8018 Wang et al.
Asian J. Chem.
that the basic nitrogen atom in MTT formazan is reactive
with HCl, yielding a cationic MTT formazan (Scheme-II).
The conjugated system of MTT formazan is therefore broken.
The change of conjugated system with pH value also provides
a reasonable explanation for the above mentioned color change
(yellowish at pH = 2.0 and deep purple at pH > 2.5). When the
pH is at 6.0 or 11.0, the absorption peak (575 nm) of MTT
formazan at the same concentration is much higher than others.
To avoid overranging the span (0-4) of microplate reader, a
lower concentration of microbial suspension must be set than
normal assay. It is well known that lower concentration of micro-
organism would bring larger experimental errors. The mecha-
nism of structural variation of MTT formazan suggested that
for achieving the best accuracy in MTT assay, the pH of sodium
dodecyl sulfate lysis buffers should be controlled at about 3.5.
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Conclusion
A quick counting method based on the curves of microbial
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ACKNOWLEDGEMENTS
The work was financed by grants from National Natural
Science Foundation (Grant Nos. 21262013 and 81273382) of
China and by a grant from National Natural Science Foun-
dation (Youth Science Funds No. 31100421) of China.