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is a differential stain which distinguishes organisms with waxy cell walls that can resist decolorization with acid alcohol.
a laboratory test usually used to determine if a sample of tissue, blood, or other body substance is infected with the bacteria that causes tuberculosis and other illnesses.
the SchaefferFulton modification (decolorize by water) Detergent method, using Tergitol 7, nonionic polyglycol ether surfactants type NP-7
Fite stain
Fite-Faraco Staining Wade Fite staining
Ellis and Zabrowarny stain (no Phenol/carbolic acid) Auramine-rhodamine stain Auramine phenol stain
All Mycobacteria - M. tuberculosis, M. leprae, M. smegmatis and atypical Mycobacterium Actinomyces (especially some aerobic ones) with Mycolic acid in their cell wall (note Streptomyces do NOT have) e.g. Nocardia Rhodococcus Gordonia (Actinomycete) Tsukamurella Dietzia Head of sperm Bacterial spores, Certain cellular inclusions e.g. Cytoplasmic inclusion bodies seen in Neurons in layer 5 of cerebral cortex neuronal ceroidlipofuscinoses (Batten disease).
Cysts of some coccidian parasites in faecal matter, such as: Cryptosporidium parvum Isospora belli Cyclospora cayetanensis A few other parasites: Taenia saginata eggs stain well but Taenia solium eggs don't (Can be used to distinguish) Hydatid cysts, especially their "hooklets" stain irregularly with ZN stain but emanate bright red fluorescence under green light, and can aid detection in moderately heavy backgrounds or with scarce hooklets.
Learning Objectives:
the biochemical basis of the acid-fast stain. 2.Perform an acid-fast stain. 3.Differentiate bacteria into acidfast and non acid-fast groups..
SAFETY CONSIDERATIONS
A volatile and flammable liquid (acid-alcohol) is used in this experiment. Do not use near an open flame. If the carbolfuchsin or methylene blue get on your clothing, they will not wash out. Note: when carbolfuchsin is heated, phenol is driven off. Phenol is poisonous and caustic. Thus, always use a chemical hood with the exhaust fan on for the hot plate or boiling water bath setup. Discard slides in a container with disinfectant. No mouth pipetting. Mycobacteria should be handled in a safety cabinet to prevent dissemination in case the human pathogen Mycobacterium tuberculosis should occur among the cultures. Infected material should be disinfected by heat because mycobacteria are relatively resistant to chemical disinfectants.
Materials to be used:
inoculating loop hot plate microscope bibulous paper paper toweling lens paper and lens cleaner immersion oil staining racks 1-ml pipettes with pipettor
Tryptic soy broth culture of Escherichia coli and nutrient agar slant culture of Mycobacterium smegmatis Ziehls carbolfuchsin prepared with either Tergitol No. 4 (a drop per 30 ml of carbolfuchsin) or Triton-X (2 drops per 100 ml of carbolfuchsin). Tergitol No. 4 and Triton-X act as detergents, emulsifiers, and wetting agents. Alkaline methylene blue Acid-alcohol Clean glass slides Commercial slides showing acid-fast Mycobacterium tuberculosis
developed by Franz Ziehl, a German bacteriologist, and Friedrich Neelsen, a German pathologist, in the late 1800s) is a very useful differential staining technique that makes use of this difference in retention of carbolfuchsin. Expected Result: Acid-fast microorganisms will retain this dye and appear red . Microorganisms that are not acid-fast, will appear blue or brown due to the counterstaining with methylene blue after they have been decolorized by the acidalcohol.
2. To kill the bacteria and let the smear adhere to the slide for easy staining.
3. Wear Goggles!!!! 3. It takes time for the primary stain to Flood the slide with carbol- penetrate the waxy cell wall at room fuchsin (3-4 drops) and place over a steaming temperature. Heat hastens the process. water bath for 5 minutes, adding stain as the edges begin to evaporate. Try not to drip stain into the boiling water. A dime sized piece of paper towel placed directly on the smear can help hold the dye on the smear.
7. Acid-alcohol is the decolorizer and thus 7. Differentiate organisms by flooding the slide differentiates between the acid-fast positive with acid-alcohol and allowing to stand for two organisms and acid-fast negative organisms. If minutes. the alcohol acts too long, it will eventually decolorize even the acid-fast cells.
9. Flood the slide with methylene blue for at least one minute.
9. This is the secondary or counterstain. It will stain the transparent non-AF cells so they may be seen. The color will be blue, much paler than the red AF+ cells. M. luteus is a non- AF coccus in packets, M. smeg. is AF+, and are tiny, branched rods.
Summary of Steps:
Results/Interpretation:
1. Heat-fix the slide as previously directed. 2. Flood the slide for 5 minutes with carbolfuchsin prepared with Tergitol No. 7 (heat is not necessary). 3. Decolorize with acid-alcohol and wash with tap water. Repeat this step until no more color runs off the slide. 4. Counterstain with alkaline methylene blue for 2 minutes. Wash and blot dry. 5. Examine under oil. Acid-fast organisms stain red; the background and other organisms stain blue.
Hints:
(1) Light (diaphragm and condenser adjustments) is critical in the ability to distinguish acid-fast-stained microorganisms in sputum or other viscous background materials.
(2) If the bacteria are not adhering to the slide, mix the bacteria with sheep serum or egg albumin during smear preparation. This will help the bacteria adhere to the slide.