The Science of

Microbiology
What is Microbiology?

•The science of microorganisms (very

small, unicellular organisms)
•The discipline is just over a century old
•Has given rise to molecular biology
and biotechnology
EVOLUTION OF
MICROBIOLOGY
 Antony van Leeuwenhoek (1632-1723)
• Inventor of the first microscope
•Leeuwenhoek earned his living as a draper, but spent much of
his spare time constructing simple microscopes composed of
double convex glass lenses held between two silver plates (figure
below)
• his microscope could magnify around 50 to 300 times,
 SPONTANEOUS GENERATION

•Redi demonstrated that organisms did not arise from

nonliving material.

•FRANCESCO REDI devised a set of experiments to

demonstrate that if pieces of meat were covered with
gauze so that flies could not reach them, no “worms”
appeared in the meat, no matter how rotten it was.
However, no maggots were observed in sealed piece of
meat (figure below)
•Despite the proof that maggots did not arise
spontaneously, some scientist still believed in spontaneous
generation.
•Louis Pasteur demonstrated that
microorganisms in the air were responsible for
food spoilage
Constructed a swan-necked flask
•Pasteur’s experiment for the competition involved his famous
“swannecked” flasks
He boiled infusion in flask, heated the glass necks, and drew
them out into long, curved tubes open at the end. Air could enter
the flask without being subjected to any of the treatments that
critics had claimed destroyed its effectiveness.

Airborne microorganisms could also enter the necks of the

flask, but they became trapped in the curves of the neck and
never reached the infusions. The infusions from Pasteur's
experiment remained sterile unless a flask was tipped so that the
infusion flowed into the neck and then back into the flask.

This manipulation allowed microorganisms trapped in the neck

to wash into the infusion, where they could grow and cause the
infusion to become cloudy.
In another experiment Pasteur filtered air through three cotton
plugs. he then immersed the plugs in sterile infusions,
demonstrating that growth occurred in the infusions from
organisms trapped in the plugs.

Pasteur, with his swan-necked flask, and John Tyndall, with his
dust- free air, finally dispelled the idea of spontaneous
generation.
 Germ Theory of Disease
KOCH’S CONTRIBUTIONS

Koch developed four postulates that aided in the definitive

establishment of the germ theory of disease. Koch’s Postulates
are follows:
 Gram staining is a method of
differentiating bacterial species into two large
groups(Gram-positive and Gram-negative).

 The Gram staining is almost always the first step in the

identification of bacteria.

 It is a valuable diagnostic tool in both clinical and research

settings, not all bacteria can be definitively classified by this
technique. This gives rise to Gram-variable and Gram-
indeterminate groups as well.
The method is named after its
inventor, the Danish scientist Hans Christian
Gram (1853–1938), who developed the technique while
working with Carl Friedländer in the morgue of the city
hospital in Berlin in 1884.

In 1884, while examining lung tissue from patients who

had died of pneumonia, Gram had discovered that
certain stains were preferentially taken up and retained
by bacterial cells.
Gram did not use a counterstain in his procedure. It was
a few years later, that the German Pathologist Carl
Weigert(1845-1904) from Frankfurt, added a final step of
staining with Safranin.

Gram himself never used the red counterstaining in order

to visualize the gram negative bacteria.
 Stained with
Bacteria All Bacteria will be stained Purple
Crystal Violet
Grams Iodine
solution
Cells will be
decolourized Add
while some Decolourizer Stain will be fixed due to formation of
cells will retain (Alcohol or Acetone) complex of Crystal Violet & Iodine
the stain

Staining with Safranin(Counter Stain)

Cells that retains the colour of Primary Stain are Gram positive.

Cells that do not retains the colour of Primary Stain and takes up the colour of
Counter Stain are Gram Negative.
Applying a primary stain (Crystal Violet) to a heat-fixed
smear of a bacterial culture.

The addition of Grams Iodine, which binds to crystal

violet and traps it in the cell.

Decolourization with Alcohol or Acetone, and

Counter staining with Safranin

 Prepare a heat fixed smear of the bacterial culture.

 Cover the smear with the Crystal Violet for 1 min.

 Add Grams Iodine, which washes the crystal violet

stain.

 Rinse the slide in running water and add

decolourizer(Alcohol).

 Again rinse the slide and cover the smear with the
Safranin for 1 min.

 Wash off the safranin with water, air dry the slide and
Observe under oil immersion lens.