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Second Shifting 2014 Microbiology Faculty 2.02L ACID FAST STAINING
Second Shifting 2014
Microbiology Faculty
2.02L
ACID FAST STAINING

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

OVERVIEW
OVERVIEW
A. BRIEF HISTORY
A.
BRIEF 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:

o Frank Ziehl: first to use carbolic acid (phenol) as mordant o Friedrich Neelsen: kept Ziehl’s mordant, but changed the primary stain to basic fuchsin (first used by Ehrlich in 1882) o Ziehl-Neelsen method uses heat to help primary stain penetrate the cell wall - hot staining method 1915: Kinyoun published cold staining method

B. MYCOLIC ACID
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
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 patient’s discharge from the

hospital, or return to gainful employment

5.

It can confirm that cultures growing on media are indeed

acid-fast

  • D. ACID FAST ORGANISMS

Mycobacteria:

o Grow at a relatively slow rate o Gram ghost or gram neutral (fail to take up both primary and counterstain on Gram staining) o Contain mycolic acid in cell wall

Second Shifting 2014 Microbiology Faculty 2.02L ACID FAST STAINING Topic Outline: I. Overview A. Brief History

Figure 1. Mycbacterium tuberculosis

Nocardia, Rhodococcus, Legionella Isospora, Cryptosporidium

METHODOLOGY
METHODOLOGY

MATERIALS o Sputum smear

o Acid fast stain:

Carbol fuchsin

Acid alcohol

Loeffler’s methylene blue

o Microscope (OIO) o Alcohol lamp

PROCEDURE

Second Shifting 2014 Microbiology Faculty 2.02L ACID FAST STAINING Topic Outline: I. Overview A. Brief History

Figure 2. Acid fast Staining Procedure

ZIEHL-NEELSEN METHOD

Also known as the 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)

2.02 Acid Fast Staining
2.02 Acid Fast Staining
2.02 Acid Fast Staining Figure 3. Ziehl-Neelsen Method KINYOUN MODIFICATION OF ZN METHOD  Also known

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

2.02 Acid Fast Staining Figure 3. Ziehl-Neelsen Method KINYOUN MODIFICATION OF ZN METHOD  Also known

Figure 4. Kinyoun Modification of ZN Method

Table 1. Ziehl-Neelsen Hot Method vs. Kinyoun Cold Method

2.02 Acid Fast Staining Figure 3. Ziehl-Neelsen Method KINYOUN MODIFICATION OF ZN METHOD  Also known
DIAGNOSIS OF TB
DIAGNOSIS OF TB

A complete medical evaluation for TB disease includes:

o

o

o

o

o

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

  • A. SPECIMEN COLLECTION

There are four (4) collection methods for pulmonary TB disease:

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

BARTLETT’S 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

Table 2. AFB Smear Classification Reporting of Results

2.02 Acid Fast Staining Figure 3. Ziehl-Neelsen Method KINYOUN MODIFICATION OF ZN METHOD  Also known

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

***