TINCIÓN DE ZIEHL NEELSEN

Se Nilsen Staining Technique Overview

Introduction to Se Nilsen Staining

• The National Autonomous University of Mexico presents the Se Nilsen staining technique, which is a differential staining process used in microbiology.

• This technique involves applying two dyes, a mordant, and a decolorizer to differentiate bacterial cell wall compositions.

Required Materials for Staining

• Essential materials include:

• A slide and biological sample.

• A burner and shallow tray for heating.

• Primary dye (carbolized fuchsin), decolorizer (acid alcohol), contrast dye (Lefler's methylene blue).

• Additional items: filter paper, tripod, beaker, tweezers, microscope, inversion oil, and bacterial culture.

Steps in the Staining Process

• The first step involves applying heat to the sample using direct or indirect methods.

• In direct heating:

• Place the slide on a bridge with filter paper soaked in primary dye.

• Heat for 5 to 10 seconds repeatedly over a total of 10 minutes while adding more dye as needed.

Decolorization Phase

• After heating, remove the filter paper and wash the slide with water until it runs clear.

• The critical decolorization step differentiates acid-alcohol fast bacteria from non-acid-fast ones by using acid alcohol; non-acid-fast cells become colorless while acid-fast remain pink.

Application of Contrast Dye

• Apply Lefler's methylene blue after decolorization for three minutes; this stains only non-acid-fast bacteria that lost their primary dye during decolorization.

• Post-staining washing is necessary until excess dye is removed before observing under a microscope.

Observations Under Microscope

• Under microscopy at 100x objective:

• Pink indicates acid-alcohol resistant bacteria; blue indicates non-resistant bacteria.

Understanding Bacterial Cell Wall Composition

• Acid-fast bacteria possess mycolic acids preventing dye passage; during heating, these waxes soften allowing carbolized fuchsin entry but not removal by bleach.

• Non-acid-fast bacteria lack such components leading to loss of primary dye and subsequent uptake of contrast dye.