Preparation of Acid Fast Reagents in Laboratory | Microbiology Practicals

PREPARATION OF ACID FAST REAGENTS IN LABORATORY

PREPARATION OF ACID FAST REAGENTS

The Acid-Fast staining technique was initially developed in 1882 by Paul Ehrlich and modified by Ziehl & Neelsen in 1890 hence called Z.N. Staining or Ziehl-Neelsen staining. Certain bacteria and Actinomycetes have many components in the cell wall and their cell wall has little permeability.

Majority of bacteria can be stained with simple stain but there are many species of Mycobacteria and Nocardia which can only be stained by a special technique known as Acid-fast staining technique.It was first used for Mycobacterium tuberculosis. Also, this staining technique can be used to stain all the species of Mycobacterium.



The Acid-Fast staining basically requires 3 reagents, A carbol Fuchsin Solution, An Acid Fast decolorizer and The Counter Stain, Methylene blue is commonly used.

In this article, I’m gonna discuss the Preparation of All the Reagents require for the Acid Fast staining, in Laboratory. Let’s Start with Carbol Fuchsin preparation.

PREPARATION OF 1 % CARBOL FUCHSIN

Materials Required for the Preparation of 1 % Carbol Fuchsin

Equipment Required –

  • 1000 ml Erlenmeyer Flask
  • Weighing Scale or Analytical balance
  • Spatula
  • Butter Paper
  • Spatula or Measuring spoon
  • Weighing paper or butter paper
  • Graduated cylinder
  • Glass or plastic funnel

Reagents Required –

ReagentsQuantity
Basic Fuchsin10 gram
Absolute Ethanol100 ml
Phenol Crystals50 gram
Distilled Water1000 ml

Procedure for the Preparation of 1% Carbol fuchsin:

Weigh Accurately 10 gram of Carbol Fuchsin on a Weighing scale or Analytical Balance.

Take a 1000 ml Clean & Sterilized Flask and pour about 30 ml of Ethanol in it.

Add the 1 gram of Carbol fuchsin to the Flask and mix well by swirling the flask.

Add the Remaining quantity of Absolute Ethanol and mix well the contents. Now, place it in a water bath at 60° C to completely dissolve the dye. Avoid direct heating of the content.

Using a Weighing scale or Analytical balance weigh accurately the 50 gm of phenol crystals and transfer it to 1000 ml flask containing the Dye-Alcohol solution.

Add about 500 ml of distilled water to the above content and dissolve the phenol crystal. Gentle heat may be required to dissolve it completely.

Add the remaining quantity of distilled water to make up the final volume to 1000 ml.

Filter the content of the flask using filter paper (Whatman No 1) and store filtered solution in an amber colored bottle.

Label bottle with the name of reagent i.e. 1% Carbol Fuchsin, lot number, date of preparation, expiration date & storage temperature and enter in the Logbook of your laboratory.

Tighten the cap of the bottle & store at room temperature.

Note: Store the reagent up to 6 months from preparation date. Occasionally filter Carbol fuchsin if precipitate forms.

Now, Let’s come to the Preparations of Acid Fast Decolorizer which varies according to the genus and species of the bacteria i.e. for different bacteria different concentrations of the Acid Fast Decolorizer is used. Also, instead of using Acid fast decolorizer we can also use the Acid-Alcohol decolorizer in some cases. Here, I’m gonna explain the preparation of 20% Acid decolorizer and 3% Acid-Alcohol Decolorizer which is commonly used for the Mycobacterium tuberculosis.



PREPARATION OF 20 % SULFURIC ACID (H2SO4) SOLUTION (20 % ACID DECOLORIZER)

Materials Required for the Preparation of 20 % Sulfuric Acid Solution (Acid-fast Decolorizer)

Equipment Required –

  • 250 ml Erlenmeyer Flask
  • Weighing Scale or Analytical balance
  • Graduated cylinder
  • Glass or plastic funnel

Reagents Required –

ComponentsQuantity
Sulfuric Acid20 ml
Distilled Water80 ml

Procedure for the Preparation of 20 % Sulfuric acid Solution (Acid-fast Decolorizer)

Measure accurately the 50ml of Distilled water in a Measuring Cylinder and Pout it into a 250 ml Erlenmeyer flask. Submerge the Erlenmeyer flask in a bowl containing cold water for the next step.

By using a measuring cylinder, measure out 20 ml of concentrated Sulfuric acid (98%) and carefully add the concentrated Sulfuric acid to the water in the Flask submerged in a cold water bowl.

NOTE: This is a highly exothermic reaction and the mixture will heat up, so always place the flask in a bowl of cold water while diluting the acids.

Add the remaining quantity of distilled water, make the volume 100 ml and gently mix the content in the flask and store it in an amber colored glass bottle with air-tight cap.

Label bottle with the name of reagent (25 % Sulfuric acid), lot number, date of preparation, expiration date & storage temperature and enter the details in Logbook of Your Lab.

Tighten the cap & store the solution at room temperature.

The 20% Sulfuric Acid is commonly used for the Mycobacterium tuberculosis. The 5% Sulfuric Acid Solution is commonly used for the Mycobacterium leprae is simply prepared by adding 5 ml of concentrated sulfuric acid in 95 ml of Distilled water in an Erlenmeyer flask.

Check out the Acid Fast staining Protocol

PREPARATION OF 3 % ACID- ALCOHOL SOLUTION (3% ACID- ALCOHOL DECOLORIZER)

Materials required for the Preparation of 3 % Acid- Alcohol Solution (3% Acid- Alcohol Decolorizer)

Equipment Required –

  • 250 ml Erlenmeyer Flask
  • Weighing Scale or Analytical balance
  • Graduated cylinder
  • Glass or plastic funnel

Reagents Required –

ComponentsQuantity
Hydrochloric Acid (HCl)3 ml
Absolute Ethanol (95%)97 ml

Procedure for the Preparation of 3 % Acid- Alcohol Solution (3% Acid- Alcohol Decolorizer)

Measure accurately the 50ml of Absolute Ethanol in a Measuring Cylinder and Pout it into a 250 ml Erlenmeyer flask. Submerge the Erlenmeyer flask in a bowl containing cold water for the next step.

By using a measuring cylinder, measure out 3 ml of concentrated Hydrochloric acid and carefully add it to the alcohol in the Flask submerged in a cold water bowl.

NOTE: This is a highly exothermic reaction and the mixture will heat up, so always place the flask in a bowl of cold water while diluting the acids.

Add the remaining quantity of Absolute Ethanol, make the volume 100 ml and gently mix the content in the flask and store it in an amber colored glass bottle with air-tight cap.

Label bottle with the name of reagent (3 % Acid-Alcohol Decolorizer), lot number, date of preparation, expiration date & storage temperature and enter the details in Logbook of Your Lab.

Tighten the cap & store the solution at room temperature.

The (3% Acid- Alcohol Decolorizer) is commonly used for the Mycobacterium tuberculosis. The 0.5% Acid- Alcohol Decolorizer is commonly used for the Mycobacterium leprae is simply prepared by adding 0.5 ml of concentrated Hydrochloric acid (HCl) in 99.5 ml of Absolute Ethanol in an Erlenmeyer flask.

PREPARATION OF 0.1 % METHYLENE BLUE SOLUTION

Materials Required for the Preparation of 0.1 % Methylene Blue Solution

Equipment Required –

  • 100 ml Erlenmeyer Flask
  • Weighing Scale or Analytical balance
  • Spatula or Measuring spoon
  • Weighing paper or butter paper
  • Graduated cylinder
  • Glass or plastic funnel

Reagents Required –

ComponentsQuantity
Methylene Blue0.1 gram
Distilled Water100 ml

Procedure for the Preparation of 0.1 % Methylene Blue Solution

Using a Weighing scale or Analytical balance, weigh out 0.1 g of Methylene blue powder.

Take out 50 ml of Distilled water in a Clean and Sterilized 250 ml Erlenmeyer flask.

Dissolve the 0.1 gm of Methylene blue in the Distilled Water in the Flask, using a stirring bar or by swirling the flask to mix the contents.

After mixing the contents, pour the solution through filter paper (Whatman No.1) and store filtered Methylene Blue solution in an amber colored glass bottle with cap.

Label bottle with the name of reagent (0. 1 % Methylene blue), lot number, date of preparation, expiration date & storage temperature and Enter in the Logbook of your laboratory

Tighten the cap of the Reagent bottle & store it at room temperature.



PRECAUTIONS TO BE TAKEN WHILE PREPARING THE ACID FAST REAGENTS

 Measure accurately the volume of concentrated Sulfuric acid, Hydrochloric acid, Alcohol & Distilled Water.

 Last few milliliters of the distilled water should be added using a pipette to avoid volume error.

 Transference of the weighed substance into an Erlenmeyer or volumetric flask should be done very carefully and not a single crystal be allowed to fall outside.

 Always add acid to water and never water to the acid as dilution of acid is a highly exothermic reaction.

 Work near a running supply of water. If the acid contacts the skin, it must be washed off rapidly with the copious amount of tap water.

 Concentrated H2SO& HCl is highly corrosive so handle with care.

SAHIL BATRA

Hi, I'm the Founder and Developer of Paramedics World, a blog truly devoted to Paramedics. I am a Medical Lab Tech, a Web Developer and Bibliophiliac. My greatest hobby is to teach and motivate other peoples to do whatever they wanna do in life.

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