Second Shifting 2014

Microbiology Faculty

ACID FAST STAINING

2.02L
Topic Outline:
I. Overview
A. Brief History
B. Mycolic Acid
C. AFB Smear
D. Acid Fast Organisms
II. Methodology
III. Ziehl-Neelsen Method
IV. Kinyoun Modification of ZN Method
V. Diagnosis of TB Disease
A. Specimen Collection
B. Sputum Collection
C. Specimen Processing
D. Extrapulmonary TB
E. AFB Smear Classification Figure 1. Mycbacterium tuberculosis

Nocardia, Rhodococcus, Legionella

OVERVIEW Isospora, Cryptosporidium

A. BRIEF HISTORY METHODOLOGY

1882: Robert Koch reported discovery of tubercle bacillus and MATERIALS
described appearance of the bacilli resulting from a complex o Sputum smear
staining procedure o Acid fast stain:
1890s: acid fast staining is also known as Ziehl-Neelsen method: Carbol fuchsin
o Frank Ziehl: first to use carbolic acid (phenol) as mordant Acid alcohol
o Friedrich Neelsen: kept Ziehls mordant, but changed the Loefflers methylene blue
primary stain to basic fuchsin (first used by Ehrlich in 1882) o Microscope (OIO)
o Ziehl-Neelsen method uses heat to help primary stain o Alcohol lamp
penetrate the cell wall - hot staining method
1915: Kinyoun published cold staining method PROCEDURE

B. 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)

C. 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
However, AFB smear should not be used in place of culture
o AF smear requires 105 acid fast bacilli per ml of sputum for
recognition by direct microscopy
o Culture detects as few as 10-100 CFU/ml of sputum
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 patients discharge from the Figure 2. Acid fast Staining Procedure
hospital, or return to gainful employment
5. It can confirm that cultures growing on media are indeed ZIEHL-NEELSEN METHOD
acid-fast Also known as the hot staining method
Heat is applied during the primary stain to increase the stain
D. ACID FAST ORGANISMS penetration and help drive the stain into the mycobacterial cell
Mycobacteria: wall
o Grow at a relatively slow rate Once stained, acid fast organisms resist decolorization with acid
o Gram ghost or gram neutral (fail to take up both primary and alcohol (hydrochloric acid-ethanol)
counterstain on Gram staining)
o Contain mycolic acid in cell wall

Transcribers: Bautista, de Castillo, Gamboa, Wong Page 1 of 2

 2.02 Acid Fast Staining
Figure 3. Ziehl-Neelsen Method
KINYOUN MODIFICATION OF ZN METHOD
Also known as the 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
Figure 4. Kinyoun Modification of ZN Method
Table 1. Ziehl-Neelsen Hot Method vs. Kinyoun Cold Method
Table 2. AFB Smear Classification Reporting of Results
DIAGNOSIS OF TB
A complete medical evaluation for TB disease includes:
o Medical history
o Physical examination
o Test for M. tuberculosis infection
o Chest radiograph
o 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
A. SPECIMEN COLLECTION
There are four (4) collection methods for pulmonary TB disease:
Transcribers: Bautista, de Castillo, Gamboa, Wong
o Coughing
o Induced sputum
o Bronchoscopy
o Gastric aspiration
B. SPUTUM COLLECTION
Coughing: most commonly used method
o Health care worker should wear personal protective
equipment and should coach and directly supervise patient
when sputum is collected
o 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
o Patient should perform deep cough, and then cough up the
specimen into a sterile container
o Container must be properly sealed, labelled and examined
immediately
Specimens with too many squamous epithelial cells and/or few
PMNs are not suitable for culture
BARTLETTS CLASSIFICATION:
1. <10 epithelial cells/LPF (Prioritized criterion; if >10 epithelial
cells, saliva was collected, not sputum)
2. >25 PMN/LPF
C. SPECIMEN PROCESSING
At least three (3) consecutive sputum specimens are needed
o Collected in 8- to 24-hour intervals
o 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)
D. 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:
o Urine
o CSF
o Pleural fluid
o Pus
o Biopsy specimens (ex. lung tissue)
E. AB SMEAR CLASSIFICATION
Culture remains the gold standard for laboratory confirmation
of TB disease, and growing bacteria are required to perform
drug-susceptibility 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
Culture examinations should be done on all diagnostic
specimens, regardless of AFB smear or Nucleic Acid Amplification
(NAA) results
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