ZIEHL NEELSEN STAINING

1. ZIEHL NEELSEN STAINING Dr Maliha Sumbul

2. Over the last century, tuberculosis (TB) has killed more than 100 million people and this has continued relatively unchanged over the last 50 years, despite the development of effective antituberculous drugs. This chapter summarizes the current status of the epidemiology, pathogenesis, diagnosis, treatment, and control of pulmonary tuberculosis. We have excluded nontuberculous mycobacterial disorders and the various forms of extrapulmonary disease, except pleural TB.

3. Historical overview • Egyptian mummies with severe skeletal deformities suggest that TB has existed since antiquity (Pott's disease). • After the plague devastated Europe during the Middle Ages, TB (the “White Plague”) began to take its heavy toll. • TB affected famous kings and political figures (e.g., King Edward VI, King Louis VIII of France, John Calvin, Cardinal Richelieu, Napoleon II). • TB, also called writer's or artist's disease, killed, among others, Nicolo Paganini, Robert Louis Stevenson, Franz Kafka, George Orwell, all five Brontë sisters, Thomas Mann, Albert Camus, and Igor Stravinsky. • The Nobel Prize for Medicine for TB-related work was given to the following: • Dr. Robert Koch for the discovery of TB bacillus (1905) • Dr. Gerhard Domagk for the discovery of the first antibacterial drug (Prontosil); also pioneered anti-TB drug development (1947) • Dr. Selman Waksman for the development of streptomycin as an anti-TB drug (1952) • In 1993, the World Health Organization (WHO) declared TB a global emergency, the only disease ever so designated. In 2003, WHO reported a continued TB pandemic

4. Dr. Robert Koch

5. Also known as the acid-fast stain • First described by two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894), a pathologist. • It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. • Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis (TB) along with some others of this genus. It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain. • It can also be used to stain few other bacteria like Nocardia. The reagents used are Ziehl-Neelsen carbol fuchsin (basic dye), acid alcohol and methylene blue / malachite green. Acid fast bacilli will be bright red after staining

6. STEP 1: Flame slides to heat fix ALCOHOL FIX IS BETTER – 1 to 2 drops of 70% v/v ethanol or methanol for 2-3 min Ziehl-Neelsen Staining Procedure

7. STEP 2: Flood the entire slide with Carbol Fuchsin Ensure enough stain is added to keep the slides covered throughout the entire staining step.

8. STEP 3: Using a Bunsen burner, heat the slides slowly until they are steaming.  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)   Caution: Using too much flame or heat can cause the slide to break Do not overheat

9. STEP 4: Rinse the slide with water

10. STEP 5: Flood the slide with 3% acid-alcohol and allow to decolorize for 5 minutes. 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.  To the right are examples of slides insufficiently and sufficiently flooded with 3% acid-alcohol.

11. STEP 6: Rinse the slide thoroughly with water and then drain any excess from the slides.

12. STEP 7: Flood the slide with the counterstain, Methylene blue. Keep the counterstain on the slides for 1-2 minutes. Malachite green

13. STEP 8: Rinse the slide thoroughly with water

14. If all steps are performed correctly you should have a slide that looks like this

15. Reporting • Negative X • No AFB seen

16. Differences b/w ZN stain methods I and II • Method I: for M.tuberculosis and M. ulcerans – strongly acid fast, 3% v/v acid solution is used to decolorize the smears • Method II:for M. leprae – only weakly acid fast, 1% acid solution is used to decolorize

17. Hot and cold ZN methods

18. 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.  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 identified and corrected, remake and stain all of the patient's slides from the problem run along with a new set of controls.

19. ACCEPTABLE RESULTS QC: Only blue background UNACCEPTABLE RESULTS QC -Remains red after decolorization -Background is not properly decolorized ACCEPTABLE QC: Red bacilli against a blue background UNACCEPTABLE QC: -Bacilli are not stained red -Background is not properly decolorized NEGATIVE QC POSITIVE QC

24. What are Mycobacteria? • Tuberculosis complex organisms are: • Obligate aerobes growing most successfully in tissues with a high oxygen content, such as the lungs. • Facultative intracellular pathogens usually infecting mononuclear phagocytes (e.g. macrophages). • Slow-growing with a generation time of 12 to 18 hours (c.f. 20-30 minutes for Escherichia coli). • Hydrophobic with a high lipid content in the cell wall. Because the cells are hydrophobic and tend to clump together, they are impermeable to the usual stains, e.g. Gram's stain. • Known as "acid-fast bacilli" because of 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". (Other bacteria which also contain mycolic acids, such as Nocardia, can also exhibit this feature.)

26. Centers for Disease Control and Prevention (CDC) Case Definition of Tuberculosis (Laboratory Criteria) • Isolation of M. tuberculosis from a clinical specimen or (when culture not obtained) • Demonstration of acid-fast bacilli in a clinical specimen Clinical Elements (All of these are needed when not confirmed by laboratory test results.) • A positive skin tuberculin test • Signs and symptoms compatible with tuberculosis or an abnormal chest radiograph • Treatment with two or more antituberculous drugs • A complete diagnostic evaluation with exclusion of other, alternative diagnoses

27. CLINICAL FEATURES MT TEST LAB TESTS (AFB STAIN, AFB C/S) RADIOLOGY (X-RAYS) DIAGNOSIS OF TB Symptoms of tuberculosis include: Fever Night-time sweating Loss of weight Persistent cough Constant tiredness Loss of appetite

28. A TB skin test is typically performed on the forearm. After sterilizing the skin, a measured amount of PPD is injected intracutaneously

29. After the PPD is injected intracutaneously, a small wheal appears on the forearm. The patient should be instructed not to scratch the wheal (but it tends to itch).

30. Within 48 to 72 hours, a positive TB skin test is marked by an area of reddish induration greater than 10 mm. It is the induration (firm bump) that is gently palpated that determines the size, not the area of redness. This reaction is slightly larger than the average positive test 17 mm in size. The positive reaction shown here was obtained with a TB skin test performed 20 years after the initial infection.

31. Dr. Gerhard Domagk (1947 Nobel Prize in Medicine for the discovery of the first antimicrobial drug) Man must want to achieve more than he is able to achieve… If we do not reach for the impossible, we shall never reach far enough to discover the possible. Our wishes should be boundless

32. QUESTIONS ?