Introduction to Microbiology

What is Microbiology?

• Biology is the study of living organisms.
• Microbiology delves into microbes, which are microscopic living organisms, and certain nonliving entities.
• Living microbes, known as cellular microbes or microorganisms, include bacteria, archaea, some algae, protozoa, and some fungi.
• Nonliving microbes, or acellular microbes/infectious particles, consist of viroids, prions, and viruses.
• Microbes exist virtually everywhere.

Acellular and Cellular Microbes

• Microbes are classified as acellular infectious agents (prions, viruses) or cellular microorganisms (prokaryotes, eukaryotes).
• Prokaryotes include archaea and bacteria, while eukaryotes consist of algae, fungi, and protozoa.
• Germs are disease-causing microbes, scientifically termed pathogens.
• Most microbes are nonpathogens.
• Pathogens cause infectious diseases (pathogen colonizes body) or microbial intoxications (pathogen produces toxin in vitro, then ingested).

Pharmaceutical Microbiology

• Ensures medications are free from harmful levels of bacteria, yeast, and molds.
• Focuses on manufacturing facilities, techniques, process controls, and product attributes.
• Microorganisms can affect patients, leading to infections, serious illness, or death, and impact companies through product recalls, sales losses, financial losses, and legal proceedings.
• Microbes play essential roles in carbon, nitrogen, oxygen, sulfur, and phosphorus cycles.
• Algae and bacteria are food for tiny animals.
• Intestinal microbes in animals aid digestion.
• Microbes act as cell models, benefiting food, beverage, chemical, antibiotic, and genetic engineering industries.
• Biotechnology involves using living organisms to make useful products or processes.

Products Requiring Microbial Participation

• Food: Dairy products, fish sauces, and fermented vegetables
• Alcoholic beverages: Ale, beer, brandy, sake, rum, sherry, vodka, whiskey, wine.
• Chemicals: Acetic acid, acetone, butanol, citric acid, ethanol etc
• Antibiotics: Amphotericin B, bacitracin, cephalosporins, etc

Pioneers: Anton van Leeuwenhoek (1632-1723)

• First to observe live bacteria and protozoa.
• He is the father of microbiology, bacteriology, and protozoology.
• Anton was a fabric merchant, surveyor, wine assayer, and a minor city official in Delft, Holland.
• He ground tiny glass lenses for single-lens microscopes.
• He accurately described human RBCs and disease agents ('animalcules').
• Shared his work with the Royal Society of London in 1684.
• Described the inhibitory effect of acetic acid on microorganisms.

Spontaneous Generation Theory (John Needham)

• John Needham supported abiogenesis by boiling meat broth and observing microorganisms after a few days.

Louis Pasteur (1822-1895)

• He was a French organic chemist
• He is the father of modern microbiology.
• He laid the foundation of bacteriology.
• He investigated fermentation, developed pasteurization, and discovered anaerobes.
• He introduced tyndallization.
• In 1880, he isolated the chicken cholera bacterium, and grew in pure culture.
• Introduced vaccines for anthrax and rabies.
• Developed the germ theory of disease

Swan Neck Experiment

• Pasteur heated medium in a swan-neck flask and prevented microbes from entering, disproving spontaneous generation.
• Microbes grew when the neck was broken.

Robert Koch (1843-1910)

• He is the father of medical microbiology.
• The German physician contributed significantly to the germ theory.
• He first isolated Bacillus anthracis.
• Discovered Mycobacterium tuberculosis in 1882 and Vibrio cholera in 1883.
• Developed methods for fixing, staining, and cultivating bacteria and demonstrated the relationship between microorganisms and diseases.

Koch's Postulates

• The microorganism must be found in diseased animals but not in healthy ones.
• The microorganism must be isolated, grown in pure culture.
• The isolated microorganism must cause the original disease when inoculated into a susceptible animal.
• The microorganism must be re-isolated from the experimentally infected animal.
• Building on Pasteur's work, Koch established requirements to demonstrate that each specific disease is caused by specific microorganism.
• The first direct demonstration which showed role of bacteria in causing disease came from the study of anthrax by Koch.
• Built test requirements based on experiments with anthrax isolated from hosts, known as Koch's Postulates.

Treatment, Assay and Testing

• The importance of the scopes of Pharmaceutical Microbiology involves but is certainly not limited to:
• Production of Antibiotics, Enzymes, vaccines, and other pharmaceutical products.
• Used as biosurfactants.
• Testing for diseases using methods such as: ELISA, Widal Test, Tuberculin Skin Test
• Treatment of Industrial Waste Material, Plant growth promotion and Sterile product preparation.
• Sterilization through methods of eliminating all forms of life.
• Steroid biotransformation, Identification of microorganisms and Testing of pharmaceutical products
• Testing the presence of E.coli, Salmonella, Pseudomonas, S. Aureus
• Microbiological assays of antibiotics (Agar diffusion and turbidometric assay).
• Evaluation of disinfectant; Eg, Phenol coefficient Test, Cup Plate method, Kelsey Sykes Test
• Antimicrobial Preservative efficacy testing
• Endotoxin testing, support of the sterility assurance system

The Cell

• Human beings start as a single cell (Fertilized ovum/zygote)
• Living things have: Cells->Tissues->Organs->Organ System
• Tissues are combinations of cells with the same function
• Organs are combinations of tissues that form a more complex functional unit
• The Organ system is a grouping of organs that have interrelated functions
• There are approximately 68 trillion human cells + bacterial cells inside the human body
• 30 trillion are human cells, where 80% are RBCs
• Glial (supporting) cells approximately consist of 1 trillion cells
• Neurons contains 100 billion cells

Eukaryotic Vs Prokaryotic cells

Eukaryotic Cell Membrane

• Cell Membrane (Plasmalemma) Functions:
• Envelops the cell, separating it from its surroundings.
• Protects the cell.
• Divides the body into extracellular and intracellular compartments.
• Regulates substance movement in/out of the cell.
• Attachment site for the cytoskeleton.
• Receptors for incoming extracellular signals.
• Cell-to-cell recognition.
• Semipermeable nature.
• Permeable to water, gases, and nonpolar substances; more permeable to K⁺ than Na⁺.
• Factors Affecting Permeability:
• Temperature.
• Type of solutes present.
• Level of cell hydration.
• Cell Membrane Structure:
• 8-10nm thick.
• Trilaminar structure.
• Main Molecular Components:
• Phospholipids.
• Polar head (hydrophilic).
• Two nonpolar tails (hydrophobic).
• Proteins.
• Integral/Transmembrane proteins span the entire thickness.
• Cholesterol.
• Polysaccharides.
• Glycocalyx:
• Carbohydrate coat on the cell surface.
• Functions in cell recognition, adhesion, and immunological responses.

Intracellular Junctions

• Occluding Junctions (Zonula Occludens or Tight Junctions):
• Form a barrier to protein movement across membranes.
• Divide cells into apical and basolateral sides.
• Adhesive Junctions:
• Macula Adherens (Desmosomes)-tight intracellular adhesion
• Zonula Adherens (Adherens Junction, Adhering Belt, Belt Desmosome, Band Desmosome)— increases the surface area for contact and is equivalent in cardiomyocytes as Fascia Adherens.

Cytoskeleton

• Determine shapes of the cell.
• Play an important role in the movements of organelles and cytoplasmic vesicles and allow the movement of entire cells
• Microfilaments (actin filaments)
• Involved in cell membrane activities (exocytosis and endocytosis).
• Associated with movement of organelles and contraction of myoepithelial cells and muscle cells
• Microtubules form the wall of centrioles, axoneme (core of cilia), tail of a sperm cell and also provides internal support of the cell
• Intermediate filaments: include:
• Keratin: Primarily for protection of epithelial cells.

Organelles- Mitochondria and Ribosomes

• Mitochondria:
• 0.5-1.0 um in diameter; 10 um long.
• Powerhouse of the cell; site for ATP synthesis.
• Motile, aggregating in areas where energy is needed.
• Present in all cells except RBCs and lens fibers.
• Release cytochrome C in stressed cells, triggering apoptosis.
• Ribosomes:
• 15-30 nm in size.
• Key site in Protein synthesis.
• Free ribosomes - Synthesizes proteins for use in cytoplasm
• Attached ribosomes - Synthesizes for internal use and for export outside cell.
• 2 Subunits- Produced in the nucleus (Nucleolus), rRNA
• 50s- prokaryotes; 60s- eukaryotes- Large subunits of Ribosomes.
  • Where tRNA gets amino acids from cytoplasm for peptides bond formation and formation of polypeptide chains
  • 30s- prokaryotes; 40s- eukaryotes - Small Subunits of Ribosomes
  • Attached to mRNA during translation
  • Peptidyl synthetase- an enzyme for peptide bond formation.

Endoplasmic Reticulum and Golgi Complex

• Endoplasmic Reticulum:
• Extensive membranous structure within the cell.
• Acts like a supporting structure for the cytoplasm.
• Rough ER (RER): contains ribosomes.
• The site of synthesis and transport of proteins and proteins transferred to Golgi Complex through vesicles.
• Smooth ER (SER): Lipid synthesis.
• Modified SER for striated muscles =sarcoplasmic reticulum for release of calcium ions which is for muscle contraction
• Golgi Complex:
• Processes, concentrates, sorts, and packages proteins from the endoplasmic reticulum.
• The secretory vesicles are used to release the proteins to the cytoplasm/used for Lysosomal development.
• O-glycosylation of proteins thru Ser and Thr

Lysosomes and Peroxisomes

• Lysosomes:
• 0.05-0.08 µm in diameter.
• With hydrolytic enzymes from Golgi
• For intracellular digestion of organic substrates and involved in heterophagy and autophagy
• Peroxisomes:
• 0.5-1.2 µm, Membrane bound-in all cells but numerous in metabolically active cells.
• The Function aids by Oxidases and catalases that produce and degrade Hydrogen peroxide

Other Cell Structures

• Centrosome:
• Microtubule Organizing Center (MTOC)
• Usually near the nucleus, surrounded by Golgi
• Centrioles are minute, short, cylindrical bodies surrounded by granular structures (centriolar satellites)
• Proteosome:
• Degraded denatured or otherwise nonfunctional polypeptides
• Remove proteins no longer needed by the cell-
• Recognizes proteins with attached molecules of ubiquitin
• Cytoskeleton:
• Determines cells shapes
• Complex array of Microtubules,Microfilaments and Intermediate filaments
• Microfilaments (actin filaments): Aids in Involved in cell membrane activities (exocytosis and endocytosisand Associated with movement of organelles : Internal support and aid in Movement of organelles and Forms wall of centrioles and tail os sperm cells
• Inclusion bodies:
• Contain accumulated metabolites or other substances with Little or no metabolic activity have the Ex: Fat droplets and Glycogen