Preparation of Solution for Ziehl Neelsen Stain - labsstudies

Preparation of Solution for Ziehl Neelsen Stain

Preparation of Solution for Ziehl Neelsen Stain

Ziehl Neelsen Stain

Is the stain used to detect Acid fast microorganism. These acid fast microorganism are Mycobacterium tuberculosis, Mycobacterium ulcerans and Mycobacterium ulcerans.

Types of Ziehl Neelsen stain techniques used in Laboratory

These are:

  1. Ziehl Neelsen stain-Hot technique
  2. Ziehl Neelsen stain-Cold technique

1. Ziehl Neelsen stain-Hot technique

Hot technique used to stain for Mycobacterium tuberculosis . Mycobacterium tuberculosis are  non-sporing,  and non-capsulated straight or slightly curved slender rod.

2. Ziehl Neelsen stain-Cold technique

Ziehl Neelsen stain-Cold technique is applied to demonstrate Mycobacterium leprae bacilli.  Mycobacterium leprae is a non-motile, non-sporing, and  straight or slightly curved rod.

 

Principle of Ziehl Neelsen stain technique

 When Mycobacterium species are stained with cold strong solution of carbol fuchsin, they resist decolourization with a solution of acid or acid ethanol and stain red. Tissues and other organisms are decolourized by the acid – ethanol solutions and are stained by methylene blue or malachite green which stain them blue or green respectively

Microscopical of Ziehl Neelsen stain :Under microscope the mycobacterium species show appearance pinkish red in microscope, Cell and background stain blue or green.You can see the image below.

Acid fast bacilli

Acid fast bacilli seen in Microscope

Equipment’s and Materials for Preparation of Solution for Ziehl Neelsen Stain

General Equipment’s and Materials Required to prepare solutions Equipment:

Equipment’s

  • Measuring graduated cylinder
  • Flasks (volumetric)
  • Pipettes
  • Analytical Balance
  • Spatula

Materials

  • Parafilm
  • Label or tape
  • Marker pen
  • Distilled water or filtered rain water
  • Concentrated sulphuric acid
  • Concentrated HCl
  • Phenol crystals
  •  Methylene blue powder

Safety precautions

Preparation of Solution for Ziehl Neelsen Stain

  • Never add acid before water. Always add acid to water.
  • Avoid using a mouth pipette.
  • Avoid getting concentrated acid on your skin, eyes, or mouth.
  • Avoid spilling on the bench or other surfaces. To absorb spilled concentrated acid, use sand or a spill kit.
  • Keep your face away from the open container and the lid on as much as possible to avoid inhaling fumes.
  • Put on PPE such as gloves, a lab coat, and closed shoes.

Make up of Solution for Ziehl Neelsen Stain

Solution 1: Sulphuric acid (25%)

  • 25 mL sulphuric acid added to 75 mL distilled water for a total volume of 100 mL
  • Using the measuring cylinder, pour out 25 mL concentrated sulphuric acid and slowly pour it down the side of the 100 mL volumetric flask containing some (about 25 mL) distilled water.
  • Swirl to combine.
  • Make a final volume of 100 mL with distilled water.
  • Inversion of film and mix
  • Use tape to label or a marker pen to label. (Reagent name, amount prepared, date prepared.) Technician in charge of preparation)

Solution 2: 3% HCl solution

  • 3 mL HCl added to a total volume of 100 mL (97mL distilled water) Examine the safety precautions for working with concentrated acids.
  • Using a glass pipet, transfer 3 mL of concentrated HCl to a 100 mL volumetric flask containing distilled water.
  • Swirl to combine.
  • Make a final volume of 100 mL with distilled water.
  • Inversion of film and mix.
  • Use tape to label or a marker pen to label. (Reagent name, amount prepared, date prepared.) Technician in charge of preparation)

Solution 3: 5% aqueous phenol

  •  Using a balance, weigh out 5 g of phenol crystals.
  • A 100 mL volumetric flask containing some distilled water is filled with 5 g phenol.
  • Swirl the mixture to dissolve it.
  • Add the remaining volume of distilled water (approximately 95 mL water in the solution.)
  • Parafilm and mix thoroughly.
  • Label using tape or label and marker pen (name of reagent, amount prepared, date of preparation. Technician who prepared)

Solution 4 :  0.3% methylene blue.

  • Using a balance, weigh out 0.3 g of methylene blue.
  • In a 100 mL volumetric flask containing some distilled water, 0.3 g methylene blue is added.
  • Swirl the mixture to dissolve it.
  • Pour in the remaining distilled water (approximately 95 mL water in the solution.)
  • Wrap in parafilm and thoroughly mix.
  • Label with tape or a label and a marker pen (reagent name, amount prepared, date of preparation). Technician in charge of preparation)

Read also: Methods of Storing Reagents and Solutions

Quality control for Ziehl Neelsen stain

Way to perform quality control for Ziehl Neelsen stain:

  • Collect two sputum smears from known positive AFB patients.
  • Stain one film with the old stain, the other with the new stain, and compare the staining reactions.

Ziehl Neelsen staining is essential for detecting acid fast bacilli( AFB) in sputum smears.

Take note that all freshly prepared reagents should be the correct color and clarity.

You may also like...

1 Response

  1. September 10, 2022

    […] Read also: Preparation of Solution for Ziehl Neelsen stain […]

Leave a Reply

Your email address will not be published.