Acid fast staining procedure for staining mycobacteria

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Transcript of Acid fast staining procedure for staining mycobacteria

  • 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.)
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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