# Presentation: 852172

## Acid-fast Smear Microscopy Details of Techniques

## Acid-Fast Organisms

- Primary stain binds cell wall mycolic acids
- Intense decolorization does not release primary stain from the cell wall of AFB
- Color of  AFB-based on primary stain
- Counterstain provides contrasting background

## Acid-Fast Stains

- Early diagnosis of mycobacterial infections
- Confirms acid-fast nature of organisms
- Monitor patients on antimyobacterial therapy
-  May be a determinant for performing other tests  such as culture or susceptibility testing

## AFB Microscopy Techniques

**Two basic techniques: same basic principle**
**Transmitted light: Carbol fuchsin staining**
**Contrast red color AFB/ on blue background**
**Fluorescence: auramine staining**
**contrast light/dark; **

## Basic Fuchsin AFB Stains

- Zeihl Neelsen’s-hot stain
- Kinyoun’s-cold stain
- Modifications
- Most of this discussion will be spent on these  techniques

## Slide 6

## Flourochrome AFB Microscopy

**Place of fluorescence in low-income ?**
**more rapid and sensitive**
**specificity : same with sufficient experience**
**equipment cost , bulbs, technical demands**
**for busy labs (> 25 slides daily) **
**External quality assessment should be done if this method is performed**
**Performing EQA PT and rechecking is a challenge  **

##  Fluorescence  and Bright-field Microscopy

## Flourochrome AFB Microscopy

_**Primary fluorochrome**_ 	         _**AFB fluoresces**_
- Auramine O 			       	Green
- Auramine O-Rhodamine B          	Yellow/orange
- Acridine Orange	       			Yellow/orange
***Note:  Color of AFB may vary with filter system on microscope***

## Auramine Stain

## Flourochrome AFB Microscopy

| Counter Stain | Background |
| --- | --- |
| Potassium permanganate
Acridine Orange
 | Dark/black
Yellow/orange
 |
|  |  |
|  |  |

## Auramine with Acridine Orange Counter stain 

## Bright-field Techniques

**Hot Ziehl-Neelsen in practice most reliable**
**more visible AFB**
**stronger color**
**Cold methods : Kinyoun, Tan Thiam Hok...**
**less laborious but also less robust**
**higher concentration fuchsin, longer staining time**
**errors !!**
**not recommended for low-income countries**

## Slide 14

## Sputum Sample

**What is a good sample?**
**What is saliva? **
**Good sample = yellow? mucous fluid? **
**Discharge from the bronchial tree**
**May contain solid or purulent substances**
**Minimal amounts of oral/ nasal material**
**May contain macrophage s and other  cells indicative of infectious disease**
**Follow-up examination samples?**

## Preparation of AFB Smears

**Smearing**
**delay: no problem (keep away from sun)**
**"good particle" **
**	****homogenization may be more reliable**
**standard size: for ease of quantification**
**thickness, evenness: find a  balance**
** ****	****sensitivity**
**	****light conditions, counter-stain**

## Sputum Concentration Methods :

**      ****-Are they worth the effort?**
**NaOH digestion + centrifugation : culture**
**Hypochlorite (NaOCl) digestion & concentration**
**homogenization, easy background**
**oxidation: staining easier; co-flocculation with proteins ?**
**concentration by sedimentation, centrifugation, filtration or flotation **
**Contradictory reports on efficiency**

## Bleach Concentration Studies

**REFERENCE****	****	****  ****	****  % SM+ / CULT+****	****      % POS. SMEARS**
**	**** ****	****	****	****	****DIRECT   BLEACH   DIRECT  BLEACH**
**GEBRE 1995, centrif.****	****	****  31%****	****       69%****	****              ****	****        + 125%**
**MIORNER 1996, sedim.****	****	****	****                        ****	****          + 25%**
**ALLWOOD 1997, centrif.****	****  ****	****  43%****	****       52%****	****       10.5%        14.8%**
**	**
**WILKINSON 1997, centrif.****	****  43%****	****       44%****	****       12.7%        12.4%**
**ÄNGEBY 2000, centrif.****	****  ****	****  57%****	****       65%****	****         9%           12.9%**
**	**
**VAN DEUN 2000, sedim.****	****	****	****	****        15.5%        16.6%**

## Danger of Cross Contamination

**If tubes re-used **
** ****Contamination (water for dilution)**
**Need  a centrifuge**
**Work : specimen preparation or reading ?**
**Useful with HIV ?**

## Fixation of AFB Smears

**Fixation **
**may kill some bacilli**
** ****makes smear stick to slide**
**by heat or alcohol**
**do not overheat**
**safe smear ?**

## Primary Staining-ZN

**Carbol fuchsin staining**
**Uses higher fuchsin concentration **
**Dissolve well !!!**
**IUATLD/WHO : 0.3%**
**	****references??**
**Heat well-apply long enough **
**Cold staining!!**
**Batch staining- great potential for cross- contamination**

## Slide 22

## Decolorization of Smears

**Decolization**
**must be complete**
**not possible to de-stain too much**
**	****	****	****repeat as needed**
**use strong acids**
**alcohol not absolutely needed**

## Counter-staining

**Provides good contrast for observation of AFB**
**background for focusing, not too strong**
**methylene blue 0.3% ?**
**	****	****diluted or < 1 min**
**use of malachite green?**

## Microscopic Reading:

**-Red slender rods on blue background**
**accept only typical shape, at least some**
**depends condition of microscope! light!**
**binocular, mechanical stage, good optics**
**100x oil immersion objective, 10x eyepieces**
**Requires: patience, sincerity**
**AFB microscopy is not difficult but tough**

## Slide 26

## Slide 27

## Slide 28

## Slide 29

## Recording and  Interpretation 

**_follow NTP instructions: no. of fields, sputa**
**quantification:**_**2 cm = 1 length = 100 HPF**_
**IUATLD/WHO scale**

## AFB Quantification Scales

| System & Quantification Scale |  |  |  |  |
| --- | --- | --- | --- | --- |
| No. of AFB per field
 | Brightf. & IUATLD/WHO Scale (1000x) | Brightf. & ATS Scale
(1000x) | Fluor. & IUATLD/WHO Scale
 (200-250x) | Fluor. & ATS Scale
 (200-250x) |
| None
1-2/300 fields
1-9/100 fields
1-9/10 fields
1-9/1 field
10-99/1field
>=100/1field | Negative
Actual
Actual
1+
2+
3+
3+ | Negative
Actual
1+
2+
3+
4+
4+ | Negative
Actual
Actual
Actual
1+
2+
3+ | Negative
Actual
Actual
1+
2+
3+
4+ |