ACID FAST STAINING Exercise No. 9 3rd Shifting Microbiology Shierline. Richmond. Clarice. Chiqui.
I. Objectives 1. The students should be able to know the principles and the techniques in acid fast staining. 2. To differentiate acid fast from non-acid fast organisms.
II. Introduction • HISTORY: – 1882: Robert Koch reported discovery of tubercle bacillus and described appearance of the bacilli resulting from a complex staining procedure – 1890s: acid fast staining is also known as Ziehl-Neelsen method: • Frank Ziehl: first to use carbolic acid (phenol) as mordant • Friedrich Neelsen: kept Ziehl’s mordant, but changed the primary stain to basic fuchsin (first used by Ehrlich in 1882) • Ziehl-Neelsen method uses heat to help primary stain penetrate the cell wall hot staining method
– 1915: Kinyoun published cold staining method
II. Introduction (cont’d) • Mycobacteria: – Grow at a relatively slow rate – Gram ghost or gram neutral (fail to take up both primary and counterstain on Gram staining) – Contain mycolic acid in cell wall
Mycolic acid • Responsible for pathogenicity by inhibiting phagocytosis by immune cells • With high lipid content; renders the cell wall waxy and impermeable to gram staining • To view cells in samples, staining requires higher concentrations of dye solution and/or a heating period • However, once the cell wall is stained with the primary stain, they resist decolorization with acid alcohol (hence, acid fast)
AFB Smear • Important role in the early diagnosis of mycobacterial infections • Microscopy: oldest, easiest, most rapid and inexpensive procedure that can be performed in laboratory to detect presence of AFB
AFB Smear (cont’d) • However, AFB smear should not be used in place of culture – AF smear requires 105 acid fast bacilli per ml of sputum for recognition by direct microscopy – Culture detects as few as 10-100 CFU/ml of sputum
AFB Smear (cont’d) • Purpose: 1. Provides presumptive diagnosis of mycobacterial disease. 2. Smear positive patients are rapidly identified and are the most infectious cases 3. It is used to follow the success of chemotherapy of tuberculous patients 4. It is of vital importance to the patient’s discharge from the hospital, or return to gainful employment 5. It can confirm that cultures growing on media are indeed acid-fast.
Acid Fast Organisms • Mycobacterium sp. • Nocardia, Rhodococcus, Legionella • Isospora, Cryptosporidium
III. Materials • Sputum smear • Acid fast stain: – Carbol fuchsin – Acid alcohol – Loeffler’s methylene blue
• Microscope (OIO) • Alcohol lamp
Expected result:
Note presence of red (acid-fast) tubercle bacilli
Ziehl-Neelsen
Kinyoun
(Hot staining)
(Cold staining)
Purpose
Primary stain
Carbol fuchsin
Carbol fuchsin (↑ concentration)
Contains phenol which solubilizes and penetrates cell wall ; all cell types will take up the primary stain
Mordant
Physical: steam
Chemical: tergitol
Increases stain penetration
Decolorizer
Acid alcohol (HCl-ethanol)
Acid alcohol (HCl-ethanol)
Decolorizes all the cells except the acid fast bacilli
Secondary/ Counter stain
Methylene blue or malachite green
Methylene blue or malachite green
Stains cells which have been decolorized
Ziehl-Neelsen Method Hot staining method
Heat is applied during the primary stain to increase the stain penetration and help drive the stain into the mycobacterial cell wall. Once stained, acid fast organisms resist decolorization with acid alcohol (hydrochloric acid-ethanol).
Kinyoun Modification of ZN Method Cold staining method
Kinyoun modification removed the heating step and instead used a higher concentration of the carbol fuchsin primary stain. This method is preferred when the AFB to be examined is in a tissue sample.
Diagnosis of TB disease www.cdc.gov/tb/education
• A complete medical evaluation for TB disease includes: – – – – –
Medical history Physical examination Test for M. tuberculosis infection Chest radiograph Bacteriologic examination of clinical specimens: • Specimen collection, processing and review • AFB smear classification and results • Direct detection of M tuberculosis in clinical specimen using nucleic acid amplification (NAA) • Specimen culturing and identification • Drug susceptibility and testing
Specimen Collection • There are four (4) collection methods for pulmonary TB disease: – Coughing – Induced sputum – Bronchoscopy – Gastric aspiration
Sputum Collection • Coughing: most commonly used method – Health care worker should wear personal protective equipment and should coach and directly supervise patient when sputum is collected – Patient should be informed that sputum is the material brought up from the lungs, and that mucus from the nose or throat and saliva are not good specimens – Patient should perform deep cough, and then cough up the specimen into a sterile container – Container must be properly sealed, labeled and examined immediately
Sputum Collection (cont’d) • Specimens with too many squamous epithelial cells and/or few PMNs are not suitable for culture • BARTLETT’S CLASSIFICATION: 1. <10 epithelial cells/LPF (Prioritized criterion; if >10 epithelial cells, saliva was collected, not sputum) 2. >25 PMN/LPF
Specimen Processing • At least three (3) specimens are needed
consecutive
sputum
– Collected in 8- to 24-hour intervals – At least one (1) being an early morning specimen
• If possible, specimens should be obtained in an airborne infection isolation (AII) room or other isolated, well ventilated area (e.g., outdoors)
Extrapulmonary TB • TB disease can occur in almost any anatomical site; thus, a variety of clinical specimens other than sputum may be submitted for examination: – Urine – CSF – Pleural fluid – Pus – Biopsy specimens (ex. lung tissue)
AFB Smear Classification Reporting of results
Diagnosis • Culture remains the gold standard for laboratory confirmation of TB disease, and growing bacteria are required to perform drugsusceptibility testing and genotyping • Positive cultures for M tuberculosis confirm the diagnosis of TB disease; however, in the absence of a positive culture, TB disease may also be diagnosed on the basis of clinical signs and symptoms alone
Diagnosis (cont’d) • Culture examinations should be done on all diagnostic specimens, regardless of AFB smear or NAA results
End. Happy aral!
P.S. Tables found in the discussion of TB diagnosis were lifted from the CDC fact sheet on tuberculosis.
