Koch and his associates prepared blocks of tuberculous tissues, and
using the newly invented microtome, shaved extremely thin layers of
samples and mounted them on glass slides. The typical approach was to
expose the slide to an alcohol-methylene blue stain at room temperature,
but this was ineffective. Through trial and error, Koch experimented with
many reagents, settling upon the addition of potassium hydroxide and
Bismarck brown, an industrial brown dye to counterstain the surrounding
tissues. The chemical sorcery was perfected by simultaneously heating the
slides to 40° C, which reduced the reaction times to only an hour.
Almost four hundred years earlier, the first voyagers to the New World
had peered through their telescopes to search for new lands as they sailed
westward. The excitement of visualizing a speck of land, magnified
through the use of glass lenses, could not have been greater than what
gripped Robert Koch when he gazed down his German microscope in his
Berlin laboratory. Harnessing his new technique, he now was faced with a
swath of chestnut-colored tissue, sparsely populated with eye-popping
cerulean bacilli, centered within the caseous tubercles.^40
Standing at the podium at the University of Berlin’s Physiology
Institute before the impromptu genius society, flanked by microscopes and
slide preparations, test tubes and culture dishes, Koch announced that he
had visualized the enemy. More significantly, he calmly proclaimed, he
had grown TB in culture.
Instead of growing the finicky bacteria in broth culture, Koch used
solidified cow or sheep serum, which had been heated and poured onto
slanted tubes to increase the surface area for growing colonies. The tubes
were then inoculated with small batches of the isolated TB samples, grown
at 37° C (human body temperature) and monitored for colony formation.
In turn, guinea pigs in his lab were inoculated with the TB cultures, and
after ten to fourteen days, were sacrificed for lung tissue microscopic
examination.
Koch was able to see the same bacilli in the guinea pig lungs, thus
completing the circle of isolating an organism, culturing the bacteria
outside an animal, infecting another animal with the germ and producing
the same sickness, and finally observing the same organism under the
microscope. These rules from Koch, the father of bacteriology, are the
gold standard of bacterial investigation, and are simply referred to as