Microbiology Essentials: Lab Techniques & Core Concepts

Posted on Sep 20, 2025 in Biology

Microscope Components & Principles

  • Ocular Lens: Remagnifies the image formed by the objective lens.
  • Objective Lens: The primary lens that magnifies the specimen.
  • Condenser: Focuses light through the specimen.
  • Diaphragm: Controls the amount of light entering the condenser.
  • Coarse/Fine Focus: Used to focus the specimen, changing the distance between the objective lens and specimen. Coarse focus is typically used only for the 4x scanning lens.

Microscope Lenses

  • Scanning Lens: 4x magnification (shortest lens).
  • Low Power Lens: 10x magnification.
  • High Power Lens: 40x magnification.
  • Oil Immersion Lens: 100x magnification (longest lens).

Microscope Principles

  • Iris Diaphragm: Holds condenser lenses and controls the amount of light entering the condenser.
  • Condenser Lenses: Focuses a cone-shaped light onto the specimen.
  • Resolving Power (RP): The ability of lenses to distinguish two points. For example, an RP of 0.4 nm can distinguish between two points if they are at least 0.4 nm apart.
  • Parfocal: Refers to a microscope with lenses that remain in focus when switching between different magnifications.

Microorganism Classification & Observation

Algae

  • Eukaryotes with cellulose cell walls.
  • Use photosynthesis for energy, producing molecular oxygen and organic compounds.

Protozoa

  • Eukaryotes that absorb or ingest organic chemicals.
  • May be motile via pseudopods, cilia, or flagella.

Microorganism Observation Techniques

  • Wet-Mount Technique: A quick and easy way to observe live microorganisms. Movement can be difficult to observe, and motility is hard to determine due to water movement and limited space.
  • Hanging-Drop Technique: Microorganisms are suspended in a drop of liquid, providing more space for movement. This method allows for observation of motility, and larger microbes are easier to observe.

Metabolic Classifications

  • Photo: Energy derived from light.
  • Chemo: Energy derived from chemical reactions.
  • Auto: Carbon source comes from an inorganic compound (e.g., CO2).
  • Hetero: Carbon source comes from an organic compound (e.g., food).

Fungal Sexual Reproduction Stages

  • Plasmogamy: Two haploid cells of different mating types (+/-) fuse to form one cell with two haploid nuclei.
  • Karyogamy: The two haploid nuclei fuse to form one diploid nucleus.
  • Meiosis: The diploid cell undergoes meiosis to produce haploid spores.

Protozoa Organelles & Staining Techniques

Protozoa Characteristics & Organelles

  • Eyespot: Helps detect light and move toward it.
  • Pellicle: Protozoa do not have cell walls; some possess pellicles, which are outer coverings of thick, elastic membranes.
  • Cytostome: The “mouth” where food enters the cell.
  • Pseudopods: Responsible for motility.
  • Cilia: Responsible for motility.
  • Contractile Vacuole: Removes excess water from the cell; it fills with water and contracts to expel it.

Microbial Staining Techniques

  • Fixing: Denatures bacterial enzymes that would cause autolysis and enhances the adherence of bacterial cells to the slide.
  • Simple Stain: Uses one reagent (e.g., crystal violet, methylene blue) to color cells.
  • Differential Stain: Uses multiple reagents to differentiate bacteria based on specific properties (e.g., Gram stain, acid-fast stain).
  • Structural Staining: Stains specific parts of the cell (e.g., flagella, capsule).

Gram Staining Procedure

  • Primary Stain: Crystal Violet (stains all cells purple).
  • Mordant: Iodine (forms a crystal violet-iodine complex within the cells).
  • Decolorizing Agent: Alcohol-Acetone (washes out the stain from Gram-negative cells).
  • Counterstain: Safranin (stains decolorized Gram-negative cells red/pink).

Gram-Positive Bacteria: Appear purple throughout the process.

Gram-Negative Bacteria: Appear purple, then purple, then colorless after decolorization, and finally red/pink after counterstaining.

ELISA, Culture Media & Oxygen Needs

Enzyme-Linked Immunosorbent Assay (ELISA)

  • Involves a primary antibody that binds to the antigen.
  • A secondary antibody then binds to the primary antibody, usually conjugated with an enzyme like horseradish peroxidase.
  • A substrate is added, which turns blue in the presence of the enzyme.
  • A blocking buffer prevents nonspecific binding.
  • A wash buffer removes unbound antibodies.

Microbial Culture Media

  • Differential Media: Differentiates colonies of desired bacteria from others, often resulting in different colors. (e.g., EMB Agar).
  • Selective Media: Suppresses the growth of unwanted bacteria. (e.g., PEA Agar).
  • EMB (Eosin Methylene Blue) Agar: A selective and differential medium used for the identification of Gram-negative bacteria.
    • Selective: Methylene blue inhibits Gram-positive bacteria.
    • Differential: Rapid fermentation of lactose lowers the pH and encourages dye absorption, causing bacteria to appear purple/black or colorless.
  • PEA (Phenylethyl Alcohol) Agar: A selective medium used to grow Gram-positive bacteria. It inhibits or reduces the growth of Gram-negative bacteria by interfering with DNA synthesis.

Bacterial Oxygen Requirements

  • Microaerophiles: Require small amounts of oxygen.
  • Obligate Aerobe: Absolutely requires oxygen for growth.
  • Facultative Anaerobe: Can grow with or without oxygen.
  • Aerotolerant Anaerobes: Tolerates oxygen but does not use it for growth.
  • Obligate Anaerobe: Inhibited or killed by the presence of oxygen.

Anaerobic Culture, Temperature & Metabolism

Key Prefixes for Oxygen Terms

  • Aero: Pertaining to oxygen.
  • Anaero: Pertaining to the absence of air/oxygen.
  • Micro: Small.
  • Tolerant: Able to withstand or endure.

Anaerobic Culture Methods

  • Thioglycolate Broth: A reducing medium that consumes oxygen when heated. It contains Resazurin, which turns pink in the presence of oxygen, indicating oxygen levels.
  • Brewer Anaerobic Jar: An airtight jar where a gas pack releases CO2 and H2, effectively removing oxygen from the environment.
  • Anaerobic Chamber/Glove Box: A sealed chamber where oxygen is purged from the environment, providing a controlled anaerobic atmosphere.

Bacterial Temperature Requirements

  • Extremophiles: Bacteria that thrive in extreme environmental conditions.
  • Psychrophiles: Cold-loving bacteria, growing optimally between -5°C and 15°C.
  • Mesophiles: Bacteria that grow at moderate temperatures, typically between 25°C and 45°C.
  • Thermophiles: Heat-loving bacteria, growing between 45°C and 70°C.
  • Hyperthermophiles: Grow at very high temperatures, with optimum growth between 70°C and 110°C.

Respiration & Fermentation

  • Respiration: Oxidizes fuels using an inorganic electron acceptor to drive ATP production.
    • Aerobic Respiration: The final electron acceptor in the Electron Transport Chain (ETC) is oxygen.
    • Anaerobic Respiration: The final electron acceptor is NOT oxygen.
  • Carbohydrate Catabolism: The breakdown of carbohydrates to produce energy. Key processes include glycolysis, the Krebs cycle, and the ETC.
  • Fermentation: Always anaerobic.
    • Releases energy from the oxidation of organic molecules.
    • Produces a range of products (e.g., acids, gases, alcohols).
    • Does not use the Krebs cycle or the ETC.
    • Uses organic electron acceptors.
  • Pyruvic Acid: A crucial intermediate needed for both respiration and fermentation pathways.

Microbiology Laboratory Tests

OF Media (Oxidation-Fermentation)

  • Determines if a microbe is oxidative or fermentative.
  • Contains high amounts of glucose and low peptone.
  • The pH indicator bromothymol blue detects changes in pH (yellow in acidic conditions, blue in alkaline conditions).
  • Typically uses two tubes: one with mineral oil (anaerobic) and one without (aerobic).

Fermentation Test

  • Phenol Red acts as a pH indicator, detecting acid production.
  • An inverted test tube (Durham tube) detects gas production.
  • Different sugars (e.g., lactose, glucose, sucrose) are used in the media to test if bacteria can ferment them.
  • Note: Not all fermentation pathways produce acids; for example, butanediol fermentation produces acetoin, a neutral product detected in the MRVP test.

MRVP Test (Methyl Red-Voges-Proskauer)

  • Consists of two tests to determine the type of glucose fermentation.
  • Cultures are split into two tubes: one for Methyl Red, one for VP reagents.
  • Methyl Red Test: Detects significant acid production (red color indicates positive).
  • Voges-Proskauer Test: Detects the neutral product acetoin. VP reagents are added to the upper part of the media, which will turn pink/red in the presence of acetoin.

MIO Deep (Motility, Indole, Ornithine)

  • Uses semi-solid agar.
  • Motility: Motile bacteria grow throughout the agar, causing a cloudy appearance.
  • Indole Production: Tested with Kovac’s reagent, which turns red in the presence of indole.
  • Ornithine Decarboxylation: This reaction makes the media less acidic (more alkaline). The pH indicator Bromocresol purple turns yellow when acidic and purple when alkaline.

Phenylalanine Slant Test

  • Agar slant supplemented with phenylalanine.
  • Tests for a bacterium’s ability to deaminate phenylalanine, producing phenylpyruvic acid.
  • Ferric chloride is added, which forms a green complex with phenylpyruvic acid, indicating a positive result.

Epidemiology & Microbial Genetics

Epidemiological Terms

  • Signs: Objective evidence of disease that can be measured or observed.
  • Symptoms: Subjective evidence of disease, described by the individual experiencing them.
  • Syndrome: A complete set of characteristic signs and symptoms associated with a particular disease.
  • Endemic: A disease constantly present in a specific geographical area or population.
  • Epidemic: A sudden increase in the number of disease cases in a short period within a specific population or area.
  • Pandemic: An epidemic that has spread across multiple countries or continents, affecting a large number of people globally.

Koch’s Postulates

  1. The suspected pathogen must be present in all cases of the disease and absent from healthy animals.
  2. The suspected pathogen must be grown in pure culture.
  3. Cells from a pure culture of the suspected pathogen must cause disease in a healthy animal.
  4. The suspected pathogen must be re-isolated from the inoculated, diseased animal and shown to be the same as the original.

Bacterial Genetic Transfer Mechanisms

  • Transformation: Genetic alteration caused by the direct uptake, incorporation, and expression of exogenous genetic material from the environment.
  • Transduction: Genetic material is transferred from one bacterium to another by a virus (bacteriophage).
  • Conjugation: Bacteria exchange plasmid DNA directly through cell-to-cell contact, typically via a sex pilus.

pGLO Plasmid Components

  • Beta-Lactamase Gene (bla): Always active, this gene confers ampicillin resistance, allowing bacteria to grow in the presence of ampicillin.
  • araC Gene: Always active, it codes for the AraC bifunctional regulatory protein, which acts as a repressor or inducer depending on arabinose availability.
    • +Arabinose: Acts as an inducer, activating gene expression.
    • -Arabinose: Acts as a repressor, inhibiting gene expression.
  • Arabinose Promoter (pBAD): A promoter that regulates the expression of GFP, controlled by the AraC protein.
  • Green Fluorescent Protein (GFP) Gene: A gene from jellyfish that produces a fluorescent glow under UV light, with its expression dependent on the presence of arabinose.