Acid Fast Staining

ACID-FAST STAINING

(ZIEHL-NEELSEN AND KINYOUN) PROCEDURES

What is Acid-Fast Stain? 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.

How does it work? Acid-fast bacteria have a waxy substance called mycolic acid in

their cell walls which makes them impermeable to many staining procedures. These bacteria are termed "acid-fast" because they are able to resist decolorization with acid alcohol.

Carbol fuchsin is the primary stain in this procedure, and it contains phenol to help solubilize the cell wall. Heat is also applied during the primary stain to increase stain penetration. All cell types will take up the primary stain.

The cells are then decolorized with acid-alcohol, which decolorizes all cells except the acid-fast ones.

Methylene blue is then applied to counterstain any cells which have been decolorized. At the end of the staining process, acid-fast cells will be reddish-pink, and non-acid fast cells will be blue. (Note: Acid-fast stains are performed on smears that have been heat-fixed.)

Some Acid Fast Staining techniques

Ziehl–Neelsen stain Kinyoun stain

Various bacterial spore staining techniques using Kenyon e.g.○ Moeller's method○ Dorners method (acid alcohol decolorizer) without the Schaeffer–Fulton

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

Notable acid-fast structures: 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 ceroid-lipofuscinoses (Batten disease).

Nuclear inclusion bodies seen in○ Lead poisoning○ Bismuth poisoning.

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: Each student should be able to

1.Understand the biochemical basis of the acid-fast stain.

2.Perform an acid-fast stain.3.Differentiate bacteria into acid-

fast 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

Materials per student: Tryptic soy broth culture of Escherichia coli and nutrient

agar slant culture of Mycobacterium smegmatis Ziehl’s 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

Why Are the Above Bacteria Used in This Exercise? One of the major objectives of this exercise is to give the student

expertise in acid-fast staining. To allow the student to differentiate between acid-fast and non-acid-fast bacteria, the authors have chosen one of the cultures from the last exercise, Escherichia coli. E. coli is a good example of a non-acid-fast bacterium.

Mycobacterium smegmatis and M. phlei are nonpathogenic members of the genus Mycobacterium.

These bacteria are straight or slightly curved rods, 1 to 10 _m in length, acid-fast at some stage of growth, and not readily stained by Gram’s method.

They are also nonmotile, nonsporing, without capsules, and slow or very slow growers. The mycobacteria are widely distributed in soil and water; some species are obligate parasites and pathogens of vertebrates.

The Ziehl-Neelsen acid-fast staining

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 acid-alcohol.

Ziehl-Neelsen (Hot Stain) ProcedureStep Rationale

1. Make a thin bacterial smear on a separate slide for each organism provided. You will especially have to work the M. smeg. with the loop. It will be helpful to add a loopful of water. Make one slide a mixed sample by adding a loop from both.

1. If you can see substance on the loop or slide surface, your smear will be too thick. The stains cannot penetrate or rinse out of thick samples M. smeg. is branched "higher" bacteria; the cells are difficult to separate.

2. Allow the smear to air dry and then heat-fix. 2. To kill the bacteria and let the smear adhere to the slide for easy staining.

3. Wear Goggles!!!! Flood the slide with carbol-

fuchsin (3-4 drops) and place over a steaming 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.

3. It takes time for the primary stain to penetrate the waxy cell wall at room temperature. Heat hastens the process.

Ziehl-Neelsen (Hot Stain) Procedure

4. Remove from bath and cool. Rinse with distilled water and blot gently with bibulous paper. Allow the slide to cool.

4. Rinse paper towel into sink and pick up at the end of class.

5. Remove the excess stain by tilting the slide and dripping acid-alcohol over it for30 seconds.

5. This pre-colorizing step stops the action of carbol-fuchsin and removes excess stain.

6. Rinse with distilled water and blot gently.6. Rinsing stops the dye action, blotting keeps the next step from being diluted. Too much pressure in blotting will break the slide.

7. Differentiate organisms by flooding the slide with acid-alcohol and allowing to stand for two minutes.

7. Acid-alcohol is the decolorizer and thus differentiates between the acid-fast positive organisms and acid-fast negative organisms. If the alcohol acts too long, it will eventually decolorize even the acid-fast cells.

Ziehl-Neelsen (Hot Stain) Procedure

8. Rinse with distilled water and blot gently. 8. Rinsing stops the action of the acid-alcohol.

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.

10. Rinse and blot dry. Observe.

Summary of Steps:

Results/Interpretation:

Kinyoun (Cold Stain) Procedure

(This may be used instead of or in addition to the Ziehl-Neelsen procedure.)

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.

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