# Presentation: 266740

## MODULE 6Smear Preparation and Staining

## Learning Objectives

**At the end of the module, participants will be able to**
**Safely prepare sputum smears**
**Prepare good quality sputum smears**
**Identify problems with smear preparation **
**Perform the Ziehl-Neelsen (ZN) method on sputum smears**
**Trouble shoot problems with the ZN method**

## Content Overview

**Labeling of slides**
**Selecting the best portion of the sample for smear preparation.**
**Techniques for preparing smears**
**Principles of the Ziehl-Neelsen method  **
**The Ziehl-Neelsen staining procedure**

## Overview of Smear Preparation

******	****Label each slide**
******	****Make smear **
******	****Air dry**
******	****Heat fix smear**

## Labeling Frosted Slides with Pencil

- Laboratory Serial Number and specimen number

**Labeling Frosted Slides with Pencil**

**Notes:**
Properly identify each slide

Properly identify each slide with the laboratory and specimen
number. Use a lead pencil to write these numbers on the frosted end of the slide. 

## Labeling Non-frosted Slides with  Diamond Pencil

- Laboratory Serial Number and specimen number

**Labeling Non-frosted Slides with  Diamond Pencil**

**Notes:**
If the slide is not frosted, engrave the laboratory serial number
using a diamond pointed stylus.

Never use a grease pencil, since markings could wash off
during the staining process.

## Specimen Appearance

## Purulent Portion

**Purulent Portion**

## Saliva Portion

**Saliva Portion**

## Consistent Quality of Smears

**Consistent Quality of Smears**

## Making Smear with Loop

**Notes:**
Using a continuous rotation, cover an oval area approximately
2 cm in length on the slide. Put only one smear on each slide. 

## Heat Fixing a Smear

- Pass the smear through the flame 2–3 times

**Heat Fixing a Smear**

**Notes:**

When dried, fix the slides using the blue flame of a Bunsen
burner. With forceps, pass the slide briefly through the flame
3 times, smear side up.

Heat fixation ensures that the sputum will stick to the glass
slide.  Excessive heating could damage the bacilli. If not
sufficiently heat fixed, the acid fast bacilli may wash off during
staining.

## Proper Thickness of Smear

**Proper Thickness of Smear**

**Notes:**
How can you tell you have the proper thickness?

If it is too thin, the specimen may yield false negative results.
If it is too thick, the smear may wash off the slide during the
staining procedure. 
Newsprint can usually be read behind a
dried smear when it is the proper thickness.
If a wire loop is used, clean it before the next use. Dip the
contaminated loop into a sand and alcohol bottle and move it
up and down to remove solid debris.

Next, heat the wire loop in the flame until red hot. Allow it to
cool before using it again.

Wet slides can create aerosols if disturbed. Place them in a
protected area where they can air dry for 15-30 minutes.

Do NOT flame them to induce drying.
This can also produce aerosols.

## 
Primary Stain (carbol fuchsin)
Decoloriser 
Counterstain (methylene blue)

**Primary Stain (carbol fuchsin)**
**Decoloriser **
**Counterstain (methylene blue)**
**  **

## Acid-Fast Principles

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

**Notes:**
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

## Principle of ZN Stain - 1

***AFB***

***Non AFB***

**Principle of ZN Stain - 1**

_**carbol fuchsin**_
**+**
**HEAT**

## AFB

***AFB***

***Non AFB***

**carbol fuchsin**

**decolouriser**

**Principle of ZN Stain - 2**

## AFB

***AFB***

***Non AFB***

**carbol fuchsin**

**decolouriser**

**Principle of ZN Stain - 3**

_**counterstain**_

## Overview of Staining Procedure

**Arrange slides in serial order smear side up**
**Keep a finger-thickness between smears**
**Flood with carbol fuchsin**
**Heat to steaming once only**
**Leave for 10 minutes **
**Rinse with water, drain**
**Apply decolorizing solution 3 min. **
**Rinse, drain**
**Apply Methylene blue counterstain, l min.**
**Rinse, drain**
**Air dry **

## Setting up Slides on Staining Rack

**Setting up Slides on Staining Rack**

## Carbol Fuchsin

**Carbol Fuchsin**

## Heating Carbol Fuchsin to Steaming

**Heating Carbol Fuchsin to Steaming**

## Rinsing Slides

**Rinsing Slides**

## Don’t Splash Adjacent Slides

**Don’t Splash Adjacent Slides**

## Decolorisation Step

**Decolorisation Step**

## Rinsing Slides

**Rinsing Slides**

## Counterstaining

**Counterstaining**

## Rinsing off Counterstaining

**Rinsing off Counterstaining**

## Drying the Stained Slides

**Drying the Stained Slides**

## Quality Control of Routine Staining

**All labs performing AFB-smear microscopy**
**Ideally daily but at least weekly**
**Positive control smear (1+)**
**in routine series for staining**
**check for the number and color of AFB**
**Record results in the laboratory register**

## Summary

**What labelling information is needed on a slide?**
**How do you select the best portion of the sample for smear preparation?**
**How can you determine the correct size and thickness of a sputum smear?**
**What are four critical steps in ZN staining?**