LAB P1

Ocular Lens: The lens you look through (10X magnification).
Objective Lenses:
- 10X: Low power.
- 40X: High power (dry).
- 100X: Oil immersion.
Condenser: Focuses the beam of light onto the slide.
Iris Diaphragm Lever: Adjusts light intensity and contrast (like a camera shutter).
Coarse Focus: Big knob. Moves stage fast. Only use on low power (10X or below).
Fine Focus: Small knob. Fine-tunes sharp focus on high power (40X/100X).
Total Magnification Formula: Ocular Lens (10X) × Objective Lens (e.g., 10 × 40 = 400X).
Resolution: The ability to see two close objects as separate and distinct.
Parfocal: The slide stays roughly in focus when you switch objective lenses.
Working Distance: The space between the lens tip and the glass slide. Higher power = less distance.
Field of View: The circle area you see. Higher power = smaller circle.
Depth of Field: How thick the layer in focus is. Higher power = thinner layer.
Prokaryote: No nucleus (e.g., bacteria).
Eukaryote: Has a nucleus (e.g., human cells, fungi).
Shapes: Bacilli (rods), Cocci (spheres), Spirilla (spirals).
Arrangements: Staphylo- (clusters), Strepto- (chains), Diplo- (pairs).
Microscope view rule: Images are always upside down and backwards.

Hanging Drop: A wet slide using petroleum jelly and a depression slide to watch live, swimming bacteria.
TTC Motility Tube: A tube stabbed with a needle. It contains a dye that turns red where bacteria breathe.
- Motile: Red color spreads everywhere (cloudy).
- Non-motile: Red color stays only on the sharp stab line.
Brownian Movement: Fake motility. It is just random vibrating caused by water crashing into the cells.

Step 1: Crystal Violet (Primary stain). Stains everything purple.
Step 2: Gram’s Iodine (Mordant). Locks crystal violet into the cell wall.
Step 3: 95% Alcohol (Decolorizer). Washes purple out of Gram-negative cells. Gram-positive stays purple.
Step 4: Safranin (Counterstain). Stains the clear Gram-negative cells pink/red.
Gram-Positive (+): Purple cells. They have a thick peptidoglycan cell wall.
Gram-Negative (-): Pink/Red cells. They have a thin wall + a lipid outer membrane.
Over-decolorizing (too much alcohol): Washes purple out of Gram-positives. Everything looks pink (false negative).
Under-decolorizing (not enough alcohol): Doesn't wash Gram-negatives. Everything looks purple (false positive).

Chemicals: Malachite Green (stains spores) and Safranin (stains cell bodies).
Role of Steam: Acts as a physical mordant to melt/open the tough spore coat so the green dye can enter.
Appearance: Green ovals (endospores) inside or outside pink/red rods (vegetative cells).
Spore Genera: Bacillus and Clostridium.
Vegetative Cell Dye: Safranin.

Chemicals: Carbolfuchsin (primary pink), Acid-Alcohol (decolorizer), Methylene Blue (counterstain).
Acid-Fast Positive (+): Bright pink/fuchsia. Their cell walls contain waxy mycolic acid (Mycobacterium).
Non-Acid-Fast (-): Blue. They lack the waxy lipid layer.
Decolorizer action: The pink stain is removed from non-acid-fast cells but retained by acid-fast cells.

Protocol: Mix cells with dark ink (Nigrosin), air dry, dye with Crystal Violet. Do NOT heat-fix.
Why no heat? Heat melts and shrinks the capsule, causing a fake halo look.
Appearance: A clear halo wrapping around a purple cell body on a dark background.
Capsule color: Clear/Colorless. It repels all dye.

Selective for: Halophiles (salt-tolerant organisms like Staphylococcus).
Selective agent: High salt (7.5% NaCl) kills everything else.
Differential for: Mannitol fermentation (eating a specific sugar).
pH Indicator: Phenol Red.
Results:
- Yellow agar: Mannitol fermenter (Staphylococcus aureus).
- Pink/Red agar: Non-fermenter (Staphylococcus epidermidis).

Selective for: Gram-negative bacteria.
Selective agents: Bile salts and crystal violet kill Gram-positive cells.
Differential for: Lactose fermentation.
pH Indicator: Neutral Red.
Results:
- Hot pink/fuchsia colonies: Lactose fermenter (E. coli).
- White/Colorless colonies: Non-fermenter.

Selective for: Gram-negative rods.
Selective agents: Eosin and Methylene Blue dyes kill Gram-positive cells.
Differential for: Lactose fermentation.
pH Indicator: The Eosin and Methylene Blue dyes themselves change color when acid is made.
Results:
- Metallic green sheen: Strong lactose fermenter (E. coli).
- Purple/Pink colonies: Weak lactose fermenter.
- Colorless colonies: Non-fermenter.

Individual Dilution: 0.1 mL added to 0.9 mL water = a 10⁻¹ (or 1/10) dilution.
Total Dilution Factor (10-1): Multiply each step by 1/10.
- Tube 1 = 10⁻¹
- Tube 2 = 10⁻²
- Tube 3 = 10⁻³
- Tube 4 = 10⁻⁴
Countable Plate rule: Pick the plate with 30 to 300 colonies. (Use Tube 3 with 169 colonies).
CFU/mL Formula (10-2):
CFU/mL=ColoniesTubeDilutionVolumePlated
CFU/mL=16910-30.1mL=16910-4=1.69106CFU/mL

Growth at the very top only: Strict (Obligate) Aerobe (needs oxygen).
Growth at the very bottom only: Strict (Obligate) Anaerobe (poisoned by oxygen).
Growth throughout the whole tube: Facultative Anaerobe (prefers oxygen but can ferment without it).
Who has SOD (Superoxide Dismutase) protective enzymes? Aerobes and Facultative Anaerobes (the ones that touch oxygen).

Minimum Temp: Lowest temperature where reproduction can start.
Maximum Temp: Highest temperature before cell enzymes denature (melt) and kill the cell.
Optimum Temp: The perfect temperature where the cell divides the fastest (most turbid/cloudy tube).
Psychrophile: Cold-loving (0°C–15°C).
Psychrotroph: Refrigerator temperature food-spoiler (20°C–30°C).
Mesophile: Body temperature pathogen (20°C–45°C).
Thermophile: Heat-loving (45°C–80°C).
Can a thermophile grow on the human body? No. Human body temperature is 37°C, which is far too cold for a thermophile's heat-activated enzymes to work.

Pour Plate: Bacteria mixed inside warm agar. Colonies grow inside, under, and on top of the agar.
Spread Plate: Bacteria spread over an already solid surface. Colonies grow only on top.
Colony: A visible pile of identical cells cloned from one single original cell.
Pure Culture: Contains only one species of bacteria (all colonies look identical).
Mixed Plate: Contains multiple different species (different colony shapes/colors).
Handwashing check: Side 1 (Before) has high, mixed colonies. Side 2 (After) has significantly fewer colonies.

Beta-Hemolysis (β): Complete clearing of red blood cells. Agar looks totally transparent/clear around colonies.
Alpha-Hemolysis (α): Partial destruction of cells. Agar turns a bruised green/brown color.
Gamma-Hemolysis (γ): No destruction. Agar stays red and normal.