# Presentation: 515321

## MODULE 5

**Preparation of ****Ziehl-Neelsen Reagents**

## Learning Objectives

**At the end of the module, participants will be able to**
**Describe the importance of using quality chemicals for reagent preparation**
**Prepare reagents required for the Ziehl-Neelsen method**
**Describe the safety requirements for reagent preparation**
**Use positive and negative control slides for the Quality Control of Ziehl-Neelsen reagents**
**Explain the use and frequency of routine quality control  procedures.**

## Content Overview

**Equipment required for staining reagent preparation**
**Reagents required for staining reagent preparation**
**Methods for staining reagent preparation**
**Quality control (QC) of freshly prepared staining reagents**
**Storage of staining reagents **

## Required Materials

**Reagents and Chemicals:**
- Basic Fuschin
- Phenol
- Alcohol
- Methylene Blue
- Sulfuric Acid
- Purified water

**Equipment:**
- Weigh Balance
- Measuring cylinders
- Stirring plate
- Magnetic stirrers

**Materials:**
- Flask
- Funnel
- Weigh paper
- Clean reagents bottles
- Labels

**Miscellaneous:**
- Gloves
- Lab coat
- Safety glasses
- QC slides
- Register

## Reagent Quality:  Specifications

| Reagent Name | Specification |
| --- | --- |
| 
Basic Fuchsin | Purchased from well known manufacturers or those certified by the Biological Stain Commission  |
| Phenol crystals | Near colorless – never black |
| Alcohol | Technical grade |
| Acid | Technical grade |
| Water | distilled or purified, not tap water |
| Methylene blue | purchased from well known manufacturers  |

**Notes:**
It is important to use high quality reagents 
Many brands do not specify quality

##  Dye Powder Specifications

## Correcting for Dye Purity

- Only needed when
-   Preparing 0.3% carbol fuchsin _AND_  basic fuchsin powder dye content below 85%
- Example:
-  	Basic fuchsin powder dye content = 75%
-  	Need 3 g basic fuchsin
-  	Decimal equivalent of 75% = 0.75
-  	3 g / 0.75 = 3.99 g = 4.0 g
-  	4.0 g of basic fuchsin powder must be weighed to have 3g of basic fuchsin available

## Weighing Balance

- Fragile, precision instrument
- Handle with care
- Must be used on level surface
- Consult  manual for operating instructions

## Using the Balance

- High precision balance not needed: ± 0.1 g is sufficient
- Keep balance (and weights, if any) clean and dry
- Always use a weighing container or paper; correct back to zero

## Water Quality

- Must be free of mycobacteria
- Use distilled or purified water
- NOT: rain water, tap water, or boiled water

## 0.3% Carbol Fuchsin

**    ****Use method recommended by the NTP**
**Contains per liter:**
- basic fuchsin powder		 3 grams
- phenol				50 grams
- 95% ethanol or methanol	100 ml
- water 				up to 1 liter
**Preparation:**
- dissolve phenol in alcohol
- dissolve the fuchsin completely in the mixture
- add the water, mix well

## 0.3% Methylene blue

**Use method recommended by the NTP**
- Contains per liter:
- methylene blue powder	3 grams
- water 				1 liter
- Preparation:
- add 500ml of water to a 2-liter flask
- add the methylene blue and mix until completely dissolved
- add remaining water, mix well

## Decolorising Solution 

**Use method recommended by the NTP**
**Contains per liter:**
**sulfuric acid (minimum 95%)****	****250 ml**
**water ****	****	****	****	****750 ml**
**Preparation:**
- Measure 750ml of water into a 2-liter flask
- Measure 250mL of concentrated sulphuric acid in a cylinder
- Pour it *slowly* into the flask containing the water, directing the flow of acid gently along the inner side of the flask
- Stop and swirl flask regularly as a lot of heat is generated until all acid is added
- Mix well and allow to cool before use

## Alternative Decolorising Solution

- Contains per liter:
- Hydrochloric acid (fuming) - 30 ml
- 95% denatured alcohol - up to 1 liter
- Preparation:
- Add one liter of 95% denatured alcohol to a 2-liter flask
- Slowly add the acid
- Mix well and allow to cool before use

## Diluting Acids Safely

**Diluting Acids Safely**

## Quality Control of New Staining Reagents

- Always needed; record results in logbook
- Positive as well as negative control slides
- Positives: 1+ TB patient sputum; exact counts
- Negatives: known AFB negative specimen
- Positives to be stained once
- Check for the number of AFB and staining quality
- Negatives:  ZN stain repeated 3 times
- Check for contaminating AFB
- Ensure background blue and clean

## Reagent Labeling and Storage

- Each bottle or container must be labeled with at least
- Name of reagent
- Date of preparation
- Store all reagents in a cool place away from direct sunlight

## Summary

- Why must quality reagents be used to prepare staining reagents?
- Why is accurate preparation of reagents necessary to obtain optimum staining results?
- What is the role of control smears in evaluating the performance of newly prepared staining solutions?
- Why should reagents be stored in clean bottles and out of direct sunlight?