Acid fast staining procedure for staining mycobacteria

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  • 1. ACID FAST STAINING PROCEDURE FOR STAINING MYCOBACTERIA
    A Presentation
    By
    Isaac .U.M.,
    Department of Microbiology & Parasitology,
    Faculty of Medicine, International Medical & Technological University,
    Dar-Es-Salaam, Tanzania
  • 2. Introduction
    Most Mycobacteria grow at a relatively slow rate; therefore, the acid-fast smear plays an important role in the early diagnosis of mycobacterial infections.  
    Microscopy is the oldest, easiest, most rapid, and inexpensive procedure that can be performed in the laboratory to detect the presence of acid-fast bacilli.  
    However,  the acid-fast smear should not be used in place of culture.  
    AFB smears require 105AFB/mLof sputum for recognition by direct microscopy; culture detects as few as 10 to 100 CFU/mLof sputum. 
    In spite of this quantitative discrepancy in sensitivity, examination of stained smears of sputum, or other clinical material, can be helpful in several ways:
    • It provides a presumptive diagnosis of mycobacterial disease.
    • 3. Smear positive patients, the most infectious cases, are rapidly identified.
    • 4. It may be used to follow the success of chemotherapy of tuberculosis patients.
    • 5. It is of vital importance to the patient's discharge from the hospital, or return to employment.
    • 6. It can confirm that cultures growing on media are indeed acid-fast.
  • Staining Methods
    The best known and distinctive property of the genus, Mycobacteria, depends upon their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. 
    Once stained the cells resist decolorizationwith acidified organic solvents and are therefore called ACID FAST. 
    Although the ability to retain arylmethane dyes such as carbolfuchsin and auramine-rhodamine after washing with alcohol or weak acids is a primary feature of this genus it is not entirely unique to the genus. 
    Other bacteria which contain mycolic acids, such as Nocardia, can also exhibit this feature. 
    The exact method by which the stain is retained is unclear but it is thought that some of the stain becomes trapped within the cell and some forms a complex with the mycolic acids. 
    This is supported by the finding that shorter chain mycolic acids or mycobacterial cells with disrupted cell walls stain weakly acid-fast.
  • 7. Figure 29-1 Mycobacterial cell wall structure. The components include the (A) plasma membrane, (B) peptidoglycans, (C) arabinogalactan, (D) mannose-capped lipoarabinomannan, (E) plasma-associated and cell wall-associated proteins, (F) mycolic acids, and (G) glycolipid surface molecules associated with the mycolic acids. (Redrawn from Karakousis et al: Cell Microbiol 6:105-116, 2004.)
    Downloaded from: StudentConsult (on 5 May 2008 05:10 PM)
    © 2005 Elsevier
  • 8. Staining Methods
    Fluorochrome staining has some advantages and disadvantages over Ziehl-Neelsen staining. 
    • One advantage is that the smear is examined under a lower magnification with a dry objective allowing a much larger area of the smear to be examined in a shorter time.  
    • 9. One of the disadvantages of fluorochrome staining is that organisms apparently dead, or rendered non-cultivable by chemotherapy may still fluoresce positive.  
    • 10. This disadvantage is caused by the superiority of the fluorochrome stain (auraminerhodamine) over the carbolfuchsin stain to bind intensely with the mycolic acids.  
    • 11. Thus in fluorescence microscopy, more bacilli are stained than would normally be when stained by the Ziehl-Neelsen method.
  • Staining Methods Fluorescent - AuramineRhodamineStaining
    STEP 1:
    • Flame slides to heat fix.
    STEP 2:
    • Flood the slide with AuramineRhodamine stain (BBL, Difco) and allow to stain for 20 minutes.
    • 12. Be sure that the stain stays on the smear. 
    • 13. Do NOT heat. 
    • 14. Do NOT use paper strips.
    STEP 3:
    • Rinse the slide with water.  
    • 15. Auramine-Rhodamine is very "sticky" and should be washed off by "peeling" the stain off the slide. 
    • 16. Aim the flow of water at the edge of the slide and slowly "peel" the stain from the slide.
  • Staining Methods Fluorescent - AuramineRhodamineStaining
    STEP 4:
    • Flood the slide with 0.5% Acid Alcoholand allow to decolorize for 5 minutes.
    • 17. Ensure that the slides are flooded thoroughly with Acid-Alcohol. 
    • 18. Continue to add acid-alcohol until NO  auramine-rhodamine stain remains visible to the naked eye.
    STEP 5:
    • Rinse off the 0.5% Acid Alcohol with water.
    STEP 6:
    • Flood each slide with Potassium Permanganate (MSDS)  and allow to quench for 1 minute.
    • 19. Note: It is critical that the Potassium Permanganate remain on the slides for no longer than 2 minutes as over quenching of fluorescence can occur.
    STEP 7:
    • Wash off the Potassium Permanganate.
  • Staining Methods Fluorescent - AuramineRhodamineStaining
    If all steps have been completed successfully then your slides when examined with 25X objective using a microscope that has an HBO L2 bulb heat filter, a BG 12 primary filter, and OG 1 barrier filter should look like this.
  • 20. Staining Methods Fluorescent - AuramineRhodamineStaining
    This smear is showing over fluorescence of the background of a slide stained with auramine-rhodamine.  The AFB in the smear are  fluorescing the same color as the debris and are blurry, making it difficult to make out the morphology.  Someone inexperienced in the  reading of fluorescent smears might mistake this as being all debris causing a false negative report to be sent.  
  • 21. Staining Methods Fluorescent - AuramineRhodamineStaining
    Any of the following mistakes during staining can contribute to this occurance:
    Insufficient washing off of the auramine-rhodamine stain.
    Insufficient decolorization of the smear because the smear was made too thick.
    The slides are not decolorized with acid-alcohol for the required length of time.
    Insufficient decolorization due to the slide not being  flooded  with acid alcohol until all visible traces of auramine-rhodamine are gone.
    Insufficient quenching with Potassium Permanganate.
  • 22. Staining Methods Fluorescent - AuramineRhodamineStaining
    Quality Control Parameters
    • A positive and negative control slideshould be included with each run of stains. 
    • 23. This will verify the correct performance of the procedure as well as the staining intensity of the acid-fast organisms.
    • 24. Control slides should be reviewed before patient smears are read to confirm that the Mycobacteria stain acid-fast. 
    • 25. If the results of the QC slides are acceptable, go on to the patient smears. 
    • 26. If, however, the control slide(s) are unacceptable, review procedures and reagent preparations. 
    • 27. When the problem has been identified and corrected, remake and stain all of the patient's slides from the problem run along with a new set of controls.
  • Staining Methods Fluorescent - AuramineRhodamineStaining
    Quality Control Parameters
  • 28. Staining Methods Ziehl-Neelsen Staining
    STEP 1:
    • Flame slides to heat fix.
    STEP 2:
    • Flood the entire slide with CarbolFuchsin.
    • 29. Ensure enough stain is added to keep the slides covered throughout the entire staining step.
    STEP 3:
    • Using a Bunsen burner, heat the slides slowly until they are steaming. 
    • 30. Maintain steaming for 5 minutes by using low or intermittent heat (i.e. by occasionally   passing the flame from the Bunsen burner over the slides)  
    • 31. Caution: Using too much flame or heat can cause the slide to break.
    STEP 4:
    • Rinsethe slide with water.
  • Staining Methods Ziehl-Neelsen Staining
    STEP 5:
    • Flood the slide with 3% acid-alcoholand allow to decolorize for 5 minutes.
    • 32. Throughout the 5 minutes, continue to flood the slides with 3% acid-alcohol until the slides are clear of stain visible to the naked eye. 
    STEP 6:
    • Rinse the slide thoroughly with water and then drain any excess from the slides.
    STEP 7:
    • Flood the slide with the counterstain,Methylene Blue. 
    • 33. Keep the counterstain on the slides for 1 minute.
    STEP 8:
    • Rinsethe slide thoroughly with water.
  • Staining Methods Ziehl-Neelsen Staining
    If all steps are performed correctly you should have a slide that looks like this.
  • 34. Staining Methods Ziehl-Neelsen Staining
    This is an example of insufficient staining with CarbolFuchsin during the Ziehl-Neelsen staining procedure and/or insufficient flaming of the CarbolFuchsin while on the slide.  The arrow is pointing to an Acid-fast bacilli that is almost non-visible.  If this step of the staining procedure is not performed correctly there is a risk of reporting a false negative result.
  • 35. Staining Methods Ziehl-Neelsen Staining
    Quality Control Parameters
    • A positive and negative control slide should be included with each run of stains. 
    • 36. This will verify the correct performance of the procedure as well as the staining intensity of the acid-fast organisms.
    • 37. Control slides should be reviewed before patient smears are read to confirm that the mycobacteria stain acid-fast. 
    • 38. If the results of the QC slides are acceptable, go on to the patient smears. 
    • 39. If, however, the control slide(s) are unacceptable, review procedures and reagent preparations. 
    • 40. When the problem has been identified and corrected, remake and stain all of the patient's slides from the problem run along with a new set of controls.
  • Staining Methods Ziehl-Neelsen Staining
    Quality Control Parameters
  • 41. Examination of Smears for Acid-Fast Bacilli
    Morphological Characteristics
    • It is critical to learn the morphological characteristics of mycobacteria since they are not the only objects that can stain as acid-fast in a smear. 
    • 42. Debris, some species of Nocardia, and some bacterial and fungal spores can also stain acid-fast.
    • 43. Acid-fast bacilli range from 1 to 10 µm in length and 0.2 to 0.6 µm in width.  
    • 44. They typically appear as slender, rod-shaped bacilli, but they may appear curved or bent.
    • 45. Individual bacteria may display heavily stained areas referred to as beads and areas of alternating stain producing a banded appearance.
    • 46. Some mycobacteria other than M. tuberculosis may appear pleomorphic, ranging in appearance from long slender rods to coccoid forms, with more uniform distribution of staining properties.
  • Examination of Smears for Acid-Fast Bacilli
    Method of Examination
    • Ziehl-Neelsen stained smears should be examined with a 54x or 100x oil immersion* objective; fluorochromestained smears with a 20x or 40x dry objective. 
    • 47. Regardless of which type of stain is being observed, each slide must be thoroughly examined for the presence or absence of acid-fast bacilli.  
    • 48. It is recommended that a minimum of 100 fields be examined before a smear is reported as negative. 
    • 49. To achieve this, you should adopt a procedure that ensures that a representative area of the smear is reviewed.  
    • 50. Three passes along the long axis of the slide or nine passes along the short axis of the slide should provide an ample area for reading and eliminate the possibility of reading the same area more than once.
  • Examination of Smears for Acid-Fast Bacilli
    Method of Examination (Continued …)
  • 51. Examination of Smears for Acid-Fast Bacilli
    Method of Examination (Continued …)
    • If you are reading slides that were made after treatment of a specimen, and it is determined that acid-fast bacilli are present, then a smear should also be made from the original untreated specimen, stained by a fluorescent stain and examined. 
    • 52. AFB should be present, ensuring that a positive report is going out on the correct patient.
    • 53. * When reading slides using an oil immersion objective, it is important to wipe the oil off the objective after reading a positive slide.  AFB can dislodge or float off a slide into the immersion oil and be transferred to one of  the next slides, possibly leading to the reporting of a false positive result.
  • Reporting Results of Acid-Fast Bacilli Smears
    When examining a slide that contains AFB, only observe enough fields to obtain a representative average of AFB present. 
    Count each AFB that is not touching another AFB as one, and because a clump represents one colony-forming unit, count clumps of AFB also as one. 
    There are several methods for the reporting of numbers of AFB seen in a smear.  
    One system recommended by the Center for Disease Control when reporting fuchsin-stained smears observed at 1000x is:
  • 54. Reporting Results of Acid-Fast Bacilli Smears
  • 55. Reporting Results of Acid-Fast Bacilli Smears
    *Since other objects can stain acid-fast (i.e. Nocardia, fungal spores, cellular debris, etc) a slide should not be reported out as being positive for acid-fast bacilli unless at least three morphologically correct AFB are seen in the smear (or per 300 fields).   Following this practice reduces the chance of reporting a false positive result.   In an instance where less than three AFB are seen in a slide it is suggested that you do one or more of the following:
    Reexamine the smear.
    Make several more smears from the specimen, stain, and examine.
    Report the questionable findings to the Doctor and ask that further specimens be submitted.
  • 56. Reporting Results of Acid-Fast Bacilli Smears
    Reports should state:
    If acid-fast bacilli were seen or not seen.
    If acid-fast bacilli were seen, state the number of AFB seen.
    Since tubercle bacilli can not be microscopically distinguished from nontuberculousmycobacteria by smear examination, do not report species.   A positive report should only state that "acid-fast bacilli were seen".
    State the method by which the smear was examined (i.e. Fluorescent Microscopy or Ziehl-Neelsen stain).
  • 57. Acid-Fast Bacilli Quality Control Slide Preparation
    Positive Control Slides
    • Collect a sputa that contains AFB in large numbers.
    • 58. Mix equal amounts of the smear positive sputa with 4% glutaraldehydein a 50 mL centrifuge tube (usually 3 - 4 mls of each).
    • 59. Vortex well.
    • 60. Let this mixture sit at room temperature for 30 minutes.
    • 61. Add sterile distilled water to yield a final volume of 40 mLs.
    • 62. Centrifuge at 3800 x g for 20 minutes.
    • 63. Decant completely, leaving only the button.
    • 64. Add sterile distilled water to yield a final small number of acid fast bacilli in the QC smear.
  • Acid-Fast Bacilli Quality Control Slide Preparation
    Positive Control Slides
    • To ensure that the organism was killed by the procedure, add a drop of the glutaraldehyde treated organism to a Lowenstein-Jensen (LJ ) slant and incubate for 3 weeks at 37°C in 5% CO2.  
    • 65. Only make slides from the glutaraldehyde treated concentrate once it has been determined that the process was successful (i.e. no growth on the LJ in 3 wks). 
    • 66. If there is growth then repeat the process until successful.
    • 67. Label slides  with "Positive QC - AFB" 
    • 68. Place a drop of the glutaraldehyde treated specimen on the slide marked positive and spread to the size of about a dime.
    • 69. Allow slides to dry and then heat fix them. 
    • 70. Slides can be stored in a dry, cool place for approximately 1   year.
    • 71. Note: Glutaraldehyde is a dangerous fixative always treat accordingly.(MSDS)
  • Acid-Fast Bacilli Quality Control Slide Preparation
    Negative Control Slides
    • Collect concentrated sputa that is negative in direct smear,and hold in the refrigerator for 7 weeks until cultures of the sputa are proven to be negative.  
    • 72. Label slides with "Negative QC - AFB"
    • 73. Place one drop of the confirmed negative concentrated sputa on each slide and spread to about the size of a dime.
    • 74. Allow slides to dry and then heat fix them. 
    • 75. Slides can be stored in a dry, cool place for approximately 1 year.
  • Acid-Fast Bacilli Stain Procedures References & Links
    For further reading on the topic of AFB staining procedures checkout the following text books:
    • Kubica, G.P., Kent, P.T.  1985.  Public Health Mycobacteriology.  A Guide for the Level III Laboratory.  U.S. Department of Health and Human Services, C.D.C., Atlanta, Georgia.
    • 76. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Volume 1, pages 3.5.1 - 3.5.11.  American Society for Microbiology, Washington, D.C., 1992.
    For more information on the preparation of stains and reagents:
    Balows, A., Hausler, W.J. Jr., Herrmann, K.L., et al.  Manual of Clinical Microbiology, Fifth Edition.  page 1308 & 1313.  American Society for Microbiology, Washington D.C. 1991.
    Kubica, G.P., Kent, P.T.  1985.  Public Health Mycobacteriology.  A Guide for the Level III Laboratory. Page 60  U.S. Department of Health and Human Services, C.D.C., Atlanta, Georgia.
    Isenberg, H.D. Clinical Microbiology Procedures Handbook, Volume 1, page 3.5.3.   American Society for Microbiology, Washington, D.C., 1992.
  • 77. THANK YOU