STLS: Preparation of Staining solutions

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.

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:

Resources

• Module for Laboratory Technicians (RNTCP), Central TB Division, MoHFW, 2005
• TB Lab Consumables Specifications 2019
• SoP for preparation of Laboratory Reagents - WHO
• Laboratory Diagnosis of Tuberculosis by Sputum Microscopy, GLI Initiative

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Reagent preparation is an important activity and it’s essential to use certified chemicals and reagents. One should always check the potency of the chemicals used and calculate the amount to be weighed accordingly.

The following are the steps for reagent (primary stain and counter stain) preparation for Florescence Microscopy:

0.1% Auramine–O (1 L), the primary stain

• Weigh 1 gm Auramine–O and transfer to conical flask.
• Add 30 gm Phenolic crystals (99.5% purity) and mix well.
• Add 100 ml absolute Ethanol (98-100% purity) and mix well.
• Add 870 ml distilled water to make up final volume of 1 L.
• Filter and transfer to amber bottle.
• Label the bottle as 0.1% Auramine–O.
• Date of preparation, date of expiry, batch no. and name of the Senior TB Lab Supervisor (STLS) who has prepared the stain should be clearly mentioned on the bottle.

1% Acid Alcohol (1 L) to decolorize

• Dissolve 5 gm Sodium chloride in 250 ml distilled water.
• Add 5 ml concentrated hydrochloric acid.
• Mix with 750 ml absolute alcohol.
• Always add acid slowly to alcohol, not vice-versa.
• Store in amber coloured bottle.
• Label the bottle as 1% Acid Alcohol.
• Date of preparation, date of expiry, batch no. and name of the STLS who has prepared the stain should be clearly mentioned on the bottle.

0.5% Potassium Permanganate, KMnO₄ (1 L), the counter stain

• Weigh 5 gm of Potassium permanganate (99.5% purity).
• Transfer to a conical flask.
• Take 1 L of distilled water.
• Add small amounts of distilled water tothe  conical flask containing KMnO₄ and mix well.
• Add the remaining distilled water to make up final volume of 1 L.
• Filter and transfer to a bottle.
• Label the bottle as 0.5% Potassium permanganate.
• Date of preparation, date of expiry, batch no. and name of the STLS who has prepared the stain should be clearly mentioned on the bottle.

Table: Preparation of Different Volumes of Stains for FM Microscopy

Resources

• TB Lab Consumables Specifications, 2019.
• SOP for Preparation of Laboratory Reagents - WHO.

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