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The National TB Elimination Program (NTEP) recommends freshly preparing reagents from commercial procured products, at the District TB units to be used for microscopy. Chemicals need to be carefully monitored for potency and the calculation needs to be accurate while preparing reagents for smear microscopy.

 

For Preparing 1% Carbol Fuchsin (500ml): The required chemicals and materials for preparing 500 ml Carbol Fuchsin are:

1. Basic Fuchsin

  • Chemical name: Pararosaniline hydrochloride 
  • Chemical structure: C19H18N3Cl
  • Molecular Wt: 323.8
  • Colour: Metallic green

Potency correction factor: Note down the dye content – this should be available on the container. The dye content should be approximately 85% - 88%. To calculate the required amount of basic fuchsin, divide the actual amount required by the dye content. For example: dye content = 85%, actual amount required = 5gms, required amount of dye = 5/0.85 = 5.88 gms.

2. Ethyl Alcohol: 50 ml (Absolute alcohol, purity must be 98-100%)

3. Carbolic acid crystals (Phenol): 25 gms

  • Chemical name: Phenol
  • Chemical structure: C6H5OH
  • Molecular Wt: 94.11
  • Melting point: 40°C+2.5

4. Distilled purified water: 500 ml

 

Steps for Preparing 1% Carbol Fuchsin (500ml)

  1. Add 25 gms of phenolic crystals to a conical flask
  2. Add 50ml ethanol
  3. Mix well until the crystals completely dissolves. Add 50 ml distilled water if required.
  4. Conical flask should be kept in water bath set at 60°C or in a trough containing warm water
  5. Weigh required amount of basic fuchsin powder and transfer it into a conical flask
  6. Mix well until the crystals dissolve well.
  7. Make the total volume to 500ml by adding distilled water
  8. Filter using Whattman filter paper and transfer into a bottle
  9. Label as 1% Carbol Fuchsin - Primary stain
  10. Date of preparation, date of expiry, batch no. and the name of the technician or STLS who has prepared the stain should be clearly mentioned on the bottle.
  11. Store it in cool place, away from direct sunlight
  12. Any time particles start to form in the Carbol Fuchsin solution, the solution must be filtered again

 

For Preparing 25% Sulphuric Acid (500ml):

  • Chemical structure: H2SO4
  • Molecular wt: 98.08
  • Purity: 95-97%
  • Colour: Clear
  1. Measure 375ml distil water into a 1L flask
  2. In a glass cylinder, measure 125ml concentrated Sulphuric Acid. Pour it slowly and gently into the conical flask containing distilled water. Note: Always add acid to water. Never add water to acid
  3. To dissipate the heat generated, place the flask in a trough of water
  4. Mix well
  5. Allow to cool
  6. Transfer into a bottle
  7. Label as 25% Sulphuric Acid - Decolorizing Solution
  8. Date of preparation, date of expiry, batch no. and the name of the technician or STLS who has prepared the solution should be clearly mentioned on the bottle.

 

For preparing 0.1% Methylene Blue (500ml):

  • Chemical name: Methylthionine chloride
  • Chemical structure: C16H18ClN3S
  • Molecular Wt: 319.9

 

Potency correction factor: Note down the dye content – this should be available on the container. The dye content should be approximately 82%. To calculate the required amount of methylene blue, divide the actual amount by the dye content. For example: dye content = 82%, actual amount required = 0.5gms, required amount of dye = 0.5/0.82 = 0.61 gms.

  • Weigh the required amount of Methylene Blue
  • Dissolve it in 500ml distilled water
  • Transfer into a bottle
  • Label as 0.1% Methylene Blue
  • Date of preparation, date of expiry, batch no. and the name of the person who prepared the solution should be clearly written on the bottle.

 

Table: Preparation of different volumes of stains for ZN microscopy
Ziehl-Neelsen method Quantity of reagent for 5L Quantity of reagent for 1L Quantity of reagent for 500 ml Quantity of regent for 100 ml

Basic fuchsin (dye content/purity if 85%)

 

 58.8 g 11.76 g 5.88 g 1.17 g
Alcohol 500 ml 100 ml 50 ml 10 ml
Phenol crystals 250 g 50 g 25 g 5 g
Distilled water To make final volume 5000 ml To make final volume 1000 ml To make final volume 500 ml To make final volume 100 ml
Carbol-fuchsin 1%        
Sulfuric acid 1250 ml 250 ml 125 ml 25 ml
Distilled water 3750 ml 750 ml 375 ml 75 ml
H2SO4 25%        

Methylene blue (dye content if 82%)

 

 6.1 g 1.22 g 0.61 g 0.12 g
Distilled water 5000 ml 1000 ml 500 ml 100 ml
Methylene blue 0.1%        

 

Always perform a quality check of the reagents prepared by using Quality Control Positive and Quality Control Negative. If purity is not available on the reagent bottle, then search the website of the company of which the reagent was procured or ask the company about the certificate mentioning about the purity of reagent

 

Please watch the video below for more information:

 

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