BacterStain™ Auramine Solution

• For Sputum:
  • Prepare the sample: Use a swab to transfer a portion of sputum containing mucus (including any yellowish material) onto a glass slide and spread it into a thin smear. The recommended area for the smear is about ½ x 1 inch (or 2 cm²).
  • Create the smear: Spread the sample using a circular motion to ensure an even distribution. Ensure the smear is of the proper thickness. Smears that are too thick may peel off during staining and may be difficult to decolorize, while smears that are too thin may not contain enough sample.
  • Allow the sample to air dry: Let the smear dry naturally in the air before proceeding.
• For Urine:
  • Prepare the sample: Create a smear from three centrifuged morning urine sediment samples. After spreading the sample, allow it to dry naturally.
  • Heat fixation: Fix the smear by using an electric slide warmer, which is the recommended method. Alternatively, the smear can be heat-fixed by passing the dried slide (with the smear facing up) through the blue cone of a flame 2-3 times.
• Staining:
  • Stain with Rhodamine-Auramine: Place the fixed slide onto a staining rack and immerse it in rhodamine-auramine solution for 15 minutes. Make sure the surface does not dry out during the staining process.
  • Remove excess color: After staining, rinse the slide with distilled water to remove excess dye.
  • Fluorescence decolorization: For 2-3 minutes, immerse the slide in acid-alcohol solution (fluorescence decolorizer).
  • Rinse thoroughly: Wash the slide with distilled water to remove any remaining acid-alcohol solution.
  • Stain with methylene blue: Immerse the sample in methylene blue solution for 3-4 minutes, ensuring the slide does not dry out.
  • Rinse and dry: Rinse with distilled water and allow the slide to dry naturally.
• Examine under the microscope: Check the slide under a fluorescence microscope using an excitation filter (K530) and a barrier filter (BG 12), or using an excitation filter (G-365) and a barrier filter (LP 420). The slide can be examined at high magnification (400X) and confirmed by immersion oil.

RESULTS

• Positive Test: AFB will fluoresce bright orange-red against a black background.
• Negative Test: Non-acid-fast bacteria will not fluoresce or may appear pale yellow, contrasting completely with the bright and acid-fast organisms.