Microscopy & Microbiology Lab Fundamentals

Pathology & Microbiology Slide Sets

• Instructor will provide permanent microscope slides for practice
  • Pathology series: human liver cancer, pyelonephritis, fibroma, myocardial infarction ("heart-attack slide")
  • “Microbiology” box (actually still pathology): hypertrophic muscle, spinal cord, Hashimoto thyroiditis, emphysema, “cocci in chains”, etc.
• Blood-typing lab postponed (kit not delivered)

Administrative / Equipment Logistics

• iPad confusion: school iPad vs. personal iPad use separate Apple IDs/subscriptions
• Clean‐up reminders for oil-immersion lenses and reused slides to avoid cross-contamination and equipment damage

The “Five I’s” (core workflow in microbiology)

1. Inoculation – introduce sample to medium
2. Incubation – controlled growth period
3. Isolation – separate individual species
4. Inspection – macroscopic & microscopic observation
5. Identification – biochemical, genetic, immunologic tests

• Memorise picture/flowchart shown in class; questions will directly reference the functions of each “I”

Culture Media

General definition

• Medium = nutrient matrix that supports microbial growth

Selective vs. Differential

Remember: selective ≈ ecological “bouncer”; differential ≈ “ID badge/colour code”.

Synthetic (Defined) vs. Complex (Undefined)

• Synthetic/Defined: every ingredient & concentration known exactly
• Complex/Undefined: contains extracts (plant, animal, yeast, etc.) → exact composition unknown
  • In exam stems, they may disguise the word “plant” or “animal” with specific examples (e.g., “sheep blood”, “dog liver broth”). Recognise that ANY natural extract = complex medium.

Size Hierarchy in Microbiology

• Always check the UNIT before the number!
  • \text{nanometer (nm)} < \text{micrometer (\mu m)} < \text{millimetre (mm)} < \text{centimetre (cm)} < \text{metre (m)}
• Study the size chart (DNA, prions, viruses, Mycobacterium, RBC, etc.)
• Viruses (nm scale) & bacteria (µm scale) are most heavily tested

Principles of Microscopy

Light Interaction & Refraction

• Magnification = complex interplay of visible-light waves + lens curvature
• Refraction: bending of light when it passes between media of differing density; larger density gap → stronger refraction → higher magnification potential

Compound Light Microscope Parts & Standard Magnifications

• Ocular (eyepiece) lens: 10\times (built-in)
• Rotating objective lenses (know all four):
  1. Scanning 4\times → total 4\times10 = 40\times
  2. Low power 10\times → 100\times total
  3. High dry 40\times → 400\times total
  4. Oil immersion 100\times → 1\,000\times total (most commonly quizzed)

Oil-Immersion Lens (100×)

• Procedure: place one drop of immersion oil on cover-slipped specimen, then slowly lower 100× lens until it almost touches oil
• Why oil?
  • Matches refractive index of glass → prevents light scatter
  • Enhances contrast & resolution; can resolve two points just 0.2\,\mu m apart
• Drawback: messy; requires meticulous lens & slide cleaning

Resolution vs. Resolving Power

• Resolution = ability to see two separate objects distinctly
• Resolving power = minimum distance at which this distinction is possible
  • Smaller value → better resolving ability

Electromagnetic Spectrum & Biological Relevance

• Visible light range: 380\,\text{nm} \text{ (violet)} \rightarrow 750\,\text{nm} \text{ (red)}
• Wavelength ↓ ⇒ Energy ↑ ⇒ Hazard ↑
  • Radio (long, safe) → Microwave (heats via molecular friction) → Infrared (remote/Bluetooth) → Visible → Ultraviolet (causes DNA \text{T–T} dimers, skin cancer) → X-ray (ionising; ~150 medical films/yr relatively safe) → Gamma (cosmic; mostly blocked by atmosphere)

Colour Perception Thought Experiment (“Blue Wall”)

• White light contains all wavelengths
• Object appears blue because it absorbs every other visible wavelength and reflects \approx 450\,\text{nm} (blue) back to retina
• In darkness (no incident photons) no colour is perceived
• Rainbows arise when droplets or prisms spatially separate (refract) the composite wavelengths

Other Microscope Types (know names only)

• Bright field, dark field, phase contrast, fluorescence, etc.
• Electron Microscope (EM) : room-sized; uses electron beams; can zoom from whole insect down to sub-cellular structures in real time; highest resolving power

Specimen Preparation Techniques

Wet Mounts

• Ideal for fluids (water, saline, broth)
• Quick, living specimen; observe size, shape, motility
• Clinical example: Vaginal wet mount (Pap smear adjunct)
  • “Whiff test” detects fishy amine odour → bacterial vaginosis or Trichomonas vaginalis
  • T. vaginalis (parasite) looks like tiny “squid” with flagella; motile; most common curable STI; treated with metronidazole
• Limitations: organisms easily damaged; slides hard to preserve

Fixed Smear & Staining Workflow

1. Heat-fix: pass air-dried smear through flame → kills & firmly attaches cells to glass
2. Stain with appropriate dye(s)

Dye Chemistry

• Basic (cationic) dyes: crystal violet, methylene blue, safranin, etc. – attract to negatively charged cell structures (DNA, membranes)
• Acidic (anionic) dyes: repelled by cell surface → stain background (negative stain)
• Positive stain colours cells; negative stain colours background

Staining Methods You Must Master

Clinical Significance of Gram Reaction

• Gram-positive examples: Staphylococcus, Streptococcus → usually susceptible to penicillin-class drugs (ampicillin, amoxicillin, nafcillin)
• Gram-negative examples: Escherichia coli → require broader/final-generation β-lactams, cephalosporins, macrolides, or fluoroquinolones (e.g., ciprofloxacin)
• Physicians start with narrow-spectrum empiric therapy based on probable Gram status, revise after culture & sensitivity results

Culture & Sensitivity (C&S)

• Always obtain cultures before first antibiotic dose whenever feasible
• Lab will plate organism, place antibiotic discs
  • No growth around disc → organism is susceptible
  • Growth up to disc edge → resistant
• Guides definitive antibiotic choice; improper C&S can be fatal

Quick Reference Numbers & Formulae

• Visible‐light window: 380 – 750\,\text{nm}
• Oil-immersion resolution limit: 0.2\,\mu m
• Compound microscope total magnifications: 40\times, 100\times, 400\times, 1\,000\times
• Safe annual diagnostic X-ray upper estimate: \approx 150 films
• Bacterial generation time (general): \approx 20\text{ min}

Ethical, Philosophical & Practical Notes

• Always drape & chaperone appropriately (especially male clinician with female pelvic exam) to respect privacy and legal standards
• Finish entire antibiotic course to prevent resistance; stopping at “feeling better” allows surviving bacteria to regrow & mutate
• Oil-immersion technique increases diagnostic accuracy but demands rigorous cleaning to protect shared lab equipment
• Colour perception & EM spectrum discussion underlines limitations of human senses and importance of instrumentation in science

Study Tips

• Memorise the 4 objective lens powers and what total magnification they yield with the 10× ocular
• Be able to assign any described medium to: selective, differential, both, synthetic, or complex
• Practise drawing quick size ladders (nm → µm → mm) and placing examples
• For stains, rote-learn: purpose, dye sequence, expected colours, and major organisms detected
• Understand WHY clinicians care (antibiotic selection, infection control)