Microbiology Lab Final Review Flashcards

Scientific Nomenclature and Identification

• Writing Scientific Names:

  • Organisms are identified by their genus and species name.

  • Genus: The first part of the name; it must always be capitalized.

  • Species: The second part of the name; it is never capitalized.

  • Form: Both names must be underlined if handwritten or italicized if typed (e.g., Escherichia coli).

• Classification Criteria (Bergey’s Manual):

  • Cell Wall Composition: Distinguishes between Gram-positive (thick peptidoglycan, teichoic acids), Gram-negative (thin peptidoglycan, lipopolysaccharide outer membrane), organisms without cell walls (e.g., Mycoplasma), and unique cell walls (e.g., acid-fast organisms).

  • Morphology and Arrangement: Includes shapes such as bacillus (rods), cocci (spheres), and spiral, as well as arrangements like diplo (pairs), strepto (chains), and staphylo (clusters).

  • Genomics: Includes the $G + C$ (guanine and cytosine) percentage within the genome.

Microscope Fundamentals and Procedures

• Compound Light Microscope Parts and Functions:

  • Fine Focusing Knob: The smaller knob on the outside used for precise focusing.

  • Coarse Adjustment/Focusing Knob: Used for initial focusing at lower powers.

  • Ocular Lens: Usually provides a magnification of $10\times$.

• Objective Lenses:

  • Scanning Objective ($4\times$): Typically indicated by a red ring.

  • Low Power Objective ($10\times$): Typically indicated by a yellow ring.

  • High Power (High Dry) Objective ($40\times$): Used for higher magnification without oil.

  • Oil Immersion Objective ($100\times$): Typically indicated by a white ring.

• Calculations and Resolving Power:

  • Total Magnification Formula: $\text{Ocular Magnification} \times \text{Objective Magnification}$.

  • Example: $\text{10} \times \text{10} = \text{100}\times$ total magnification.

  • Resolving Power: The ability to distinguish detail and structure. For the compound light microscope, it is $0.2\,\mu m$ or $200\,nm$.

Smear Preparation and Basic Staining

• Heat Fixed Smear Preparation:

  • Smears should be prepared from broth or slant cultures.

  • The smear must be thin to allow light to penetrate; thick smears may require looking at the outer edges for clarity.

  • Procedure: Allow the smear to air dry completely, then heat fix by passing it through the Bunsen burner flame $3$ times.

• Simple Staining vs. Negative Staining:

  • Simple Stain: Uses a basic (positive) dye where the chromophore is a positively charged ion (cation). Bacterial cells typically have a negative charge and attract the positive dye, staining the cell while the background remains bright or colorless. Example dye: Methylene blue.

  • Negative Stain: Uses an acidic (negative) dye where the chromophore is a negatively charged ion (anion). The dye is repelled by the negative bacterial cell, resulting in colorless cells against a stained background. Example dye: Congo red.

Differential Staining Techniques

• Four Main Steps: Primary stain, mordant, decolorizer, and counterstain.

• Gram Staining (Cell Wall Classification):

  • Primary Stain: Crystal Violet (applied for $1\,min$, rinses to leave all cells purple).

  • Mordant: Iodine (applied for $1\,min$, helps the primary stain stick).

  • Decolorizer: $95\%$ Ethanol (added drop by drop until run-off is colorless; critical step).

  • Counterstain: Safranin (applied to colorize Gram-negative cells).

  • Results: Gram-positive cells appear purple; Gram-negative cells appear pink or red.

• Endospore Staining (Exercise 4):

  • Primary Stain: Malachite Green (applied using steam as the mordant).

  • Mordant: Steam (from a light boil).

  • Decolorizer: DI Water.

  • Counterstain: Safranin.

  • Results: Endospores appear green; vegetative cells appear pink.

• Acid-Fast Staining (Exercise 6):

  • Identifies organisms with waxy mycolic acid in their cell walls.

  • Primary Stain: Carbolfuchsin (applied with steam).

  • Mordant: Steam.

  • Decolorizer: Acid Alcohol.

  • Counterstain: Methylene Blue.

  • Results: Acid-fast organisms (e.g., Mycobacterium, Nocardia) appear pink red; non-acid-fast appear blue/purple.

  • Pathogenic Examples: Mycobacterium tuberculosis (TB), Mycobacterium leprae (leprosy), and Nocardia (nocardiosis).

• Capsule Staining:

  • Primary Stain: Congo red.

  • Mordant: Acid alcohol.

  • Decolorizer: DI Water.

  • Counterstain: Carbolfuchsin.

Biochemical Testing (Metabolic Identification)

• Carbohydrate Fermentation:

  • Uses a pH indicator.

  • Positive (Acid): Yellow.

  • Negative (Alkaline): Purple.

  • Gas: Indicated by a bubble in the Durham tube.

• Starch Digestion:

  • Tests for the enzyme amylase.

  • Requires adding iodine to the plate. Clearing around the growth indicates starch has been digested (positive).

• DNA Digestion:

  • Tests for the enzyme DNAse.

  • Clearing around colonies indicates a positive result.

• Catalase Production:

  • Tests for catalase, which breaks down hydrogen peroxide ($H_2O_2$).

  • Positive: Bubbling upon addition of peroxide.

• SIM Tube (Sulfur, Indole, Motility):

  • Sulfur ($H_2S$): Black precipitate indicates positive hydrogen sulfide production.

  • Indole: Requires Kovacs reagent. A pinkish-red ring at the top indicates positive indole production from tryptophan breakdown.

  • Motility: Cloudiness or turbidity spreading away from the initial inoculation line indicates the organism is motile.

• Urease Digestion:

  • Tests for the enzyme urease.

  • Positive: Hot pink color change.

• Other Tests:

  • Oxidase: Positive result is a purple color on filter paper within $30\,seconds$.

  • Citrate (Simmons Citrate): Tests if citrate is the sole carbon source. Positive result is a change from green to blue.

Culture Media and Body Specimens

• Blood Agar (Differential):

  • Used to distinguish hemolytic patterns.

  • Alpha Hemolysis: Partial hemolysis; appears as a greenish tint (e.g., normal throat flora).

  • Beta Hemolysis: Complete hemolysis; appears as clear zones around colonies (e.g., Streptococcus pyogenes / Group A Strep).

  • Gamma Hemolysis: No hemolysis; no change in the blood (e.g., Staphylococcus epidermidis).

• Mannitol Salt Agar (MSA) (Selective and Differential):

  • Selective: Contains high salt ($NaCl$), favoring halophiles/halotolerant organisms like Staphylococcus.

  • Differential: Contains mannitol and pH indicator. Mannitol fermenters (e.g., Staphylococcus aureus) turn the media yellow. Non-fermenters (e.g., Staphylococcus epidermidis) leave it pink.

• MacConkey Agar (Selective and Differential):

  • Selective: Contains crystal violet and bile salts to inhibit Gram-positives; selects for Gram-negative enteric.

  • Differential: Contains lactose. Lactose fermenters appear bright pink.

• Eosin Methylene Blue (EMB) (Selective and Differential):

  • Selective: For Gram-negative enteric.

  • Differential: Distinguishes lactose fermenters. Strong fermenters like E. coli often show a metallic green sheen.

• Triple Sugar Iron (TSI) Agar:

  • Yellow Slant/Yellow Butt: Ferments glucose plus lactose and/or sucrose.

  • Red Slant/Yellow Butt: Ferments glucose only.

  • Red Slant/Red Butt: No fermentation.

  • Black Precipitate: $H_2S$ production.

  • Cracks/Lifting Media: Gas production.

• IMViC:

  • Indole: Positive test indicated by red ring at the top of the tube after adding Kovac's reagent.

  • Methyl Red: Positive test shows a red color indicating acidic environment (pH < 4.4).

  • Voges-Proskauer (VP): Positive test indicates acetoin production (Usually opposite result of MR test)

  • Citrate: Positive test shows color change in the medium from green to blue, indicating utilization of citrate as the sole carbon source. Blue is indicative of a positive citrate test.

Microbial Diversity: Eukaryotes vs. Prokaryotes

• Yeast and Molds (Fungal Organisms):

  • Yeast: Larger than bacteria, oval-shaped, often show budding. Example: Candida albicans.

  • Molds: Characterized by branch-like structures called hyphae and attached spores.

• Protozoa (Single-celled Eukaryotes):

  • Identified by specific forms like the trophozoite (motile stage) or ring stage (in RBCs).

  • Examples: Giardia lamblia, Plasmodium (causes malaria), Entamoeba histolytica, Balantidium.

• Helminths (Multicellular Parasites):

  • Includes worms and flukes.

  • Example: Schistosoma mansoni (blood fluke).

Antimicrobial Agents and Serology

• Physical Agents (Exercise 21): Includes UV light and heat. Resistance varies by structure (e.g., endospores).

• Chemical Agents (Exercise 22):

  • Antiseptics: Used on living tissue.

  • Disinfectants: Used on inanimate objects.

• Antibiotic Sensitivity (Kirby-Bauer Method):

  • Measures the zone of inhibition in millimeters ($mm$).

  • Results are compared to a chart to determine if an organism is Susceptible (S), Intermediate (I), or Resistant (R).

• Serology Assays:

  • Antigen-Antibody Binding: Very specific; used for diagnosis.

  • Immunodiffusion: Lines of precipitation form on an agar plate.

  • ELISA (Enzyme-linked Immunosorbent Assay): Uses enzymes for detection.

  • Fluorescent Microscopy: Uses fluorochrome-labeled antibodies.

Dilution Series and Plate Counts

• Dilution Principles:

  • A $1:100$ dilution is made by adding $1\,ml$ of sample to $99\,ml$ of water ($10^{-2}$).

  • A $1:10$ dilution is made by adding $1\,ml$ of sample to $9\,ml$ of water ($10^{-1}$).

  • Total Dilution is calculated by multiplying steps (ex., $10^{-2} \times 10^{-1} \times 10^{-1} = 10^{-4}$).

• PFU and CFU Calculations:

  • Countable Range: Between $30$ and $300$.

  • TFTC: Too Few To Count (< 30).

  • TMTC/TNTC: Too Many To Count / Too Numerous To Count (> 300).

• Colony Forming Units (CFU) Calculation:

  • $\frac{\text{Colony Count}}{\text{Dilution factor of plate}} = \text{CFU/ml}$.

  • Example: $37$ colonies on a $10^{-8}$ plate:

  • $\frac{37}{10^{-8}} = 3.7 \times 10^9\,CFU/ml$.