Most Mycobacteria grow at a relatively slow rate; therefore, the acidfast 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 acidfast bacilli. However, the acidfast smear should not be used in place of culture. AFB smears require 105 AFBmL of sputum for recognition by direct microscopy; culture detects as few as 10 to 100 CFUmL of 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. Smear positive patients, the most infectious cases, are rapidly identified. It may be used to follow the success of chemotherapy of tuberculosis patients. It is of vital importance to the patients discharge from the hospital, or return to employment. It can confirm that cultures growing on media are indeed acidfast.
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 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 105 AFB/mL of sputum for recognition by direct microscopy; culture detects as few as 10 to 100 CFU/mL of 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. Smear positive patients, the most infectious cases, are rapidly identified. It may be used to follow the success of chemotherapy of tuberculosis patients. It is of vital importance to the patient's discharge from the hospital, or return to employment. 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 decolorization with acidified organic solvents and are therefore called ACID FAST. • Although the ability to retain arylmethane dyes such as carbol fuchsin 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. 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 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. One of the disadvantages of fluorochrome staining is that organisms apparently dead, or rendered non-cultivable by chemotherapy may still fluoresce positive. This disadvantage is caused by the superiority of the fluorochrome stain (auramine rhodamine) over the carbol fuchsin stain to bind intensely with the mycolic acids. Thus in fluorescence microscopy, more bacilli are stained than would normally be when stained by the Ziehl-Neelsen method. Staining Methods Fluorescent - Auramine Rhodamine Staining • STEP 1: Flame slides to heat fix. • STEP 2: Flood the slide with Auramine Rhodamine stain (BBL, Difco) and allow to stain for 20 minutes. Be sure that the stain stays on the smear. Do NOT heat. Do NOT use paper strips. • STEP 3: Rinse the slide with water. Auramine-Rhodamine is very "sticky" and should be washed off by "peeling" the stain off the slide. Aim the flow of water at the edge of the slide and slowly "peel" the stain from the slide. Staining Methods Fluorescent - Auramine Rhodamine Staining • STEP 4: Flood the slide with 0.5% Acid Alcohol and allow to decolorize for 5 minutes. Ensure that the slides are flooded thoroughly with Acid-Alcohol. 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. 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 - Auramine Rhodamine Staining • 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. Staining Methods Fluorescent - Auramine Rhodamine Staining • 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. Staining Methods Fluorescent - Auramine Rhodamine Staining • Any of the following mistakes during staining can contribute to this occurance: 1. Insufficient washing off of the auramine-rhodamine stain. 2. Insufficient decolorization of the smear because the smear was made too thick. 3. The slides are not decolorized with acid-alcohol for the required length of time. 4. Insufficient decolorization due to the slide not being flooded with acid alcohol until all visible traces of auramine-rhodamine are gone. 5. Insufficient quenching with Potassium Permanganate. [...]... controls Staining Methods Ziehl-Neelsen Staining • Quality Control Parameters 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 Debris, some species of Nocardia, and some bacterial and fungal spores can also stain acid- fast Acid- fast. .. Health mycobacteriology: a guide for the level III laboratory, US Department of Health and Human Service, Centers for Disease Control, Atlanta, 1985 * AFB, Acid Fast Bacilli 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.. .Staining Methods Fluorescent - Auramine Rhodamine Staining • Quality Control Parameters A positive and negative control slide should be included with each run of stains This will verify the correct performance of the procedure as well as the staining intensity of the acid- fast organisms Control slides should be reviewed before patient smears are read to confirm that the Mycobacteria stain acid- fast. .. 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 Staining Methods Ziehl-Neelsen Staining • Quality Control Parameters A positive and negative control slide should be included with each run of stains This will verify the correct performance of the procedure as well as the staining. .. Keep the counterstain on the slides for 1 minute • STEP 8: Rinse the slide thoroughly with water Staining Methods Ziehl-Neelsen Staining • If all steps are performed correctly you should have a slide that looks like this Staining Methods Ziehl-Neelsen Staining • This is an example of insufficient staining with Carbol Fuchsin during the ZiehlNeelsen staining procedure and/or insufficient flaming... submitted Reporting Results of Acid- Fast Bacilli Smears • Reports should state: 1 If acid- fast bacilli were seen or not seen 2 If acid- fast bacilli were seen, state the number of AFB seen 3 Since tubercle bacilli can not be microscopically distinguished from nontuberculous mycobacteria by smear examination, do not report species A positive report should only state that "acid- fast bacilli were seen" 4 State... 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 Isenberg, H.D Clinical Microbiology Procedures... the possibility of reading the same area more than once Examination of Smears for Acid- Fast Bacilli • Method of Examination (Continued …) 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... performance of the procedure as well as the staining intensity of the acid- fast organisms Control slides should be reviewed before patient smears are read to confirm that the mycobacteria stain acid- fast If the results of the QC slides are acceptable, go on to the patient smears If, however, the control slide(s) are unacceptable, review procedures and reagent preparations When the problem has been... x g for 20 minutes Decant completely, leaving only the button 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 . ACID FAST STAINING PROCEDURE FOR STAINING MYCOBACTERIA A Presentation By Isaac .U.M., Department of Microbiology. correct performance of the procedure as well as the staining intensity of the acid- fast organisms. Control slides should be reviewed before patient smears are read to confirm that the Mycobacteria. as well as the staining intensity of the acid- fast organisms. Control slides should be reviewed before patient smears are read to confirm that the mycobacteria stain acid- fast. If the