Microbiology – Chapter 3: Culturing, Media & the Five I’s

Inoculation
- Collecting a specimen (swabs, fluids, etc.) and introducing it to growth material.
- Exactly what the class did when they “went around swabbing things.”
- Requires sterile tools (plates, swabs, water) to prevent cross-contamination.
Incubation
- Placing inoculated material in a controlled environment.
- Common temperature settings: $25^{\circ}C$ (room temp, minimizes odor), $30^{\circ}C$ , $37^{\circ}C$ (human body temp), $42^{\circ}C$ .
- Most human pathogens grow between $20^{\circ}C$ – $45^{\circ}C$ ; outside this range organisms are usually non-pathogenic to humans.
- Incubators also regulate O(2) / CO(2) to encourage growth.
Isolation
- Separating individual species on/within media.
- Goal: obtain single, well-separated colonies (small dots) rather than confluent growth.
- Enables pure-culture work.
Inspection
- Macroscopic: colony color, size, shape, margin, odor (e.g., Pseudomonas smells “fruity”).
- Microscopic: cell morphology after staining.
- May include metabolic or biochemical traits.
Identification
- Confirming the organism’s identity by:
- Stains (Gram, acid-fast, etc.)
- DNA/protein/antibody assays (PCR, immunoassays, fluorescent tags).
- Biochemical panels, enzymatic tests.
- Viruses cannot be cultured on agar; require PCR or immunologic assays because they lack cell walls/metabolism.

Culture = To grow microorganisms intentionally.
- Either “something grows” or “nothing grows.”
- Example: suspected UTI → urine is cultured on plates and analyzed by dipstick/microscope.
Pure culture myth: A plate may contain multiple species even if it looks like one biofilm.
Viruses cannot be grown on standard plates because they need living host cells.

Typical turn-around for bacterial cultures: $48$ – $72$ h.
Fast growers (e.g., Listeria, Pseudomonas, Streptococcus): $1$ – $2$ days.
Slow growers (e.g., Mycobacterium tuberculosis): up to $30$ days → culture is no longer first-choice diagnostic.

Liquid (broths)
Semi-solid (slants; jelly-like)
Solid (plates)
Agar
- Complex polysaccharide from red algae.
- Solid at room temp; melts above $100^{\circ}C$ and solidifies near $42^{\circ}C$ .
- Made like gelatin: melt → pour → cool.

Synthetic (chemically defined): every ingredient & concentration is known.
Complex (undefined): contains extracts from plants, animals, or yeasts—exact composition partially unknown (e.g., nutrient broth, blood agar, MacConkey).

General-purpose – support wide range of microbes (e.g., TSA – Tryptic Soy Agar).
Enrichment – supplemented with growth enhancers (vitamins, amino acids, blood) to favor fastidious organisms.
Selective – ingredients inhibit unwanted groups and allow a select group to grow (e.g., high-salt MSA for staphylococci; Gram-positive vs Gram-negative plates).
Differential – multiple species grow, but each displays a distinguishable trait (color change, hemolysis).
- One plate can be both selective and differential, e.g., Mannitol Salt Agar (MSA):
  - $7.5$ % NaCl selects Staphylococcus spp.
  - Mannitol fermentation turns phenol-red indicator yellow for S. aureus.

Contains $5$ % sheep blood; used for many pathogens.
Hemolysin enzymes digest RBCs → reveals zones around colonies.
1. Alpha (α) hemolysis: partial/greenish clearing = incomplete RBC lysis.
2. Beta (β) hemolysis: clear, wide halo = complete RBC lysis; typically more virulent (e.g., Strep pyogenes).
3. Gamma (γ) hemolysis: no hemolysis (e.g., Enterococcus spp.).

Anaerobic vs aerobic tubes: presence/absence of gas bubbles indicates O(_2) usage.
pH indicators: media change color correlating to acidity/alkalinity.
Immunologic or molecular tests (e.g., interferon-γ release assay “Quantiferon Gold” for TB) complement or replace slow cultures.

Plate divided into $4$ quadrants; loop sterilized between streaks.
Each successive quadrant dilutes cells, leading to single colonies in the final sector (seen around $7$ – $8$ o’clock on diagram).
Critical for obtaining pure culture for further testing.

Phenotypic (macroscopic/microscopic) tests – colony/cell morphology, staining.
Biochemical panels – sugar fermentation, enzyme activities.
Immunologic assays – antibody binding.
Genotypic assays – PCR, DNA probes, sequencing.
Viruses – detected only by molecular/immunologic techniques, not plate culture.

Units used: meters → cm → mm → μm (micrometers) → nm (nanometers).
Viruses: $20$ – $800\,\text{nm}$ (smallest).
Small bacteria: $200$ nm.
Typical bacteria: up to $700\,\mu\text{m}$ .
Yeasts: $3$ – $4\,\mu\text{m}$ (classified with parasitology from therapeutic standpoint).
Protozoa (true parasites): $100$ – $300\,\mu\text{m}$ —largest under standard microscopy.

Culture turn-around time drives hospital result timelines (“couple of days”).
M. tuberculosis slow growth explains need for faster diagnostics (skin test, Quantiferon, chest X-ray).
BCG vaccine (given outside U.S.) → false-positive skin tests.
Hemolysis patterns correspond to pathogenic potential; β-hemolytic strep more aggressive.
Selective/differential media streamline searches from “needle-in-a-haystack” to precise ID.
Sterility of supplies is non-negotiable; contaminated plates would show growth before use.

High cost of ultrapure sterile supplies (e.g., $10$ – $15$ for a small vial of biomedical-grade water).
Reliance on biologically derived reagents: embryonic bovine serum, blood, or algal agar base—reminds us lab science still depends on living sources.
Historical ingenuity: many traditional media/techniques originated long ago yet remain foundational, though molecular tools now expand capabilities.