Acid Fast Staining Sample

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Tuberculosis ( TB ) is the taking cause of decease in the universe from a bacterial infective disease. The disease affects 1. 8 billion people/year which is equal to tierce of the full universe population.

In the United States TB is on the diminution. In 2007 a sum of 13. 293 instances were reported. The TB rate declined to 4. 4 instances per 100. 000 population. the lowest recorded rate since national coverage began in 1953. Despite this overall betterment. advancement toward TB riddance has slowed in recent old ages ; the mean one-year per centum diminution in the TB rate slowed from 7. 3 % per twelvemonth during 1993–2000 to 3. 8 % during 2000–2007. Besides. since 1993 there has been a gradual diminution in the figure of TB patients with coinfection with HIV. and the figure of instances of multiple drug-resistant Terbium has bit by bit dropped.

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On the other manus. the proportion of TB instances contributed by nonnative individuals has increased each twelvemonth since 1993. In 2007 the TB rate in nonnative individuals in the United States was 9. 7 times higher than in U. S. -born individuals. In many provinces. particularly in the West. the upper Midwest. and the Northeast. most new instances of TB now occur in persons who are foreign born.

Mycobacteria TB

Mycobacteria TB is the etiologic agent of TB in worlds. Worlds are the lone reservoir for the bacteria. Mycobacterium bovis is the etiologic agent of TB in cattles and seldom in worlds. Both cattles and worlds can function as reservoirs. Worlds can besides be infected by the ingestion of unpasteurised milk. This path of transmittal can take to the development of extrapulmonary TB. exemplified in history by bone infections that led to hump dorsums. Other human pathogens belonging to the Mycobacterium genus include Mycobacterium avium which causes a TB-like disease particularly prevailing in AIDS patients. and Mycobacterium leprae. the causative agent of Hansen’s disease. General Characteristics

Mycobacteria TB is a reasonably big nonmotile bacillar bacteria distantly related to the Actinomycetes. Many non infective mycobacteriums are constituents of the normal vegetations of worlds. found most frequently in dry and greasy venues. The rods are 2-4 microns in length and 0. 2-0. 5 um in breadth. Mycobacterium TB is an obligate aerobe. For this ground. in the authoritative instance of TB. MTB composites are ever found in the well-aerated upper lobes of the lungs. The bacteria is a facultative intracellular parasite. normally of macrophages. and has a slow coevals clip. 15-20 hours. a physiological feature that may lend to its virulency. Two media are used to turn MTB Middlebrook’s medium which is an agar based medium and Lowenstein-Jensen medium which is an egg based medium. MTB settlements are little and fan colored when grown on either medium. Both types of media contain inhibitors to maintain contaminations from out-growing MT. It takes 4-6 hebdomads to acquire ocular settlements on either type of media.

Colonies of Mycobacterium TB on Lowenstein-Jensen medium. CDC. Ironss of cells in vilifications made from in vitro-grown settlements frequently form typical serpentine cords. This observation was foremost made by Robert Koch who associated cord factor with deadly strains of the bacteria. MTB is non classified as either Gram-positive or Gram-negative because it does non hold the chemical features of either. although the bacteriums do incorporate peptidoglycan ( murein ) in their cell wall. If a Gram discoloration is performed on MTB. it stains really decrepit Gram-positive or non at all ( cells referred to as “ghosts” ) . Mycobacteria species. along with members of a related genus Nocardia. are classified as acid-fast bacteriums due to their impermeableness by certain dyes and discolorations.

Despite this. one time stained. acid-fast bacteriums will retain dyes when heated and treated with acidified organic compounds. One acid-fast staining method for Mycobacterium TB is the Ziehl-Neelsen discoloration. When this method is used. the MTB. vilification is fixed. stained with carbol-fuchsin ( a pink dye ) . and decolorized with acid-alcohol. The vilification is counterstained with methylene-blue or certain other dyes. Acid-fast B appear pink in a contrasting background. In order to observe Mycobacterium TB in a sputum sample. an surplus of 10. 000 organisms per milliliter of phlegm are needed to visualise the B with a 100X microscope aim ( 1000X mag ) . One acid-fast bacillus/slide is regarded as “suspicious” of an MTB infection.

Mycobacteria TB. Acid-fast discoloration. CDC.

Cell Wall StructureThe cell wall construction of Mycobacterium TB deserves particular attending because it is alone among procaryotes. and it is a major determiner of virulency for the bacteria. The cell wall composite contains peptidoglycan. but otherwise it is composed of complex lipoids. Over 60 % of the mycobacterial cell wall is lipid. The lipid fraction of MTB’s cell wall consists of three major constituents. mycolic acids. cord factor. and wax-D. Mycolic acids are alone alpha-branched lipoids found in cell walls of Mycobacterium and Corynebacterium. They make up 50 % of the dry weight of the mycobacterial cell envelope. Mycolic acids are strong hydrophobic molecules that form a lipid shell around the being and affect permeableness belongingss at the cell surface.

Mycolic Acids are thought to be a important determiner of virulency in MTB. Probably. they prevent onslaught of the mycobacterium by cationic proteins. muramidase. and oxygen groups in the phagocytic granule. They besides protect extracellular mycobacterium from complement deposition in serum. Cord Factor is responsible for the serpentine cording mentioned above. Cord factor is toxic to mammalian cells and is besides an inhibitor of PMN migration. Cord factor is most copiously produced in deadly strains of MTB. Wax-D in the cell envelope is the major constituent of Freund’s complete adjuvant ( CFA ) . The high concentration of lipoids in the cell wall of Mycobacterium TB have been associated with these belongingss of the bacteria: Impermeability to discolorations and dyes

Resistance to many antibioticsResistance to killing by acidic and alkalic compoundsResistance to osmotic lysis via complement depositionResistance to lethal oxidizations and survival interior of macrophages The Ziehl–Neelsen discoloration. besides known as the acid-fast discoloration. was first described by two German physicians: the bacteriologist Franz Ziehl ( 1859–1926 ) and the diagnostician Friedrich Neelsen ( 1854–1898 ) . It is a particular bacteriological discoloration used to place acid-fast beings. chiefly Mycobacteria. Mycobacterium TB is the most of import of this group because it is responsible for TB ( TB ) . Other of import Mycobacterium species involved in human disease are Mycobacterium kansasii. Mycobacterium marinum. and members of the Mycobacterium avium composite. Acid fast organisms like Mycobacterium contain big sums of lipid substances within their cell walls called mycolic acids.

These acids resist staining by ordinary methods such as a Gram discoloration. [ 1 ] It can besides be used to stain a few other bacteriums. such as Nocardia. The reagents used are Ziehl–Neelsen carbolfuchsin. acerb intoxicant. and methylene blue. Acid-fast B will be bright ruddy after staining. A fluctuation on this staining method is used in mycology to differentially stain acid-fast encrustations in the epidermal hyphae of certain species of Fungi in the genus Russula. [ 2 ] [ 3 ] It is besides utile in the designation of some Protozoa. viz. Cryptosporidium and Isospora. The Ziehl–Neelsen discoloration can besides impede diagnosing in the instance of paragonimiasis because the eggs in an egg cell and parasite phlegm sample ( OnP ) can be dissolved by the discoloration. and is frequently used in this clinical scene because marks and symptoms of paragonimiasis closely resemble those of TB. Procedure…

1. Drop suspension onto slide2. Air dry slide 10 proceedingss at 60 °C. heat-fix slide 10 proceedingss at 90 °C3. Flood slide with Carbol Fuchsin4. Keep a flame beneath the slide until steam appears but do non let it to boil5. Let hot slide to sit for 3 to 5 proceedingss. rinse with tap H2O6. Flood slide with 30 % hydrochloric acid in isopropyl intoxicant7. Let to sit 1 minute. rinse with tap H2O8. Flood slide with Methylene Blue9. Let to sit 1 minute. rinse with tap H2O10. Blot dry11. Position under oil submergence lensSurveies have shown that an AFB discoloration without a civilization has a hapless negative prognostic value. An AFB Culture should be performed along with an AFB discoloration ; this has a much higher negative prognostic value. Alterations

•5 % sulphuric acid is used for destaining Mycobacterium leprae alternatively of the 20 % used for Mycobacterium TB. •Kinyoun alteration ( or cold Ziehl–Neelsen technique ) is besides available. •A protocol in which a detergent is substituted for the extremely toxic phenol in the fuchsin staining solution. [ 4 ] •PROCEDURE ( Ziehl-Neelsen Method )

•1. Heat-fix a vilification of a bacteria as follows:•a. Using the dropper bottle of distilled H2O found in your staining rack. topographic point 1/2 a bead of H2O on a clean slide by touching the dropper to the slide. •b. Aseptically take a little sum of the bacteria from the agar surface and blend it with the H2O. Flame the cringle and allow it chill. •c. Using the cringle. distribute the mixture over the full slide to organize a thin movie. •d. Let this thin suspension to wholly air dry.

•e. Pass the slide ( film-side up ) through the fire of the Bunsen burner 3 or 4 times to heat-fix. •2. Cover the smear with a piece of blotting paper and inundation with carbol fuchsin. •3. Steam for 5 proceedingss by go throughing the slide through the fire of a gas burner. •4. Let the slide to chill and rinse with H2O.

•5. Add the acid-alcohol decolorizing easy dropwise until the dye no longer runs off from the vilification.•6. Rinse with H2O.•7. Counterstain with methylene blue for 1 minute.•8. Wash with H2O. smudge dry. and observe utilizing oil submergence microscopy.


In 1882 Robert Koch reported the find of the tubercle B ( 4 ) and described the visual aspect of the B ensuing from a complex staining process. During the same clip period several other research workers ( Ehrlich. Ziehl. Rindfleisch. and Neelsen ) . meaning to better on Koch’s method. introduced alterations to the reagents and the process. Franz Ziehl was the first to utilize carbolic acid ( phenol ) as the mordant. Friedrich Neelsen kept Ziehl’s mordant. but changed the primary discoloration to the basic fuchsin ( first used by Ehrlich in 1882 ) . This method became known as the Ziehl-Neelsen method in the early to mid 1890s. In this method heat is used to assist drive the primary discoloration into the waxy cell walls of these difficult-to-stain cells. The usage of heat in this method has been the ground that this technique is called the “hot staining” method.

The Ziehl-Neelsen method has endured as a dependable and effectual manner to show the acid-fast bacteriums.

In 1915. Kinyoun published a method that has become known as the “cold staining” method because the heating measure was removed in favour of utilizing a higher concentration of the carbolfuchsin primary discoloration.


The acid-fast discoloration is performed on samples to show the feature of acerb speed in certain bacteriums and the cysts of Cryptosporidium and Isospora. Clinically. the most of import application is to observe Mycobacterium TB in phlegm samples to corroborate or govern out a diagnosing of TB in patients.


There are three common acid-fast staining methods. Ziehl-Neelsen ( hot ) . Kinyoun ( cold ) . and Auramine-Rhodamine Fluorochrome ( Truant method ) . The accent in this Atlas-Protocol undertaking will be on the Ziehl-Neelsen and the Kinyoun methods because the slides produced by these methods can be visualized utilizing a standard bright-field microscope. The fluorochrome method is used by big research labs that have a fluorescent ( UV ) microscope. For comparing intents. the formula for the reagents and the protocol for all three methods are included below. but images for the fluorochrome method will non be a portion of the Atlas at this clip.

Many bacterial cells are easy stained with simple discolorations or utilizing the Gram discoloration. A few types of bacteriums. such as the mycobacterium and Nocardia species. do non stain utilizing these techniques or. if stained. they produce a variable reaction because their walls are non permeable to the rosaniline dyes in common staining regimens ( 12 ) . The cell walls of the mycobacterium contain mycolic acids giving the cell walls a high lipid content. This characteristic is thought to be the ground ( 5. 10 ) these bacteriums are hard to stain. To see these cells in samples staining requires higher concentrations of the dye solution and/or a warming period ( 4 ) . However. one time a discoloration is introduced into the cell wall. taking it with a decolorizer is even more hard. The look “acid fast” is derived from the observation that even with the add-on of hydrochloric acid to the intoxicant decolorizer. some of the stained cells retain the primary discoloration ( carbolfuchsin ) . Cells that let go of the primary discoloration ( carbolfuchsin ) with decolorizing will be seeable after the counterstaining measure is complete. Bacteria described as acerb fast will look ruddy when analyzing specimens utilizing bright-field microscopy. Non-acid-fast cells and field dust will look blue.

Acid speed is a characteristic that is shared by merely a few beings. so staining to find if organisms possess this trait is utile in microbic designation strategies.

RECIPES ( 6. 7. 15 )

  1. A. Ziehl-Neelsen method for acid-fast staining ( 6. 7 )
  2. Carbolfuchsin discoloration:
  3. Basic fuchsin. 0. 3 g
  4. Ethanol. 95 % ( vol/vol ) . 10 milliliter
  5. Phenol. heat-melted crystals. 5 milliliter
  6. Distilled H2O. 95 milliliter
  7. Dissolve the basic fuchsin in the ethyl alcohol ; so add the phenol dissolved in the H2O.
  8. Mix and allow stand for several yearss. Filter before usage.
  9. Bleaching dissolver:
  10. Ethanol. 95 % ( vol/vol ) . 97 milliliter
  11. Hydrochloric acid ( concentrated ) . 3 milliliter
  12. Counterstain:
  13. Methylene bluish chloride. 0. 3 g
  14. Distilled H2O. 100 milliliter
  15. B. Kinyoun method for acid-fast staining ( 15 )
  16. Kinyoun carbolfuchsin solution:
  17. Solution A. Dissolve 4 g of basic fuchsin in 20 milliliter of ethyl intoxicant.
  18. Solution B. Dissolve 8 g of phenol ( melted ) in 100 milliliter of distilled H2O.
  19. Mix solutions A and B together and let to stand for a few yearss.
  20. Acid-alcohol bleaching agent:
  21. Ethanol. 95 % ( vol/vol ) . 97 milliliter
  22. Hydrochloric acid ( concentrated ) . 3 milliliter
  23. Methylene bluish counterstain:
  24. Methylene bluish chloride. 0. 3 g
  25. Distilled H2O. 100 milliliter
  26. Dissolve by agitating.
  27. C. Truant method for acid-fast staining ( 6. 7 )
  28. Fluorescent staining reagent:
  29. Auramine O. CI 41000. 1. 50 g
  30. Rhodamine B. CI 749. 0. 75 g
  31. Glycerol. 75 milliliter
  32. Phenol ( heat melted crystals ) . 10 milliliter
  33. Distilled H2O. 50 milliliter
  34. Mix the two dyes good with 25 milliliter of the H2O and the phenol. Add the staying H2O and glycerin and mix once more. Filter the ensuing staining fluorescent reagent through glass wool and shop at 4oC or room temperature.
  35. Bleaching dissolver:
  36. Ethanol. 70 % ( vol/vol ) . 99. 5 milliliter
  37. Hydrochloric acid ( concentrated ) . 0. 5 milliliter
  38. Counterstain:
  39. Potassium permanganate. 0. 5 g
  40. Distilled H2O. 99. 5 g
  41. PROTOCOLS ( 1. 6. 7 )

Smear PreparationNormally. fixing a vilification for staining involves using a really little sample to the centre of a carefully cleaned glass slide. The microbic sample is normally taken from a broth civilization or a suspension of micro-organisms produced by blending a bantam sum of solid affair from settlements with H2O. The suspension can be made straight on the slide or it can be mixed in a tubing and transferred to the slide.

Since many bacteriums cleaving to each other in civilization ( both broth and colonial signifier ) . vigorous manual or mechanical commixture may be required to bring forth a proper distribution of the beings for microscopic rating. Clumps of beings make it hard to detect the features of single cells.

The quality of the staining for acid-fast bacteriums will be affected by the quality of the vilification. A good quality smear will hold a thin movie of the specimen or civilization. leting single cells to react to the staining protocol. The waxen nature of the mycobacterium organisms causes them to drive H2O so fluids should be added to the slide after distributing the sample in a thin movie over the slide.

Organisms grown in media incorporating complex lipoids will stain better. and normally. turn better.

Basic Smear Preparation

1. Clean a glass slide ( some labs will supply pre-cleaned slides ; be certain to take any dust or crushed glass dust ) harmonizing to instructions provided by your teacher.

2. Fix the sample harmonizing to instructions provided by your teacher. Make certain that an aerosol is non generated during this procedure.

3. Using a unfertile pipet or microbiological cringle. use a little sample of the specimen to the slide by easy distributing the liquid to do a thin movie ; If you are utilizing solid affair from a settlement. be certain to take a really infinitesimal sample and distribute it into a really thin movie. Using the cells before adding H2O ( or other blending fluid ) will assist the cells adhere to the slide. The size of the movie should be about 1 centimeters in diameter. Avoid any actions that would spill droplets of the sample in the encompassing country.

4. Let the smear to dry wholly.

5. Repair the vilification at 80oC for 15 proceedingss or for 2 hours on a hot home base set for 65oC to 70oC.

6. Continue to the staining protocol of your pick.

Note: Reports of the endurance of mycobacterium at these temperatures have been made. Follow the proper handling safeguards of your laboratory/institution.

A. Ziehl-Neelsen method for acid-fast staining ( 1. 6. 7 )

1. Heat fix an air dried vilification at 80oC for at least 15 proceedingss or for 2 hours on an electric hot home base at 65oC – 70oC

2. Put a slide with an air-dried and heat-fixed vilification on suited staining device. Cut a piece of absorptive paper to suit the slide and saturate the paper with the carbolfuchsin discoloration.

3. Carefully heat the bottom of the slide by go throughing a fire under the rack or by puting the slide on a hot home base until steam rises ( withoutboiling! ) . Keep the readying moist with discoloration and steaming for 5 proceedingss. reiterating the warming as needed.

( Caution: overheating causes splattering of the discoloration and may check the slide ) .

4. Wash the movie in a soft and indirect watercourse of tap water* until no colour appears in the wastewater.

5. Keeping the slide with forceps. rinse the slide with the bleaching dissolver. Immediately wash with tap water* . as above. Repeat the decolorizing and the lavation until the stained smear appears faintly pink and the fluid rinsing off the slide runs clear.

6. Flood the vilification with the methylene blue counterstain for 20 to 30 seconds. and wash with tap water* . as above.

7. Gently smudge. or air dry the vilification.

8. Analyze under oil submergence. ** Acid-fast bacteriums appear ruddy. and non-acid-fast bacteriums ( and other beings and cellular stuffs ) appear bluish.

* Note: Most labs use deionized or distilled H2O for all lab processs. See note in Tips and Comments subdivision sing the usage of tap H2O.

** Note: For clinical samples. scrutiny of at least 300 Fieldss is required before declaring a specimen negative for acid-fast bacteriums. ••FIG. 1. Mycobacteria TB ( 5 ) .

•B. Kinyoun method for acid-fast staining ( 1 )

1. Heat fix an air dried vilification at 80oC for at least 15 proceedingss or for 2hours on an electric hot home base at 65oC – 70oC

2. Flood slides with Kinyoun’s carbolfuchsin reagent and let to stain for 5 proceedingss at room temperature.

3. Rinse with deionized H2O and tilt slide to run out.

4. Bleach with acid-alcohol for 3 proceedingss and rinse once more with deionized H2O. *

5. Redecolorize with acid-alcohol for 1-2 proceedingss or until no more ruddy colour runs from the vilification. *

6. Rinse with deionized H2O and drain standing H2O from the slide surface by tipping the slide.

7. Flood slide with methylene bluish counterstain and let to stain for 4 proceedingss.

8. Rinse with distilled H2O and let to air prohibitionists.

9. Examine under high prohibitionist ( 400X ) magnification. and confirm acid-fast constructions under oil submergence ( 1000X ) .

* Note: Most practicians apply decolorizer until the fluid rinsing off the slide runs clear.

C. Truant method for acid-fast staining ( 1. 6. 7 )

1. Heat fix an air dried vilification at 80oC for at least 15 proceedingss or for 2 hours on an electric hot home base at 65oC – 70oC

2. Wash the slide with a soft and indirect watercourse of distilled H2O until no colour appears in the wastewater.

3. Flood the vilification with the decolorizing agent for 2 to 3 proceedingss. and so wash with distilled H2O as above.

4. Flood the vilification with the permanganate counterstain for 2 to 4 proceedingss.

5. Wash the slide with distilled H2O as above. smudge with absorptive paper. and dry. •6. Analyze the slide with a fluorescence microscope equipped with a BG-12 exciter filter and an OG-1barrier filter. Acid-fast bacteriums appear as brilliantly fluorescent. yellow-orange cells in a dark field ; non-acid-fast

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