Week 6 – Selective & Differential Media, Streak for Isolation

Course & Lab Logistics

• Week: 6 of BIOL-107
• Today’s in-lab assessments:
  • Lab Quiz 5 (covers Streak for Isolation, Special Media: EMB & MSA, Microscopy Skills)
  • Microscopy Skills Test
• Next-week deliverables:
  • Pre-Lab 7 (complete online)
  • Lab Quiz 5 retake / make-up if needed

Core Vocabulary

• Selective medium
  • Formulated to prevent or strongly inhibit the growth of certain taxa while allowing others to thrive.
• Differential medium
  • Contains indicators that detect observable metabolic differences among organisms growing on the same plate, most often expressed as a color change.
• Enterobacteriaceae ("enterics")
  • Large family of Gram-negative (G—) bacilli typically isolated from the gut or soil.
  • Characteristic trait: ability to ferment lactose and/or other simple sugars.

Eosin Methylene Blue Agar (EMB)

• Selective Components
  • Eosin Y + Methylene Blue dyes create an environment that inhibits Gram-positive (G+) bacteria.
• Differential Components
  • Lactose is the fermentable carbohydrate.
  • The dyes act as pH-responsive indicators.
• Reading EMB plates
  • Metallic green, black, or deep purple colonies = vigorous acid production ⇒ strong lactose fermenters (e.g., Escherichia coli).
  • Pinkish/"fish-eye" colonies = weak acid production ⇒ slow/partial fermenters (e.g., some Enterobacter spp.).
  • Colorless or transparent colonies = non-fermenters (e.g., Pseudomonas aeruginosa).
• Representative organisms used in lab
  • Escherichia coli (G— rod, strong fermenter → metallic green sheen)
  • Staphylococcus epidermidis (G+, growth inhibited; serves as negative control)
  • Pseudomonas aeruginosa (G— rod, non-lactose-fermenter → colorless)

Mannitol Salt Agar (MSA)

• Selective Component
  • High NaCl concentration 7.5\% (w/v)
  • Halotolerant Gram-positive staphylococci survive.
  • Most other bacteria (including G— enterics) are killed or severely inhibited.
• Differential Components
  • Carbohydrate: mannitol.
  • Indicator: Phenol Red (yellow in acid, red at neutral pH, hot pink in basic).
• Reading MSA plates
  • Yellow medium around colonies = mannitol fermentation → acidification (diagnostic for Staphylococcus aureus).
  • No color change (medium remains red) = non-fermenter (e.g., Staphylococcus epidermidis).
• Representative organisms used in lab
  • Escherichia coli (G— rod, growth inhibited)
  • Staphylococcus epidermidis (growth +, no yellow zone)
  • Staphylococcus aureus (growth +, bright yellow zone)

Concept Connections & Significance

• Both EMB and MSA combine selective and differential properties, enabling a two-step logic:
  \text{(1) Can it grow?} \rightarrow \text{(2) If it grows, what does the color say about its metabolism?}
• Rapid preliminary identification assists in:
  • Clinical diagnosis (e.g., differentiating pathogenic S. aureus from commensal staphylococci).
  • Environmental or food-safety testing (detecting fecal contamination via enterics).
• Demonstrates the importance of physiological ecology—knowing salt tolerance, dye tolerance, and metabolic pathways to inform isolation strategies.

Streak for Isolation (T-Streak Method)

• Goal: obtain isolated single colonies ⇒ genetically pure culture for downstream tests.
• Principle: serial dilution on solid medium; each successive sector receives fewer bacteria.
• Plate layout (T-streak)
  • Sector 1: heavy inoculum
  • Sector 2: light streaks coming off sector 1 (after flaming loop)
  • Sector 3: lightest streaks coming off sector 2 (after flaming again)
• Detailed Protocol
  1. Gather sterile loop, labeled agar plate (EMB or MSA), don PPE.
  2. Aseptically pick colony / mix culture; apply to Sector 1.
  3. Flame loop; cool.
  4. Drag through edge of Sector 1 3–4 times, then streak into Sector 2.
  5. Flame loop; cool.
  6. Drag through edge of Sector 2 and streak into Sector 3.
  7. Flame loop; invert plate (agar up); place on class incubation tray.
• Alternatives: Quadrant streak (four sectors) for even greater dilution when cells are very dense.
• Analytical Endpoint: observe morphology & color changes on special media after incubation (usually 24–48 h at 37\,^{\circ}\mathrm{C}).

Example Plate Interpretations

• EMB plate showing metallic-green colonies isolated in Sector 3 → presumptive E. coli.
• MSA plate with golden-yellow halo in Sector 2 → presumptive S. aureus.
• Lack of growth on EMB but profuse pink colony growth on MSA (no yellow) → likely S. epidermidis (G+ staph, non-mannitol-fermenting).

Practical, Ethical & Safety Notes

• Correct isolation and identification are critical for antibiotic stewardship (choosing proper therapy).
• Biosafety Level 2 procedures apply to S. aureus and P. aeruginosa; improper technique can lead to lab-acquired infections.
• Disposal: plates with potential pathogens must be autoclaved before discarding.

Quick-Reference Color Key

• EMB
  • Metallic green / black / deep purple → strong acid \Downarrow strong lactose fermenter
  • Pink “fish-eye” → weak acid \Downarrow slow fermenter
  • Colorless → no fermentation
• MSA
  • Yellow medium → acid \Downarrow mannitol fermented (likely S. aureus)
  • Red / unchanged → no fermentation (other staph or halotolerant genera)

Equations & Concentrations Mentioned

• Salt concentration of MSA: 7.5\%\ \text{NaCl}
• Typical incubation: 24–48\ \text{h} at 37\,^{\circ}\mathrm{C}
• Dilution pattern in T-streak approximates powers of 10 (each sector reduces CFU by (~10^1–10^2)).

Study Tips for Lab Quiz 5

• Memorize selective vs differential definitions verbatim; quiz questions often rephrase textbook language.
• Be able to match organism ↔ expected plate appearance (e.g., E. coli on EMB, S. aureus on MSA).
• Diagram the T-streak from memory; label flame steps.
• Review previous microscopy skills: magnification calculation \text{Total Magnification} = \text{Ocular} \times \text{Objective}.
• Practice interpreting mixed-culture streak plates: decide which colony to pick for further testing.