BacterStain™ Methylene Blue

A reagent is recommended for use in clinical diagnosis of human clinical specimens and for bacteriological and histological research purposes using human-derived samples.
Code: 04006
Packaging: 100ml/bottle or according to the customer’s request..
Storage Conditions: 18 – 25°C.
Transportation: Ambient temperature.
Shelf Life: 24 months from the date of manufacture.

INTENDED USE

BacterStrain™ Methylene blue is used in cell staining and microbiological research.

PRINCIPLES

BacterStrain™ Methylene blue serves as a counterstain in the Ziehl Neelsen method. After bacteria are stained with Carbon Fuchsin and decolorized with acid alcohol, acid-fast bacteria retain the red color, while non-acid-fast bacteria lose the red color and are stained blue with Methylene blue. Methylene blue is not affected by the decolorization process, helping to differentiate between acid-fast and non-acid-fast bacteria.

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.

BacterLab ensures the quality of each product batch by testing with ATCC reference strains.

Reference strains	Conditions	Expected results
Positive specimen	oil immersion lens (100x)	AFB stains red on a light blue background, with no staining residue visible
Negative specimen	oil immersion lens (100x)	no AFB is observed

For 1 liter of strain

Methylen Blue	3 g
DW (qsp)	900 mL

*Note: This ingredient is for reference only our company will formulate the ingredient according to requests or tender specifications.

Product Name	Code	Packaging
BacterStrain™ Methylene blue	04006	100ml/bottle or according to the customer’s request..