Preparing an Acid-Fast Stain using the Ziehl-Nielsen Method

This four and half minute video shows how to prepare an Acid-Fast Stain using the Ziehl-Nielsen Method. This resource was developed by Charles Adjei Osei of the Kwame Nkrumah University of Science and Technology and Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009. Copyright 2009-2010, Kwame Nkrumah University of Science and Technology and Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommerical 3.0 License http://creativecommons.org/licenses/by-nc/3.0/. Video transcribed by Andrea Matsumoto (University of Michigan).

(English captions by Andrea Matsumoto, University of Michigan.)

Prepare sputum smear by taking the sputum
from the sputum container using the loop,

which has been sterilized.

Spread it on the microscope slide very wide,
about two to three centimeter diameter and

then flame the loop again.

Flame the sputum smear by fixing it.

These are the spirit lamp and then the stains
required for the staining procedure.

Cover the smear with Carbol Fuchsin.

And heat it with the spirit lamp intermittently
until it steams but should not boil or the

steam should not get dried and should be done
intermittently for five minutes.

Tip the steam off and allow it to cool before
washing the excess stain from the slide of

the smear.

Cover the smear with the decolorizer is twenty
percent sulfuric acid for two minutes.

After two minutes tip off the decolorizer
and wash it under the tap water or distilled

water.

Cover the smear with the counterstain with
methylene blue for two minutes.

No heating.

Wash it under the tap water or distilled water.

And air dry it on the draining rack.

Put oil immersion on the smeared slide and
then examine it under the times hundred (100X) objective

of the microscope.

And you are expected to see reddish or pinkish
bacilli, which can appear in a form of a cocci

or it can also appear as a spiral or it can
appear as a dot.