Microbiology Lab Exam 1 Study Guide: Key Concepts and Procedures

Lab #1: The Compound Microscope

Parts and Features of the Compound Light Microscope

The compound light microscope is an essential tool in microbiology, allowing for the visualization of microorganisms and cellular structures. Understanding its components and their functions is crucial for accurate observation and analysis.

• Objective Lenses: Scanning (4x), Low Power (10x), High Power (40x), Oil Immersion (100x).

• Resolution: The ability to distinguish two points as separate; higher resolution means finer detail.

• Magnification: The process of enlarging the appearance of an object.

• Image Inversion: Images appear upside down and reversed when viewed through the microscope.

• Field Diameter: The visible area under the microscope; decreases as magnification increases.

• Parfocal: The ability to switch objectives with minimal refocusing.

• Working Distance: The space between the objective lens and the specimen; decreases with higher magnification.

• Condenser and Iris Diaphragm: Adjust these to improve resolution at higher magnifications.

• Oil Immersion: Used with the 100x objective to increase resolution by reducing light refraction.

• Numerical Aperture (NA): Indicates the light-gathering ability of the lens; increases with magnification.

• Resolving Power Calculation: $\text{Resolving Power} = \frac{\lambda}{2 \times \text{NA}}$ (where $\lambda$ is the wavelength of light).

• Biological Stains: Enhance contrast, making cellular structures visible.

• Centering the Image: Ensures the specimen remains in view when switching to higher magnification.

• Shapes of E. coli and Staphylococcus aureus: E. coli is rod-shaped (bacillus); Staph. aureus is spherical (coccus).

• Micron: 1 micron ($\mu$m) = $1 \times 10^{-6}$ meters.

• Virus Size: Most viruses are 0.02–0.3 microns; too small for light microscopes.

• Distinguishing RBCs and WBCs: RBCs lack nuclei; WBCs have nuclei and are larger.

Microscope Usage and Safety

• Never adjust the coarse focus when objectives other than scanning are over the stage.

• Proper adjustment of condenser and iris diaphragm is essential for optimal resolution.

Lab #2: Simple Positive and Negative Staining

Principles and Procedures of Staining

Staining techniques are used to enhance visibility and differentiate cellular structures. Understanding the chemistry and application of stains is fundamental in microbiology.

• Advantages of Staining: Increases contrast, reveals cellular structures (e.g., cell wall, nucleus).

• Direct vs. Differential Stains: Direct stains color all cells; differential stains distinguish between cell types.

• Stains as Salts: Composed of positive and negative ions; dissociate in water.

• Chromophore: The colored ion in a stain.

• Acidic Dyes: Carry a negative charge; stain background, not cells.

• Basic Dyes: Carry a positive charge; bind to negatively charged cell surfaces.

• Bacterial Cell Surface Charge: Negative; human cells also generally negative.

• Positive vs. Negative Staining: Positive stains color cells; negative stains color background.

• Human Cells in Smears: Presence may indicate contamination or infection.

• Slide Preparation: Use saline to prevent cell lysis; fix slides by heat or chemicals to preserve cells.

• Rinsing Slides: Excessive rinsing can wash away cells; use Bibulous paper to dry slides.

• Negative Stain: Slides are not fixed or rinsed; stain must dry completely; cells appear clear against dark background.

• Identification: Staining alone does not identify genus/species; further tests required.

• Stain Color Mixing: Sequential staining with different colors can result in mixed or layered colors depending on cell uptake.

Lab #3: Differential Stains and Gram Staining

Gram Staining and Its Importance

Gram staining is a differential technique that classifies bacteria based on cell wall structure, aiding in diagnosis and treatment decisions.

• Differential vs. Simple Staining: Differential stains distinguish cell types; simple stains color all cells.

• Types of Differential Stains: Gram stain, Acid-fast stain.

• Primary vs. Counter Stain: Primary stain colors all cells; counter stain colors cells that lost primary stain.

• Gram Stain Groups: Gram-positive (purple), Gram-negative (pink/red).

• Structural Differences: Gram-positive: thick peptidoglycan, no outer membrane; Gram-negative: thin peptidoglycan, outer membrane.

• Staining Colors: Gram-positive: purple (crystal violet); Gram-negative: pink/red (safranin).

• Staining Steps:

  1. Crystal violet (basic dye)

  2. Iodine (mordant)

  3. Acetone alcohol (decolorizer)

  4. Safranin (counter stain, basic dye)

• Purpose of Each Step:

  • Crystal violet: stains all cells

  • Iodine: forms complex with dye, fixes color

  • Acetone alcohol: removes dye from Gram-negative cells

  • Safranin: stains decolorized cells

• Mordant: Enhances dye binding; omission results in weak staining.

• Critical Step: Decolorization; over-decolorizing removes dye from all cells, under-decolorizing leaves dye in Gram-negative cells.

• Human Cells: Do not retain Gram stain; appear colorless or faintly stained.

• Incorrect Gram Stain: Misidentification can lead to improper treatment.

• SECs and WBCs: SECs (squamous epithelial cells) indicate contamination; WBCs indicate infection.

• Yeast Staining: Yeast cells stain purple (like Gram-positive).

• Identification Limitations: Gram stain does not identify genus/species; further testing required.

Lab #4: The Microbial World

Microbial Diversity and Identification

Microbiology encompasses a wide range of organisms, from bacteria to parasites. Recognizing their structures and classification is essential for understanding their roles and impacts.

• Taenia Scolex: Contains hooks and suckers for attachment.

• Bacterial Internal Structures: Not visible at 100x due to small size and lack of organelles.

• Best Objective for Bacteria: Oil immersion (100x) provides highest resolution.

• Bacterial Classification: Domain: Bacteria; Kingdom: Monera.

• Ring-shaped MO in RBCs: Plasmodium (malaria).

• Long, Thin MO in Blood: Trypanosoma (African sleeping sickness).

• MO with Cilia and Nuclei: Paramecium; Domain: Eukarya; Kingdom: Protista.

• MO with Pseudopods: Amoeba; Domain: Eukarya; Kingdom: Protista.

• Spirogyra Structures: Spiral chloroplasts; Domain: Eukarya; Kingdom: Plantae.

• Sexual and Asexual Stages: Many protozoa and fungi; structures include spores, cysts.

• Flagella: Motility structure; seen in bacteria and protozoa (e.g., Giardia).

• Biological Vector: Organism that transmits pathogens (e.g., mosquito for Plasmodium).

• Dysentery: Severe diarrhea; caused by Entamoeba, Shigella, etc.

• Bacterial Shapes: Bacillus (rod), Coccus (sphere).

• Anabaena Structures: Heterocysts (nitrogen fixation), vegetative cells.

• MO Location in RBCs: Plasmodium inside; Trypanosoma between RBCs.

• Organisms with Complete Body Systems: Taenia, Ascaris.

• Adult Ascaris: Large roundworm; males have curved tail, females straight.

• Identification: Unique structures (e.g., cilia, flagella, nuclei) help identify MOs.

• Motility Structures: Flagella, cilia, pseudopods.

• Prokaryotic vs. Eukaryotic Cells: Prokaryotes lack nucleus/organelles; eukaryotes have both.

• Parasite vs. Saprophyte: Parasites feed on host; saprophytes feed on dead matter.

Lab #5: Wet Mounts of Live Microbes

Wet Mount Preparation and Observation

Wet mounts allow observation of live microorganisms, their motility, and cellular structures. Proper technique is essential to prevent contamination and ensure accurate results.

• Wet Mount vs. Smear: Wet mounts show live cells and motility; smears are stained and fixed.

• Preparation: Use saline to maintain cell integrity; pick up MO before adding to slide.

• Biological Hood: Used for handling pathogenic MOs to prevent exposure.

• Brownian Movement: Random motion caused by water molecules; distinguish from true motility.

• Bacteria vs. Yeast: Bacteria are smaller, lack nuclei; yeast are larger, have nuclei.

• Pond Water Specimens: Require oxygen and nutrients to remain healthy.

• LCPB Stain: Enhances mold visibility; may kill cells (disadvantage).

• Objective Lenses for Wet Prep: Low and high power; oil immersion not used due to cover slip.

• Saline Use: Prevents osmotic shock; water may lyse cells.

• Bread Mold Identification: Look for sporangia, hyphae.

• Transfer Tool: Use sterile loop or needle.

• Prokaryote vs. Eukaryote: Eukaryotes have visible nuclei/organelles; prokaryotes do not.

• Sterile Technique: Prevents contamination; improper technique affects results.

• Bunsen Burner: Used for sterilization.

• Lab Safety: Know location of fire extinguisher and fire blanket.

• Wet Prep Applications: Used for diagnosing infections, observing motility, identifying parasites, etc.

• Limitations: Wet preps lack staining detail; harder to identify structures.

• Yeast Reproduction: Asexual budding; visible as small outgrowths.

Comparison Table: Gram-Positive vs. Gram-Negative Bacteria

Feature	Gram-Positive	Gram-Negative
Cell Wall Thickness	Thick peptidoglycan	Thin peptidoglycan
Outer Membrane	Absent	Present
Stain Color	Purple (crystal violet)	Pink/red (safranin)
Teichoic Acids	Present	Absent
Susceptibility to Penicillin	High	Low

Additional info:

• Some explanations and context were inferred to ensure completeness and clarity for exam preparation.

• Scientific names are italicized according to academic convention.

• Equations are provided in LaTeX format as required.