Microbiology Introduction and Bacteria Overview

Prions and Zoonotic Pathogens in the Environment

• Prion Diseases: These are not caused by bacteria but by prions. They are characterized by extremely long incubation periods, ranging from $20,$30, to $40 \text{ years}$. Identifying a specific exposure source is difficult unless there is an obvious event, such as a transplant.

• Environmental Observations:

  • Marine Phenomena: Events like the leaching of whales, dolphins, and schools of fish are often poorly understood. While some are suspected to be caused by viruses or specific infections, many marine organisms cannot be cultured properly in a lab, leaving the exact cause unknown.

  • Zoonotic Diseases: Humans primarily know the zoonotic diseases that affect domestic animals because those are the ones studied. There is a vast amount of unknown pathogens in wild animals, including birds and other organisms, that remain unidentified.

Fundamental Concepts of Microbiology and Cell Biology

• Roles of Microorganisms: Microorganisms are responsible for a wide array of ecological and biological processes, including the production of oxygen. However, they are NOT responsible for the production of smog.

• Classification of Fungi: The fungi/yeast family is unique because it is the only family that contains both unicellular organisms and multicellular organisms. A mushroom, though a fungus, is not typically categorized as a microbe.

• Prokaryotic Cell Characteristics: The defining characteristic of prokaryotic cells is the absence of membrane-bound organelles. They do not contain a nucleus or mitochondria.

• Viral Characteristics: Viruses are considered non-living because they cannot reproduce themselves independently. Their structures may include:

  • Capsids: Protein coats.

  • Genetic Material: Either DNA or RNA.

  • Envelope: Some viruses possess an outer lipid layer.

  • Spikes: Protein structures used for attachment.

• Protozoal Motility: Protozoa move using specific structures: cilia, flagella, and pseudopods.

Clinical and Pathological Considerations

• Diphtheria and Pseudomembranes: Diphtheria (part of the DPT or Tdap vaccine: Diphtheria, Pertussis, and Tetanus) causes the formation of "pseudomembranes" in the respiratory tract. These are not true membranes but layers of tissue and debris that can slough off and physically block the airway, leading to suffocation. Emergency intervention is often required to clear the airway.

• Anatomical Distinctions in Cilia:

  • Human respiratory tracts and fallopian tubes contain cilia.

  • The human intestines do NOT contain cilia; they contain villi.

  • Bacteria do NOT contain cilia; cilia is a eukaryotic cell structure.

• Infectious Agents: The human immunodeficiency virus (HIV) is the infectious agent responsible for AIDS.

Immunology: Vaccines vs. Antibiotics

• Targeting Bacteria: Bacterial infections are primarily treated with antibiotics. Because antibiotics are effective at clearing these infections, vaccinations for bacteria are less common, though they do exist (e.g., pneumococcus).

• Targeting Viruses and Toxins: Most common vaccinations are directed against viruses (e.g., Measles, Mumps, Rubella, Hepatitis, COVID, Flu, Shingles, Rabies) or bacterial toxins (e.g., Tetanus, Botulism).

• The Tetanus Vaccine: This does not eliminate the bacteria but rather neutralizes the toxin released by the bacteria.

• Challenges in Vaccine Development:

  • Genetic Drift/Strains: Organisms that shift or drift genetically (changing from strain A to B to C) are difficult to vaccinate against.

  • Parasites and Protozoa: Malaria is the most common protozoal disease worldwide and a leading cause of death. Despite a billion-dollar annual investment from the Bill Gates Foundation, an FDA-approved vaccine remains in development due to the complexity of the parasite.

  • HIV: Billions have been spent on HIV research, but a vaccine does not exist because individuals can be infected with multiple strains and the virus hides/mutates effectively.

  • PrEP (Pre-Exposure Prophylaxis): This is a daily medication containing antivirals used to prevent HIV infection, necessitated by the lack of a permanent vaccine.

Microscopy Principles and Techniques

• Sterilization vs. Disinfection:

  • Alcohol: This is a disinfectant, NOT a sterilizing agent. It cannot kill spores, certain fungi, or prions.

  • Autoclave: This is the only reliable method for sterilization.

  • Historical Context: In the early years of the HIV epidemic, reusing needles and cleaning them only with alcohol accidentally spread the virus.

• Microscope Components and Path of Light:

  1. Light Source: Provides illumination.

  2. Condenser: Concentrates light onto the specimen.

  3. Iris Diaphragm: Located in the condenser area, it controls the amount of light.

  4. Specimen: The object being viewed on the stage.

  5. Objective Lens: The first lens the light enters after exiting the specimen.

  6. Prism: Bends the light toward the eye.

  7. Ocular Lens (Eyepiece): The final lens light passes through to the viewer.

• Magnification Calculations:

  • Total Magnification: $Ocular \times Objective$.

  • Calculation: If the ocular lens is $10 \times$ and the objective is $45 \times$, the total magnification is $450 \times$.

  • Oil Immersion: A $100 \times$ objective with a $10 \times$ ocular results in $1000 \times$ magnification. Immersion oil is required to prevent light scattering (refraction) and ensure light passes directly into the objective.

• Resolution and Parfocal Properties:

  • Resolution: The ability to distinguish two distinct points. The resolution of a standard light microscope is approximately $0.2 \text{ micrometers } (\text{μm})$.

  • Parfocal: A property where lenses are calibrated to stay in focus when switching between different objectives, requiring minimal adjustment.

  • Wavelength: Shorter wavelengths (like UV light in Fluorescence Microscopy) provide higher resolution. Electron microscopes have the highest resolution and magnification.

• Microscopy Types:

  • Dark-Field Microscopy: Visible light is scattered, showing bright organisms against a dark background.

  • Fluorescence Microscopy: Uses UV light for illumination.

Laboratory Procedures and Microbial Growth

• Smear Preparation Order:

  1. Create a smear of the organism.

  2. Air dry (to avoid aerosolizing the bacteria into the face).

  3. Heat fix (to adhere the bacteria to the slide).

  4. Stain.

• Negative Staining: The background is darkened while the organism remains bright. This is used specifically to determine cell size and cell shape.

• Culture Characteristics:

  • Turbid Culture: Indicates microbial growth in a liquid medium (broth).

  • Colony: A population of cells growing on a solid surface that originated from a single cell.

  • Five Descriptors for Colony Morphology: Form, Margins, Elevation, Size, Color.

  • Agar: A solidifying agent derived from seaweed. It boils at $100^\text{°}C$ and solidifies at approximately $40 \text{ °C}$. It is not a nutrient source and is preferred over gelatin because it remains solid at higher incubation temperatures.

• Inoculation Tools: A loop is used for liquid transfers; a pick (needle) is used for solid/solvent transfers.

Taxonomy and Nomenclature

• Naming Rules: Scientific names are either italicized or underlined, but never both. The first word is the Genus (capitalized) and the second is the specific epithet (lowercase). Bacteriology follows an encyclopedic book of rules, and naming new species requires committee approval.

• Origins of Names:

  • Microscopic Appearance: e.g., Staphylococcus (clusters of cocci).

  • Plate Appearance: e.g., S. aureus (looks golden on a plate).

  • Location on Body: e.g., S. epidermidis (found on the skin).

  • Honorary: e.g., Escherichia coli (named after the Spanish scientist Escherich).

  • Disease Symptoms: Names can reflect the condition caused (e.g., the species name dysentery).

  • Discovery Location: e.g., Trypanosoma gambiense (discovered in Gambia).

• Abbreviations and Ambiguity: E. coli can refer to both the bacterium Escherichia coli and the protozoan Entamoeba coli. Context is required to distinguish them.

Historical Figures in Microbiology

• Robert Hooke: Established Cell Theory; first to observe the "cell."

• Antonie van Leeuwenhoek: First to observe living microorganisms ("animalcules").

• The Debate Over Spontaneous Generation:

  • Biogenesis: The theory that life comes only from life.

  • Spontaneous Generation: The theory that life arises from non-living matter.

  • Key Figures: Francesco Redi (maggot experiments), John Needham (claimed spontaneous generation), Lazzaro Spallanzani (disproved Needham), and Louis Pasteur (definitively proved biogenesis).

• Louis Pasteur: Famous for his work on fermentation and the Pasteur Institute. He was considered the "Bill Gates" of his era due to his wealth and influence.

• Ignaz Semmelweis: Introduced handwashing in clinical settings.

• Joseph Lister: The first to use chemicals (disinfectants like phenol) for surgery. The product "Listerine" is named after him. "L-forms" refer to bacteria that lose their cell walls after disinfectant treatment.

• Robert Koch: Developed Koch's Postulates, a set of steps to prove a specific microbe causes a specific disease.

• Edward Jenner: Developed the first vaccination (smallpox).

• Paul Ehrlich: Developed the first synthetic drug (salvarsan, an arsenic derivative) to treat syphilis.

• Alexander Fleming: Discovered Penicillin (the first antibiotic).

• Rebecca Lancefield: Identified and classified different strains of Streptococcus (e.g., Strep A) in the 1930s; these are known as "Lancefield groups."

Industrial Microbiology and Genetic Engineering

• Recombinant DNA: Important for drug production (e.g., insulin, EPO, vaccines).

• Agriculture and Tomatoes: In the early 1990s, scientists discovered the gene responsible for rotting in tomatoes. By genetically engineering tomatoes to turn this gene off, they could be preserved longer for nationwide transport. This is applied to most commercial tomatoes today, except for heirloom varieties.

• Nutritional Enhancement: Probiotics (bacteria) and yeast can be added to foods to increase vitamins (like $B_{12}$) and protein content.

Emerging Infectious Diseases (EIDs)

• COVID-19: A global modern pandemic.

• Zika Virus: Transmitted by mosquitoes and discovered to be present in sperm/ejaculate. During the 2013-2016 Brazillian Olympics era, it was linked to microencephalopathy (small heads in babies) and retardation. It has since spread to Florida and the Caribbean.

• West Nile Virus: Causes encephalitis.

• Bovine Spongiform Encephalopathy (BSE): Also known as Mad Cow disease, caused by prions.

• Ebola: A viral disease causing severe hemorrhagic fever.

• Legionnaires' Disease: An emerging bacterial disease.

• Cryptosporidiosis: A protozoal disease.

• Historical Impact: The 1918-1919 Spanish Flu epidemic killed massive numbers of teenagers and young adults; the Bubonic Plague in the 1400s killed nearly half the population of Europe.

Questions & Discussion

• Student Question: "How does PrEP work?"

• Response: PrEP contains antivirals. Unlike a vaccine which you might take once, PrEP must be taken continually because an HIV vaccine doesn't exist. HIV strains are complex; you can even be infected with multiple strains simultaneously.

• Student Question: "I thought increasing nutritional value wasn't a part of recombinant DNA?"

• Response: It can be. For example, adding genes to produce vitamin $B_{12}$ or adding yeast/bacteria to enhance protein content increases the nutritional profile of the food products.

• Student Question: "Why are we not vaccinating against bacteria as much as viruses?"

• Response: The primary reason is that we have antibiotics to kill bacteria. Viruses generally act faster, and our antivirals are not as effective at totally clearing an infection compared to antibiotics.