BacterStain™ Alkaline Methylene Blue

Used in the Ziehl Neelsen staining kit

• Slide Preparation:
  • Prepare a clean, scratch-free slide, immerse it in 95% ethanol, dry it, then heat over a flame.
  • Label the slide and mark the smear position on the underside of the slide.
  • Collect the clinical specimen (such as sputum, fluid, tissue) or colony from culture, centrifuge to collect the sediment.
  • Use a sterile inoculating loop to take one loopful (3mm in diameter) or place one drop of the specimen onto the slideAllow it to air dry and fix by gentle heat
• Fixing the Slide: Allow the slide to air dry naturally in a biosafety cabinet or dry it at 60°C.
• Staining:
  • Flood slide with BacterStain™ Carbon Fuchsin (Code: 04004) and heat 2-3 times until steam rises (Do NOT allow stain to boil or to dry on the slide), let it cool naturally for 5 minutes.
  • Wash with water
  • Apply several changes of BacterStain™ Alcohol acid (Code: 04009) until no more colour seems to come from the preparation
  • Rinse with water and counterstain with BacterStain™ Methylene blue (Code: 04006) for approximately 1 – 2 minutes.
  • Decolorize using acid alcohol solution, rinse thoroughly.
  • Stain with Methylene blue solution for 1-2 minutes, rinse well.
• Rinse with water, drain until dry.
• Examine under the microscope, using the oil immersion lens

RESULTS

• Mycobacteria: Red/pink color.
• Other background fields: Blue color.
• AFB Morphology from the specimen: AFB appears as thin, slightly curved rods, staining red, either isolated or clustered together, easily visible against the blue background.