Background Coloring or Counter-staining
Flood the entire slide with the methylene blue solution. Leave it on for one minute. This will set the typical blue color for the background allowing the red-pink bacilli to contrast with it and be more easily visible in the microscope analysis of the sample.
Step 9: Third Wash and Set to Dry
Wash the surface of the slide carefully with a low pressure stream of water to completely eliminate the methylene blue solution. Tilt the slides to remove water excess and clean the back of the slide with cotton fabric in case there are any reagents on it.
Let the slides dry either at room temperature or for about a minute in a stove, supporting them upright in a holder on absorbent paper. No absorbent paper support on the smear.
Once the slides are dry that's it! They have been stained with the Ziehl Neelsen Technique and are ready to analyse in the microscope and detect acid fast bacteria.
Note: The effectiveness of the stain highly depends on the smear making. As long as the smear was properly made as a thin uniform layer the results will be clearly visible, if not the very thickness of the layer or the effect of the reagents can lead to see only clusters or fuchsin and methylene blue precipitates which can be confused with acid fast bacteria.