Page 11 - Flow Cytometry Protocols Fourth Edition

Howard M. Shapiro
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expand into pharmacologic research. The company from which he
obtained methylene blue is still in business, and is now known as
Hoechst.
The year 1891 also saw the emergence of diphtheria antitoxin,
the first successful immunotherapy, developed by Emil Behring
(von Behring after he won the first Nobel Prize in medicine, in
1901), a longtime colleague of Ehrlich. Immunoprophylaxis had
been around longer. Variolation, which intentionally infected its
subject with what was hoped would be a mild case of smallpox in
hopes of preventing infection with the virulent natural form, had
been known for centuries but often proved fatal. Beginning around
1800, it was replaced by vaccination, introduced by Edward Jenner,
who had noted that milkmaids who had contracted cowpox, which
rarely caused serious illness, were thereafter immune to smallpox.
Pasteur decided to honor Jenner by calling the immunoprophylaxes
for anthrax and rabies he himself later developed "vaccines." The
list of vaccine targets continues to increase.
It was not until the 1890s that it was accepted that eukaryotic
cells gave rise to new cells only by mitotic division, and the role of
the chromosomes ( the name of which provides the clue that they
were not readily visible without staining) in heredity was not eluci-
dated until the next century. By 1900, the chemistry of proteins was
beginning to be understood, but the two types of nucleic acid then
recently found to comprise "nuclein" had not yet been named and
would not be called DNA and RNA for decades. Although pho-
tography permitted more objective recording of microscope images
than did drawing, the enhanced visual sense given to observers only
allowed them to describe the sizes, shapes, colors, and textures of
cells and their components, and motility and growth in culture
offered the only indications of viability. Most microscopists still
relied on sunlight as an illumination source; electric light and bright
mantle lamps fueled by oil or gas only arrived on the scene late in
the nineteenth century. Detection, characterization, and counting
of cells were dependent on fallible human observers who in almost
all cases had no objective means to confirm their findings and no
alternative to manual data input and analysis.
Advanced darkfield microscopy techniques requiring only sun-
light illumination had, by the early 1900s, permitted visual obser-
vation of light scattered by particles below the resolution limit;
"ultramicroscopes" documented the Brownian motion of large
colloid molecules and confirmed Einstein's predictions. Fluores-
cence microscopy, introduced around 1915, allowed observation of
viruses stained with fluorescent dyes decades before the physico-
chemical bases of the phenomenon were clarified. By the 1930s, the
development of photoelectric sensors and electronics allowed spec-
trophotometers and microspectrophotometers to be built. There
were already cytometric problems for them to solve.