THE MICROBIOLOGY SECTION

I. DEFINITION OF TERMS:
A. Microbiology
- A branch of science that studies microorganisms

B. Microorganisms (Microbes)
- Organisms too small to be seen by the unaided eye
- Classification:
a. Acellular Infectious Agents: Viruses
b. Cellular Microbes
i. Prokaryotes: Archaea, Bacteria
**Prokaryotes: No membrane-bound organelles; No nuclear membrane
ii. Eukaryotes: Algae, Fungi, Protozoa
**Eukaryotes: Presence of membrane-bound organelles; Presence of nuclear membrane
- Includes:
1. Viruses
- Acellular infectious agents; Incapable of surviving outside host cells
- Structure:
i. Core
 Nucleic acid
 Either DNA or RNA (Never both)
ii. Capsid
 Proteinaceous material that protects the nucleic acid core
 Made up of structural subunits known as capsomeres
* Nucleocapsid: Unit made up of the nucleic acid core and the proteinaceous capsid
iii. Envelope
 Not present in all viruses unlike the core and the capsid
 Made up of glycoproteins and lipids
 Naked viruses do not have envelopes while enveloped viruses are equipped with envelopes
* Due to its acellular nature, some textbooks do not classify viruses as true microorganisms.
2. Bacteria
- Unicellular, prokaryotic microbes
- Include the eubacteria and the cyanobacteria (formerly blue-green algae)
- Cell wall is unique due to presence of peptidoglycan
- Taxonomic classification: Kingdom Monera, Domain Bacteria

3. Archaea
- Unicellular, prokaryotic microbes that live in extreme conditions
- Taxonomic classification: Kingdom Monera, Domain Archaea

4. Protozoa (Protozoan)
- Unicellular, eukaryotic organisms that are either free-living or parasitic; Non-photosynthetic
- Taxonomic classification: Kingdom Protista, Domain Eukarya

5. Fungi
- Non-photosynthetic eukaryotic microbes
- Microscopic fungi include the unicellular yeasts and the multicellular molds
- Capable of carrying out both sexual and asexual reproduction
o Yeast cells reproduce via budding
o Molds grow via apical extension
- Cell wall is predominantly made up of chitin
- Taxonomic classification: Kingdom Fungi, Domain Eukarya

6. Algae
- Photosynthetic eukaryotic microbes; Some are unicellular while others are multicellular
- Cell wall is predominantly made up of cellulose
- Taxonomic classification: The microscopic, unicellular algae are placed under Kingdom Protista; Domain Eukarya
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C. Pathogen
- Disease-causing microorganism

Branches of Microbiology
1. Bacteriology: Study of bacteria
2. Mycology (as a branch of microbiology) : Study of microscopic fungi (yeast and molds)
3. Phycology/ Algology: Study of algae
4. Virology: Study of viruses
5. Protozoology: Study of the protozoans

II. THE DEVELOPMENT OF MICROBIOLOGY

Early Beginnings: Discovery of Cells and Very Small Organisms

A. Zacharias Janssen
- One of the several individuals who discovered that if two convex lenses (two-lens instrument) were put together,
small objects would appear larger
-
B. Giovanni Faber
- Coined the term ‘microscopio’ or microscope to refer to the two-lens system

C. Robert Hooke
- Used a 25x microscope
- 1665: Published the book Micrographia which detailed his studies using the 25x microscope
o Studied a piece of cork: He used the word “cella” to describe the ‘great many little boxes’ he observed
o He became the first person to describe and draw a microorganism (mold)

D. Anton van Leeuwenhoek

- Came up with a 200x microscope
- Described hundreds of tiny, living animals (probably protozoa and algae) which he called animalcules

Understanding Disease Transmission: Uprise of Epidemiology

*Epidemiology: Study of the distribution and determinants of a disease in a specified population

A. Naturalists
- Believed in the theory of miasma (“bad air”) which stated that the origin of epidemics such as cholera, malaria,
and plague were due to ‘bad air’, emanating from rotting organic matter

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 B. Ignaz Semmelweis
- Was a Hungarian obstetrician
- Observed the mortality of pregnant women due to puerperal fever (childbed fever)
o Determined that the disease was more prevalent in the ward handled by medical students than in the
ward run by midwifery students (29% death vs 3% death)
o He concluded that the source of contagion must be from cadavers on which the medical students
previously had been performing autopsies before entering the wards. Midwifery students, on the other
hand, did not work on cadavers
o He directed his staff to wash their hands in chlorine water before entering the maternity ward leading to
fewer fatalities due to childbed fever

C. John Snow
- Father of Epidemiology
- Investigated the 1854 London cholera epidemic: Concluded that cholera was waterborne and not spread by
miasma
o Spot-mapping: Snow interviewed the sick and healthy Londoners and plotted the location of each
cholera case on a district map
o He later found out that most cholera cases were clustered and were getting their water source from the
street pump in Broad Street
o Requested the parish Board of Guardians to remove the street pump handle, effectively breaking the
spread of the disease

The Classical Golden Age of Microbiology:

- Beginning around 1854, microbiology blossomed and continued until the advent of World War I

A. Louis Pasteur
- Considered by many as the father of bacteriology
- Was the first to correctly explain that fermentation involves converting sugar into alcohol (wine)by yeasts in the
absence of oxygen
- Concluded that souring and spoilage of wine and dairy products is caused by the presence of bacteria
- Came up with the pasteurization technique as a practical solution for the “wine disease” (wine souring)
o Original method proposed by Pasteur involved heating the wine to 55 0C after fermentation but before
aging
- Proposed the GERM THEORY OF DISEASE
o Specific pathogens are responsible for specific infectious disease

* To completely validate the germ theory, what was missing was the ability to isolate a specific bacterial species from a
diseased individual and demonstrate the isolated organism caused the same disease. This validation was given by Robert
Koch as Louis Pasteur was unable to isolate any causative agents

B. Robert Koch
- Verified the germ theory of disease by coming up with the Koch’s postulates
o Postulate 1: The same microorganisms are present in every case of the disease
o Postulate 2: The microorganisms are isolated from the tissues of a dead animal and a pure culture is
prepared. (The microorganisms are then later identified)
o Postulate 3: Microorganisms from the pure culture are inoculated into a healthy, susceptible animal. The
disease (same disease in postulate 1) is reproduced.
o Postulate 4: The suspected pathogen must be re-isolated from the tissue specimens of the experimental
animal and shown to be the same as the original

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COMPETITION BETWEEN THE KOCH LAB AND THE PASTEUR LAB

The Pasteur Lab

Focused on mechanism of infection & immunity
Notable scientists
Louis Pasteur with Charles Chamberland
Louis Pasteur
Louis Pasteur with Emile Roux
Alexander Yersin and Emile Roux
Elie Metchnikoff
Attenuated (weakened) the bacterial cells of chicken cholera
Applied the principle of attenuation to anthrax and demonstrated
that he could protect sheep against the disease
Emile Roux: Began testing of an experimental rabies vaccine
against dogs
Louis Pasteur: Gave the untested (in humans) rabies vaccine to a
9-year-old boy, Joseph Meister, who had been bitten and mauled
by a rabid dog. The boy recovered and remained healthy.
Identified the diphtheria toxin
Discovered phagocytosis

The Koch Lab

Focused on procedural methods for isolation, cultivation, & identification of pathogens
Notable scientists
Robert Koch Identified and cultured Mycobacterium tuberculosis
Discovered Bacillus anthracis
Isolated Vibrio cholerae and confirmed John Snow’s suspicion
that water is the key to transmission
Friedrich Loeffler Isolated Corynebacterium diphtheriae (diphtheria bacillus)
Georg Gaffky Cultivated the typhoid bacillus
Paul Ehrlich Suggested some dyes might control bacterial infections
Shibasaburo Kitasato Isolated Clostridium tetani
Emil von Behring Developed the diphtheria antitoxin

INDEPENDENT SCIENTISTS DURING THE CLASSICAL GOLDEN AGE OF MICROBIOLOGY

Gerhard Hansen Observed bacterial cells in leprosy patients
Albert Neisser Discovered Neisseria gonorrhoeae
Charles Laveran Discovered that malaria is caused by a protozoan
Theodore Escherich Discovered the bacterium responsible for infant diarrhea
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Richard Pfeiffer Identified a bacterial causative agent of meningitis (Haemophilus influenzae)
Shibasaburo Kitasato and Alexander Independently discovered the bacterium causing plague (Yersinia pestis)
Yersin
Jules Bordet and Octave Gengou Cultivated Bordetella pertussis (pertussis bacillus)

III. COMMON TECHNIQUES IN MICROBIOLOGY SECTION

1. Gram Stain
- Classifies bacteria into two broad groups based on their cell wall structure
o Gram-positive bacteria: purple stain
o Gram-negative bacteria: pink/ red stain
- Also allows observation and further classification of bacteria according to shape:
o Cocci: Spherical-shaped bacteria
o Bacilli: Rod-shaped bacteria
- Classification of bacteria based on both gram-staining reaction and shape:
o Gram-positive cocci
o Gram-negative cocci
o Gram-positive bacilli
o Gram-negative bacilli

2. Acid-Fast stain
- Allows differentiation of acid-fast bacilli (red stain) from non-acid fast organisms
- Is most useful for detection of the acid-fast bacilli Mycobacterium tuberculosis

3. KOH preparation
- A test that utilizes 10% (routine concentration) potassium hydroxide for detection of fungal elements
- Especially useful for detection of fungal elements in keratinous samples such as skin scrapings, nails, and hairs

4. Culture
- Involves isolating the pathogen from the actual site of infection and growing the isolate in vitro using an artificial
environment
- Allows the microbiologist to come up with a sufficient population of the pathogen which will be used for testing and
eventual identification

5. Sensitivity Testing/ Antimicrobial Susceptibility Testing

- After isolation and identification of the pathogen during culture, the sensitivity test is performed to determine the
most appropriate antibiotics that can be used for treating the infection caused by the pathogen
- Most routinely used method: Kirby-Bauer Technique (Disk Diffusion Susceptibility Test)

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