Domain: Healthcare — Microbiology-Immunology
Target concept: Pseudomonas aeruginosa Detection

Video generation prompt: Inoculate Pseudomonas aeruginosa culture into a citrate agar tube containing bromothymol blue indicator, and incubate at 37°C for 72 hours, observing changes in the color of the medium.

Evaluation guide:
Target concept: Citrate utilization by Pseudomonas aeruginosa as a diagnostic biochemical trait, mediated by enzymatic citrate transport and metabolism yielding alkaline end-products (e.g., NH₃, Na₂CO₃), detected via bromothymol blue (BTB) pH indicator shift. Minimum mechanistic assumptions: (i) BTB is a reliable, stoichiometric pH reporter with defined green (pH ≤6.8) and blue (pH ≥7.6) endpoints; (ii) P. aeruginosa expresses functional citrate permease and oxidative citrate cycle enzymes; (iii) alkalization requires viable, metabolically active cells, not passive diffusion or contamination. Phased storyline: [Phase 1: Baseline] Citrate agar tube shows uniform emerald-green color (BTB at pH ~6.8). [Phase 2: Intervention] Sterile loop streaks surface with P. aeruginosa culture. [Phase 3: Incubation] Tube placed in 37°C incubator; timer advances to 72 h. [Phase 4: Outcome] Medium transforms completely to homogeneous cobalt-blue, with no green remnants, confirming citrate-positive alkalization. Visibility constraints: Must show side-by-side control/test comparison under consistent, color-accurate lighting; agar surface must be fully in-focus; blue hue must be discriminable from purple or turquoise using standardized color reference; no occlusion of slant surface by condensation or fingerprints.

Manual:
I. Basic Demonstration Information
Demonstration Title: Pseudomonas aeruginosa Citrate Utilization Test (Simmons Citrate Agar with Bromothymol Blue) Demonstration
Subject: Healthcare - Microbiology-Immunology - Bacterial Biochemical Identification
Demonstration Purpose: To visually demonstrate the positive citrate utilization capability of Pseudomonas aeruginosa via alkaline pH shift-induced color change in Simmons citrate agar containing bromothymol blue indicator; to reinforce recognition of the key diagnostic phenotype (green → blue transition); and to master interpretation of biochemical test results in clinical microbiology.

II. Demonstration Carrier/Equipment
Core Carrier: Simmons citrate agar slant tube (1.5% agar, 0.2% sodium citrate, 0.008% bromothymol blue, pH 6.8), pre-incubated control tube (green), and test tube inoculated with pure P. aeruginosa culture (ATCC 27853 or equivalent)
Auxiliary Equipment: Sterile inoculating loop, 37°C incubator with visible temperature/time display, white backdrop, calibrated color reference card (for BTB green/blue), digital timer overlay (0 h / 72 h)

III. Demonstration Content Description
Layout: Two identical citrate agar tubes placed side-by-side on white background under uniform daylight-spectrum LED lighting. Left tube: uninoculated control (green). Right tube: inoculated with P. aeruginosa, labeled ‘+Pae’. Both tubes oriented vertically, caps closed during incubation, then opened for final observation.
State Control: Tubes maintained at 37°C for exactly 72 h (timer-verified); no condensation allowed to pool on agar surface (slant angle ≥30°); ambient CO₂ excluded (incubator not candle/CO₂-enriched).
Comparison Baseline: Identical lot number, expiration date, and batch-manufactured tubes; same lot of bromothymol blue; identical lighting and viewing angle to eliminate chromatic bias.

IV. Observation of Core Phenomena
Control Tube Color: Uniform, translucent emerald-green (pH ~6.8), no blue tinge, no growth.
Test Tube Color Change: Complete, homogeneous transition from initial green to vivid cobalt-blue (pH ≥7.6); color change affects entire slant surface, including upper and lower regions; no mottling, yellow zones, or precipitation.
Growth Correlation: Thin, translucent, non-pigmented bacterial growth visible as faint sheen or fine line along stab/streak — confirming viability and metabolic activity.
Stability & Specificity: Blue color persists upon gentle agitation; no reversion to green; no acid production (no yellow halos); confirms citrate-positive, alkaline metabolism unique to P. aeruginosa among common non-fermenters.

V. Summary of Key Knowledge Points and Principles
Core Concept Definition: The citrate test detects an organism’s ability to use citrate as the sole carbon and energy source, producing alkaline byproducts (e.g., sodium carbonate, ammonia) that raise medium pH, causing bromothymol blue to shift from green (pH 6.0–7.6) to blue (pH >7.6).
P. aeruginosa Significance: A definitive positive citrate test is a key differential trait distinguishing P. aeruginosa (citrate+) from other Gram-negative rods like Acinetobacter spp. (citrate−) or Enterobacter spp. (variable), supporting identification in clinical isolates.
Indicator Chemistry: Bromothymol blue is a sulfonephthalein dye with pKa = 7.1; its green color reflects mixed protonation states at neutral pH; deprotonation above pH 7.6 yields intense blue dianion — a stoichiometric, irreversible visual signal of sustained alkalization.

VI. Demonstration Precautions
Tube Integrity: Avoid scratching agar surface; ensure slant depth ≥2 cm to prevent false negatives from insufficient citrate exposure.
Inoculum Standardization: Use light, single stab-and-streak (not heavy inoculum) to avoid carryover of acidic metabolites from prior medium.
Timing Rigor: Strict 72 h incubation required — shorter durations yield weak/incomplete blue; longer durations risk overgrowth artifacts or CO₂ absorption reversing pH.
Color Verification: Always compare against fresh control tube and reference card under D65 lighting; avoid fluorescent or yellow-tinted ambient light that distorts blue perception.

Prompt Grounding rubric:
level_1: No citrate agar tube, no bromothymol blue color, and no inoculation action appear; there's no incubator or time label, the medium's color never changes (or shows a color that isn't green or blue at all), and there's no before/after comparison shown.
level_2: A citrate agar tube appears but its starting color isn't the expected green; the inoculation gesture is vague or aimed away from the medium; there's no labeled 37°C incubator or time stamp visible; any color change that occurs doesn't follow an alkalinization pattern (e.g., green turning yellow).
level_3: The tube is recognizable as green citrate agar, and a loop briefly touches the surface, but there's no clear time progression shown (no '0h' to '72h' label change); any color shift is ambiguous (a faint bluish tinge rather than a full green-to-blue change), and the incubator, if shown, is unlabeled or shows the wrong temperature.
level_4: The tube is labeled and starts clearly green; a sterile loop visibly contacts the medium surface; a labeled 37°C incubator and explicit time markers (e.g., '0 h' to '72 h') appear; the medium turns from green to a distinctly different blue, though a minor flaw remains, such as the blue looking slightly purple-tinged, the change happening with no visible intermediate teal tone, or small illegible label text.
level_5: Every element matches the prompt precisely: the tube is labeled 'Citrate Agar + Bromothymol Blue'; the first frame shows a uniform emerald-green medium; a sterile loop visibly streaks the surface; the incubator display reads '37°C' and '72 h'; the final frame shows an even, unambiguous blue medium with no patchiness or contamination marks; the before/after states are shown side by side or in a clearly time-stamped sequence.

Scientific and Causal Correctness rubric:
level_1: The color change contradicts the biology: e.g., green turns to yellow or red instead of blue; the organism is mislabeled (e.g., called 'E. coli' or shown as round, purple-staining cocci instead of a rod); there's no visible link between the organism and the color shift; the medium looks liquid or cracked rather than a solid agar slant.
level_2: The label reads 'P. aeruginosa,' but the depicted shape is wrong (chains, clusters, or spore-like forms instead of single rods); the color changes but breaks the pH logic: e.g., blue appears before any incubation time has passed, or before inoculation; the agar looks unstable (melting, bubbling, or fogged with condensation).
level_3: The organism label and the general idea that citrate metabolism should turn the medium blue are both present, but not reinforced visually: the medium starts green and ends blue, yet the blue looks patchy or washed-out, and there's no visible growth line or sheen along the streak to confirm the bacteria actually grew.
level_4: The full causal chain holds: inoculation introduces the labeled organism; the medium's only carbon source is citrate; metabolism raises the pH; the color shifts from green to blue with a crisp, even edge; the one gap remaining is the absence of a subtle growth cue (such as a faint sheen or growth line along the streak) even though the color change itself is scientifically sound.
level_5: The causality is both stated and shown: on-screen text or narration notes that citrate metabolism raises the pH; the medium starts as a uniform emerald green; after incubation it becomes a uniform, vivid blue with no green left and no yellow tinge; a thin, translucent growth line is visible along the streak, confirming the bacteria actually grew; nothing else competes for attention: no yellow patches, no gas bubbles, no mold.

Spatiotemporal Consistency rubric:
level_1: Severe spatiotemporal breaks: the tube turns from green to blue with no incubation period ever shown; the incubator is visible but the tube sits outside it; a hand holds the tube upside down; the room lighting flickers erratically; the tube spins in ways a hand wouldn't produce; or the loop looks bigger than the tube itself.
level_2: Major flaws: the '0 h' and '72 h' shots don't share the same background, angle, or tube position, so the time jump has no visual anchor; the tube moves or rotates noticeably between shots; the lighting shifts from warm to cool white in a way that would change how the color reads; the hand's motion during inoculation looks anatomically wrong; or the agar surface ripples like a liquid instead of holding as a gel.
level_3: Most spatial relationships hold: same background, fixed camera angle, consistent tube orientation; the incubation period is implied through a label or split screen but without a 'placing into the incubator' motion actually shown; lighting stays steady but with a slightly uneven shadow on one side; sped-up time is acceptable as long as the green-to-blue order is never reversed.
level_4: High fidelity: the clip reads as one continuous shot or a clean cut with matching framing and focus throughout; the tube's position and framing stay visually stable across frames; the lighting stays even and consistent in color so the green-to-blue shift reads clearly; the hand's motion (flaming the loop, removing the cap, streaking the surface) looks smooth and natural; the on-screen timer moves cleanly from 0h to 72h with no jumps or reversals.
level_5: Full consistency end to end: the recording holds one fixed framing with no cuts, the tube centered and evenly lit with no distracting reflections; the color shown is clearly a vivid cobalt/Prussian blue at the end versus a clear emerald green at the start, matching a reference color card rather than drifting toward purple or turquoise; the hand's grip, loop angle, and streaking motion all look natural and unforced; nothing floats or defies gravity, and the agar surface never deforms unnaturally; the timer overlay matches the stated incubation time and stays legible throughout.