Domain: Natural Science — Microbiology
Target concept: The phenomenon of immediate production of a large number of bubbles in hydrogen peroxide-positive reactions

Video generation prompt: Take a colony of *Escherichia coli* and add 3% hydrogen peroxide solution.

Evaluation guide:
Target concept: Catalase-mediated decomposition of hydrogen peroxide in Escherichia coli, producing immediate, macroscopic oxygen gas evolution. Minimum mechanistic assumption: E. coli expresses active catalase enzyme that rapidly converts H₂O₂ → H₂O + O₂(g), and this reaction is both necessary and sufficient to generate the observed bubbling under 3% H₂O₂. Phased storyline: (1) Static frame shows isolated off-white E. coli colony on sterile surface; (2) Pipette descends and delivers single colorless H₂O₂ droplet onto colony center; (3) At moment of contact (t=0), bubble nucleation erupts *exclusively at colony surface*; (4) Within ≤0.3 s, bubbles expand radially with vigorous coalescence and upward buoyancy; (5) Peak effervescence (t=1–2 s) shows dense, frothy, dynamic bubble field occupying >3× initial droplet area; (6) Bubbling subsides by t=4 s, leaving wet colony unchanged. Visibility constraints: Camera must maintain tight focus on colony-H₂O₂ interface; lighting must highlight bubble transparency, curvature, and motion; no occlusion by hand or equipment; perspective must be perpendicular to surface to verify spatial origin and radial spread.

Manual:
I. Basic Demonstration Information
Demonstration Title: Catalase Test for Hydrogen Peroxide-Positive Reaction in Escherichia coli
Subject: Natural Science - Microbiology - Biochemical Identification
Demonstration Purpose: To visually demonstrate the immediate, vigorous bubbling caused by catalase enzyme activity in E. coli when exposed to 3% hydrogen peroxide; to enable recognition of the catalase-positive phenotype through characteristic gas evolution; and to reinforce the principle that this rapid effervescence is diagnostic for aerobic/facultative bacteria possessing catalase.
II. Demonstration Carrier/Equipment
Core Carrier: Fresh, isolated colony of Escherichia coli (18–24 h growth on nutrient or blood agar, off-white, smooth, 1–2 mm diameter)
Auxiliary Equipment: Sterile glass slide or agar plate section; calibrated dropper or 10-μL loop; 3% hydrogen peroxide solution (freshly prepared, colorless, aqueous); white/light background for contrast; bright, diffuse illumination.
III. Demonstration Content Description
Layout: Place E. coli colony centrally on clean glass slide or cut agar square. Position under even lighting with white background. Hold dropper vertically 1–2 cm above colony.
State Control: Use colony grown under standard aerobic conditions (no anaerobic stress); avoid over-aged or dried colonies. Ensure H₂O₂ is unexpired and stored cold to preserve reactivity.
Comparison Baseline: No control colony needed for this demonstration—focus is on *presence* and *immediacy* of bubbling as the sole positive indicator.
IV. Observation of Core Phenomena
Expected Phenomenon: Within ≤0.3 seconds of H₂O₂ droplet contact, a sudden, dense, and expanding cloud of small, rapidly rising bubbles emerges exclusively from the colony surface. Bubbles coalesce, overflow droplet boundary, and sustain for 2–4 seconds with vigorous effervescence. No bubbling occurs on adjacent agar or slide surface.
Visual Feature Recognition: Key identifiers are (1) temporal immediacy (no latency), (2) spatial confinement (originates only at colony interface), (3) morphological signature (small, spherical, fast-rising, coalescing gas bubbles), and (4) intensity (≥100 visible bubbles, >3× area expansion).
V. Summary of Key Knowledge Points and Principles
Core Concept Definition: Catalase is an endogenous bacterial enzyme that decomposes hydrogen peroxide (H₂O₂) into water and oxygen gas. A positive catalase test is defined by *immediate, copious bubbling* upon exposure to 3% H₂O₂—indicating presence of functional catalase.
Scientific Significance: This reaction protects aerobic bacteria from oxidative damage by neutralizing toxic H₂O₂ generated during respiration. E. coli is catalase-positive, distinguishing it from catalase-negative species (e.g., Streptococcus). The visual intensity and speed directly reflect catalase concentration and activity level.
VI. Demonstration Precautions
Colony Freshness: Use 18–24 h culture—older colonies may show reduced activity; never use boiled or heat-killed controls unless explicitly comparative.
H₂O₂ Concentration: Strictly use 3% (v/v); higher concentrations cause nonspecific oxidation; lower concentrations yield weak or delayed reactions.
Contamination Avoidance: Do not touch colony with loop before adding H₂O₂; avoid carryover of blood (from blood agar) which causes false positives.
Timing Emphasis: Video must capture the first 3 seconds—critical window for confirming immediacy. Delayed or gradual bubbling indicates false negative or technical error.

Prompt Grounding rubric:
level_1: No visible colony of E. coli and no hydrogen peroxide solution added; no pipette, no agar plate or culture medium; no liquid droplet contact event; completely unrelated scene (e.g., plant, animal, abstract graphic).
level_2: A microbial colony-like object (e.g., speckled white blob) is present, and a clear liquid is dispensed, but not onto the colony; or liquid is added to an empty surface; no spatial co-location of colony and H₂O₂; timing order reversed (e.g., bubbles appear before addition).
level_3: A visible bacterial colony (small, off-white/cream-colored circular spot) is placed on a solid surface (e.g., agar plate or sterile slide); 3% H₂O₂ solution is visibly added via dropper/pipette *onto* the colony, but bubble generation is delayed (>2 s), weak (<5 bubbles), or localized only at edge; colony size or color deviates significantly from typical E. coli (e.g., large, yellow, or fuzzy).
level_4: Correct colony: small (1–2 mm), smooth, convex, off-white/creamy, circular, isolated colony on sterile surface (agar or glass slide); 3% H₂O₂ solution (colorless, slightly viscous droplet) is delivered precisely onto center of colony; immediate (≤0.5 s) onset of bubbling observed, but bubble volume or density is suboptimal (e.g., ~20–50 bubbles, moderate rise, no vigorous overflow); all core objects and spatial relationships correct.
level_5: Perfect prompt grounding: (i) clearly identifiable *Escherichia coli* colony: 1–2 mm diameter, smooth, glistening, off-white, circular, isolated on sterile agar plate or clean glass slide; (ii) labeled or contextually unambiguous 3% hydrogen peroxide solution (colorless, aqueous droplet); (iii) pipette/dropper delivers one discrete droplet *directly onto colony center*; (iv) within ≤0.3 s of contact, vigorous, dense, and sustained bubbling erupts: ≥100 visible bubbles, rapid expansion (>3× original droplet area), frothy effervescence with continuous gas evolution; (v) strict temporal sequence: colony → pipette approach → droplet contact → instantaneous bubble surge.

Scientific and Causal Correctness rubric:
level_1: No bubbling occurs at all; or bubbles appear spontaneously without H₂O₂ addition; or bubbles are static, non-gaseous (e.g., soap-like foam with no motion); no causal link between addition and reaction.
level_2: Bubbling occurs but is slow (>3 s delay), sparse (<10 bubbles), and inert (no upward movement or expansion); bubbles resemble condensation or water droplets, not gas-driven; colony changes color or dissolves unrealistically; no catalase logic implied.
level_3: Bubbling begins within 1–2 s after addition; moderate number (~30–60 bubbles); bubbles rise and coalesce but lack vigor and continuity; colony remains morphologically intact, with no lysis or discoloration; causal link is perceptible but weak (e.g., no visual contrast between pre- and post-addition states).
level_4: Bubbling initiates ≤0.8 s post-contact; vigorous, dynamic, and continuous CO₂/O₂ gas release visible as rising, merging, bursting microbubbles; colony remains stable (no dissolution, swelling, or pigment change); visual contrast sharply highlights ‘before’ (calm, wet colony) vs. ‘after’ (effervescent eruption); catalase-mediated decomposition (2H₂O₂ → 2H₂O + O₂) is plausibly implied by scale, speed, and morphology of bubbles.
level_5: Instantaneous (≤0.3 s), explosive, high-density bubbling: bubbles originate *exclusively from colony surface*, expand radially outward in <1 s, occupy >4× initial droplet footprint, show turbulent coalescence and bursting dynamics consistent with O₂ gas evolution; colony retains precise shape, texture, and color throughout, with zero degradation or artifact; causal chain is visually unambiguous: droplet contact → immediate enzymatic catalysis → macroscopic gas release; no competing phenomena (e.g., splashing, evaporation, condensation) obscure the core catalase-positive reaction.

Spatiotemporal Consistency rubric:
level_1: Severe spatiotemporal breaks: colony appears/disappears mid-sequence; droplet floats in air without gravity; bubbles form before pipette motion; abrupt cuts or frame jumps >0.5 s; inconsistent lighting/shadow direction across frames.
level_2: Noticeable temporal discontinuity: ≥2 frame drops or stutters; pipette motion jerky or non-linear; bubbles appear with unnatural delay or freeze mid-rise; inconsistent perspective (e.g., colony shifts position between shots without camera motion); lighting changes abruptly.
level_3: Minor temporal gaps: one brief stutter or slight frame jitter; pipette approaches smoothly but stops abruptly before contact; bubbles rise with mild deceleration or uneven density; colony remains fixed in frame, but background subtly shifts; lighting stable but shadows lack directional consistency.
level_4: Smooth, continuous timeline: pipette descends steadily at natural speed (≈0.2–0.4 s descent), contacts colony with visible impact (micro-splash), and bubbling begins immediately without lag; bubbles evolve continuously (nucleate, rise, merge, burst) with consistent upward vector and gravity-aligned motion; colony stays centered and stable; lighting uniform, with soft directional highlight enhancing bubble contrast.
level_5: Flawless spatiotemporal fidelity: (i) pipette tip moves in smooth parabolic descent (accelerated then decelerated), makes precise contact with audible *soft click* (optional but reinforcing), and retracts cleanly; (ii) bubble nucleation starts *at exact contact point* and propagates radially outward in <0.2 s; (iii) bubble field maintains coherent upward buoyancy, consistent bubble size gradient (smaller at base, larger at top), and realistic turbulence; (iv) zero frame drops, flicker, or perspective shift; (v) lighting remains constant with specular highlight on droplet pre-contact and diffuse reflection off froth post-contact, fully physically grounded.