Page 10 - Flow Cytometry Protocols Fourth Edition

Flow Cytometry: The Glass Is Half Full 5
former tending to stain cytoplasm and the latter to stain nuclei. He
had also used the blue basic dye methylene blue to stain bacteria.
In 1882, Ehrlich joined forces with Koch, and developed a stain
that identified Mycobacterium tuberculosis (Mtb ), newly discovered
by Koch, by its ability ("acid-fastness") to retain stains after being
washed in strongly acidic alcohol solutions. Slight modifications by
others yielded the Ziehl- Neelsen (ZN) stain, which has remained
the standard for detecting Mtb by transmitted light microscopy
since 1883, with over 50 million slides analyzed annually. Ehrlich's
work also inspired Christian Gram's initial work on staining
bacteria.
Ironically, Europe's burgeoning dye industry had had its begin-
nings in the 1850s; a British chemistry student's failed attempt to
synthesize quinine, which could be used to treat malaria and was
much in demand, serendipitously yielded the dye mauve, made
fashionable by Queen Victoria. Quinine itself had been isolated in
1820 from cinchona bark; it had been known as an antimalarial
( and one of the only effective drugs against any disease) since the
1600s.
In the 1630s, after European invaders and their African slaves
brought malaria to the Americas, Jesuits brought cinchona, a native
Peruvian folk remedy for chills back to Rome, based on the unsci-
entific but correct suspicion that it might cure malaria, a disease
known since ancient times and common enough in Rome to have
killed several popes. "Jesuit Powder" became an effective, though
scarce and expensive, remedy for the disease, which until the 1950s
was a problem in Northern as well as Southern regions of the world.
No similarly effective treatment for any other infectious disease
appeared before 1890.
The discovery of malaria parasites by Alphonse Laveran in 1880
motivated an intensive search for dyes which facilitated identifica-
tion of these organisms in the blood of infected patients, who could
then be treated with quinine. Laveran, a French military physician
in Algeria, had examined the unstained blood of a malaria patient
and found motile particles containing a blackish-brown pigment
(now called hemozoin) known to be associated with the disease,
but his findings would not be widely accepted until the pathogen's
morphology was better characterized by staining. It took over 20
years to come up with "the new black" for parasites. Gustav Giem-
sa's stain, developed in 1904 and containing the red acid dye cosin,
methylene blue, and the blue basic dye azure B, quickly became and
has remained the "gold standard" for blood smear microscopy.
Noting in 1891 that methylene blue by itself stained malaria
parasites, Ehrlich had procured a supply from a dye company and
successfully treated two malaria patients with it, anticipating his
later success in curing syphilis with two of the over 900 compounds
he tested against that disease. Ehrlich coined the term chemother-
apy and his demonstrations of it prompted many dye companies to