Unit 1: General Microbiology

1. History of Microbiology

Discovery of Microorganisms (Prokaryotes to Eukaryotes)

Microbiology officially began with the invention of the microscope. While the term "microbe" is broad, its discovery was staggered.

• Antonie van Leeuwenhoek (1670s): A Dutch cloth merchant, he built high-quality single-lens microscopes. He was the first to observe and meticulously describe single-celled organisms, which he called "animalcules" (now known as bacteria and protozoa), from rainwater, dental plaque, and other sources.
• Louis Pasteur (1860s): Often called the "Father of Microbiology," he disproved the theory of spontaneous generation with his famous swan-neck flask experiment. He demonstrated that microbes are present in the air and are responsible for fermentation and spoilage. His work led to the development of pasteurization and vaccines.
• Robert Koch (1870s-80s): A German physician, he provided the first direct proof of the Germ Theory of Disease. He developed techniques to grow microbes in pure culture on solid media (using agar, an idea from Angelina Hesse). He established Koch's Postulates, a set of criteria to link a specific microbe to a specific disease.

Discovery of Viruses

Viruses were discovered as entities smaller than bacteria that could cause disease.

• Dmitri Ivanovsky (1892): A Russian botanist, he studied Tobacco Mosaic Disease (TMD). He found that the sap from an infected plant could transmit the disease even after it was passed through a filter fine enough to trap all bacteria. He called it a "filterable agent."
• Martinus Beijerinck (1898): He independently replicated Ivanovsky's work and gave this filterable agent a name. He called it contagium vivum fluidum (contagious living fluid) and coined the term "virus."
• Wendell Stanley (1935): He crystallized the Tobacco Mosaic Virus (TMV), showing it was part particle and part chemical, blurring the line between living and non-living.

2. Status of Microorganisms in the Living World

The classification of life, especially microscopic life, has evolved significantly.

Haeckel's System (1866)

Ernst Haeckel proposed a three-kingdom system:

1. Plantae: Multicellular plants.
2. Animalia: Multicellular animals.
3. Protista: A "catch-all" kingdom for all "simpler" life forms, including bacteria, algae, fungi, and protozoa. This was the first system to formally recognize microorganisms.

Whittaker's Five Kingdom System (1969)

Robert Whittaker's system became the standard for decades. It classified organisms based on cell structure (prokaryotic/eukaryotic), cellular organization (unicellular/multicellular), and mode of nutrition.

1. Monera: All prokaryotes (bacteria, cyanobacteria). Unicellular.
2. Protista: Unicellular eukaryotes (protozoa, simple algae).
3. Fungi: Eukaryotic, multicellular (mostly), saprotrophic (absorptive nutrition).
4. Plantae: Eukaryotic, multicellular, autotrophic (photosynthetic).
5. Animalia: Eukaryotic, multicellular, heterotrophic (ingestive nutrition).

In this system, microorganisms are found in Monera, Protista, and Fungi.

Carl Woese's Three Domain System (1977)

Carl Woese revolutionized classification by using 16S ribosomal RNA (rRNA) sequencing. This molecular approach revealed a fundamental split in the prokaryotes.

1. Domain Bacteria: "True" bacteria. Prokaryotic.
2. Domain Archaea: Prokaryotes that are genetically and biochemically distinct from bacteria (e.g., extremophiles). They are more closely related to Eukarya than to Bacteria.
3. Domain Eukarya: All eukaryotes (Protista, Fungi, Plantae, Animalia are now kingdoms *within* this domain).

3. Aseptic Techniques

Aseptic techniques refer to all methods used to prevent contamination of cultures, sterile equipment, or the experimenter with unwanted microorganisms.

Sterilization

Definition: Sterilization is the complete removal or destruction of *all* forms of microbial life, including vegetative cells, spores, and viruses. This is an absolute term; something is either sterile or it is not.

Physical Methods of Sterilization

Chemical Methods of Sterilization

These agents are used to disinfect or sterilize. Disinfectants are used on inanimate objects, while antiseptics are used on living tissue.

• Alcohols (Ethanol, Isopropanol): Denature proteins, dissolve lipids. Good for skin antiseptics and disinfecting surfaces. (Typically 70% concentration).
• Halogens (Iodine, Chlorine): Oxidizing agents. Iodine (Betadine) is an antiseptic. Chlorine (bleach) is a disinfectant for water and surfaces.
• Phenols (e.g., Lysol): Disrupt cell membranes, denature proteins. Stable and effective disinfectants.
• Gases (Ethylene Oxide): Highly penetrating gas used for heat-sensitive materials like plastic petri dishes and catheters. It's an alkylating agent.

4. Culture Media

Definition: Culture Medium (plural: media) is a nutrient-rich substance (liquid or solid) prepared in the lab to support the growth and multiplication of microorganisms.

Types of Culture Media

Media can be classified based on their consistency, composition, or purpose.

1. Based on Consistency:
   • Liquid (Broth) Media: No gelling agent. Used to grow large quantities of microbes.
   • Solid Media: Contains a gelling agent (usually 1.5-2.0% agar). Used for isolation, enumeration, and observing colony morphology.
   • Semi-solid Media: Contains a low concentration of agar (e.g., 0.5%). Used to study motility.
2. Based on Composition:
   • Defined (Synthetic) Media: The exact chemical composition is known (e.g., Czapek's medium).
   • Complex (Undefined) Media: The exact composition is unknown. Contains complex ingredients like peptone, yeast extract, or beef extract (e.g., Nutrient Broth).
3. Based on Purpose:
   • Basal Media: Simple media that supports the growth of non-fastidious microbes (e.g., Nutrient Agar).
   • Enriched Media: Basal media supplemented with extra nutrients like blood or serum (e.g., Blood Agar) for growing fastidious (picky) microbes.
   • Selective Media: Contains substances (e.g., antibiotics, dyes) that inhibit the growth of unwanted microbes and *select* for the desired ones (e.g., MacConkey Agar selects for Gram-negative bacteria).
   • Differential Media: Contains indicators that allow for the differentiation of two types of microbes based on a biochemical property (e.g., MacConkey Agar differentiates lactose fermenters (pink) from non-fermenters (colorless)).

Preparation of Media (for Bacteria, Fungi, Actinomycetes)

The general steps are:

1. Weighing: Accurately weigh the powdered components as per the formula.
2. Dissolving: Dissolve the components in the required volume of distilled water, usually with gentle heating. If it's an agar medium, it must be boiled to fully dissolve the agar.
3. pH Adjustment: Check the pH with a pH meter and adjust it to the required level (usually 6.8-7.2 for bacteria, 5.0-6.0 for fungi) using dilute HCl or NaOH.
4. Dispensing: Pour the medium into final containers (flasks, test tubes).
5. Sterilization: Sterilize the medium in an autoclave (121°C, 15 psi, 15-20 min).

Specific Media Examples:

• For Bacteria: Nutrient Agar (NA) / Nutrient Broth (NB). A complex, basal medium.
• For Fungi: Potato Dextrose Agar (PDA) or Sabouraud Dextrose Agar (SDA). These media have a high sugar content and a low pH, which inhibits bacterial growth.
• For Actinomycetes: Actinomycete Isolation Agar (AIA) or Starch Casein Agar (SCA). These may contain antibiotics like cycloheximide to inhibit fungi.

5. Isolation of Pure Cultures

A pure culture contains only one species or strain of microorganism. Isolation is crucial for studying a specific microbe.

Streak Plate Method:

This is the most common technique used to obtain isolated colonies.

1. Take a sterile inoculating loop and pick up a sample (inoculum) from a mixed culture.
2. Streak the inoculum back and forth in one quadrant of an agar plate.
3. Sterilize the loop in a flame.
4. Turn the plate 90 degrees. Drag the loop from the first quadrant into the second, spreading the bacteria.
5. Sterilize the loop again.
6. Turn the plate 90 degrees. Drag the loop from the second quadrant into the third.
7. Sterilize the loop again and repeat for a fourth quadrant.
8. Incubate the plate. The process mechanically dilutes the bacteria, so that in the final quadrants, individual cells are deposited. Each cell will grow into a visible, isolated colony, which is a pure culture.

Other Methods:

• Spread Plate: A small volume of a *diluted* liquid sample is pipetted onto the surface of an agar plate and spread evenly with a sterile "hockey stick" (glass spreader).
• Pour Plate: A diluted sample is mixed *with* molten (warm) agar and then poured into a sterile petri dish. Colonies grow both on the surface and embedded within the agar.

6. Vaccines

Definition: A vaccine is a biological preparation that provides active acquired immunity to a particular infectious disease. It "trains" the immune system to recognize and fight a pathogen.

Principle: A vaccine introduces a safe form of the pathogen (or a part of it), called an antigen, into the body. The immune system mounts a response, creating memory cells (T-cells and B-cells). If the body later encounters the *actual* pathogen, these memory cells launch a rapid and strong attack, preventing or (more commonly) reducing the severity of the disease.

Types of Vaccines: