This document provides detailed procedures for staining mycobacteria using acid-fast staining methods, including Ziehl-Neelsen and fluorescent auramine-rhodamine staining. It describes each step of the staining process, highlights potential issues, and emphasizes the importance of quality control with positive and negative control slides. The document also covers examining stained smears, reporting results, and preparing quality control slides to validate acid-fast staining procedures.

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

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.

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

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.

12. Be sure that the stain stays on the smear.

13. Do NOT heat.

15. Auramine-Rhodamine is very "sticky" and should be washed off by "peeling" the stain off the slide.

17. Ensure that the slides are flooded thoroughly with Acid-Alcohol.

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.

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.

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)

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.

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.

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.

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.

52. AFB should be present, ensuring that a positive report is going out on the correct patient.

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).

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.

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.

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.

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.

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