Microbiology Lab Exam 1 Study Guide: Microscopy, Staining, Aseptic Technique, and Bacterial Identification

Microscopy

Parts and Functions of the Microscope

• Ocular Lens (Eyepiece): Magnifies the image, usually 10x.

• Objective Lenses: Provide additional magnification (commonly 4x, 10x, 40x, 100x).

• Stage: Platform where the slide is placed.

• Coarse and Fine Focus Knobs: Adjust the focus of the image.

• Light Source: Illuminates the specimen.

• Condenser: Focuses light onto the specimen.

• Diaphragm: Regulates the amount of light reaching the specimen.

Microscope Use and Care

• Carry with both hands (one on arm, one under base).

• Clean lenses with lens paper only.

• Store with lowest objective in place and stage lowered.

Total Magnification Calculation

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

• Example: 10x ocular and 40x objective: $10 \times 40 = 400\times$ total magnification.

Ubiquity of Microorganisms

Growth on Plates and in Broth

• Colony Morphology: Shape, margin, elevation, color, and texture of colonies on solid media.

• Broth Growth Patterns: Turbidity (cloudiness), pellicle (surface growth), sediment (bottom growth), flocculent (clumps).

• Interpretation: Different organisms produce characteristic growth patterns.

Aseptic Technique

Principles and Methods

• Aseptic Technique: Procedures to prevent contamination of cultures and the environment.

• Transfer Methods:

  • Broth to broth: Use sterile loop, avoid touching sides of tubes.

  • Broth to solid media: Streak or inoculate agar with sterile instrument.

• General Rules: Flame loop before and after use, minimize exposure of sterile media, work near flame if possible.

Staining Techniques

Negative Stain

• Basic vs. Acidic Dyes:

  • Basic dyes (e.g., crystal violet, methylene blue): Positively charged, stain cell structures.

  • Acidic dyes (e.g., nigrosin, India ink): Negatively charged, stain background, not cells.

• Simple vs. Negative Stains:

  • Simple stain: Colors the cells directly.

  • Negative stain: Colors the background, leaving cells clear.

• Steps of Negative Staining:

  1. Place a drop of acidic dye on slide.

  2. Mix in bacteria.

  3. Spread with another slide.

  4. Air dry (do not heat fix).

• Appearance: Cells appear clear against a dark background.

• Purpose: Useful for observing cell morphology and size without distortion.

Smear Preparation and Simple Stain

• Smear Prep from Liquid Media:

  1. Place a drop of culture on slide.

  2. Spread to thin film.

  3. Air dry, then heat fix.

• Smear Prep from Solid Media:

  1. Add drop of water to slide.

  2. Mix in small amount of colony.

  3. Spread, air dry, heat fix.

• Simple Staining: Apply basic dye (e.g., methylene blue), rinse, blot dry.

• Appearance: Cells are colored, background is clear.

• Information Provided: Reveals cell morphology (shape: cocci, bacilli, spirilla) and arrangement (chains, clusters, pairs).

• Example Bacteria: Escherichia coli, Staphylococcus aureus (must know genus and species).

Gram Stain

• Purpose: Differentiates bacteria into Gram-positive and Gram-negative based on cell wall structure.

• Steps:

  1. Crystal violet (primary stain)

  2. Iodine (mordant)

  3. Alcohol (decolorizer)

  4. Safranin (counterstain)

• Interpretation:

  • Gram-positive: Purple (thick peptidoglycan retains crystal violet).

  • Gram-negative: Pink/red (thin peptidoglycan, loses crystal violet, takes up safranin).

• Example Bacteria: Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative).

Acid-Fast Stain

• Purpose: Identifies acid-fast bacteria with waxy cell walls (mycolic acid).

• Steps:

  1. Carbol fuchsin (primary stain, heat applied)

  2. Acid-alcohol (decolorizer)

  3. Methylene blue (counterstain)

• Interpretation:

  • Acid-fast: Pink/red (retain carbol fuchsin).

  • Non-acid-fast: Blue (take up methylene blue).

• Cell Components: Mycolic acid in cell wall resists decolorization.

• Example Bacteria: Mycobacterium tuberculosis, Mycobacterium smegmatis.

Isolation Techniques

Streak Plate and Pour Plate Methods

• Streak Plate: Used to isolate pure colonies by spreading bacteria over agar surface in a pattern.

• Pour Plate: Serially diluted sample is mixed with molten agar and poured into a plate; colonies grow within and on surface.

• Serial Dilutions: Stepwise dilution of a sample to reduce concentration of organisms for isolation.

• Purpose: Obtain isolated colonies for further study.

• Example Bacteria: Escherichia coli, Bacillus subtilis.

Unknown Morphological Identification

Using a Dichotomous Key

• Gram Stain: First step in identification; determines Gram reaction and morphology.

• Dichotomous Key: Stepwise tool to identify organisms based on observable traits (e.g., Gram reaction, shape, arrangement).

Oxygen Requirements

Growth Patterns and Tests

• Growth Patterns in Thioglycollate Medium:

  • Obligate aerobes: Growth at top (require O2).

  • Obligate anaerobes: Growth at bottom (killed by O2).

  • Facultative anaerobes: Growth throughout, more at top (use O2 when present).

  • Aerotolerant anaerobes: Even growth throughout (ignore O2).

  • Microaerophiles: Growth just below surface (require low O2).

• Medium Used: Thioglycollate broth (contains reducing agents to create O2 gradient).

• Example Bacteria:

  • Obligate aerobe: Pseudomonas aeruginosa

  • Obligate anaerobe: Clostridium sporogenes

  • Facultative anaerobe: Escherichia coli

  • Aerotolerant anaerobe: Enterococcus faecalis

  • Microaerophile: Helicobacter pylori

• Catalase Test: Detects presence of catalase enzyme (breaks down H2O2 to H2O and O2).

• Interpretation: Bubbling upon addition of H2O2 indicates positive result (e.g., Staphylococcus spp.).

Summary Table: Staining Techniques Comparison

Stain Type	Primary Dye	Counterstain	Purpose	Result
Simple Stain	Methylene blue, crystal violet, etc.	None	Cell morphology	Cells colored, background clear
Negative Stain	Nigrosin, India ink	None	Cell morphology (no heat fix)	Cells clear, background dark
Gram Stain	Crystal violet	Safranin	Gram reaction	Purple (Gram+), Pink (Gram-)
Acid-Fast Stain	Carbol fuchsin	Methylene blue	Acid-fastness	Red (acid-fast), Blue (non-acid-fast)

Additional info:

• Knowing genus and species names is essential for lab identification and reporting.

• Lab report questions often focus on interpretation of results and understanding of procedures.