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Borax methylene blue: a spectroscopic and staining study

P T Donaldson, A Russo, C Reynolds

    Stain Technology
    |July 1, 1978
    PubMed
    Summary
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    Borax methylene blue stability depends on temperature and pH. Adjusting pH to 6.5 significantly enhances its stability, crucial for effective blood staining of malaria parasites.

    Area of Science:

    • Biochemistry
    • Histology
    • Parasitology

    Background:

    • Borax methylene blue is a staining solution used in microscopy.
    • Its stability at different temperatures and pH levels is critical for reliable results.
    • Previous studies have shown varying outcomes, necessitating further investigation.

    Purpose of the Study:

    • To investigate the stability of borax methylene blue at various temperatures and pH levels.
    • To determine the optimal conditions for its use as a blood stain.
    • To explain historical discrepancies in its application.

    Main Methods:

    • Borax methylene blue solutions were incubated at temperatures ranging from 30°C to 60°C.
    • Absorption peaks were measured over time to assess polychroming.

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  • pH was adjusted to 6.5 using a borax-acid phosphate buffer.
  • A modified staining protocol was developed and tested on malaria parasites (Plasmodium falciparum, P. vivax, P. berghei).

    • Main Results:

    • Borax methylene blue is stable at room temperature (22-25°C).
    • Polychroming occurs slowly at 30°C and rapidly at 60°C, altering the absorption peak.
    • Adjusting the pH to 6.5 maintains the absorption peak at 665 nm even at 60°C.
    • The optimized staining protocol yielded excellent Nocht-Giemsa type staining of malaria parasites with distinct morphological features.

    Conclusions:

    • pH 6.5 is critical for the stability of borax methylene blue, preventing degradation at elevated temperatures.
    • The study validates historical findings and clarifies reasons for previous experimental failures.
    • The optimized staining method provides a reliable tool for visualizing malaria parasites.